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https://openalex.org/W2980971688	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0223650&type=printable	English	null	Urban commuting dynamics in response to public transit upgrades: A big data approach	PloS one	2,019	cc-by	9,165	RESEARCH ARTICLE Qi-Li Gao1, Qing-Quan LiID1,2,3, Yan Zhuang1, Yang Yue2,3, Zhen-Zhen Liu4, Shui- Quan Li4, Daniel Sui5 1 State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, Wuhan University, Wuhan, P.R. China, 2 Shenzhen Key Laboratory of Spatial Smart Sensing and Services, Department of Urban Informatics, School of Architecture and Urban Planning, Shenzhen University, Shenzhen, P.R. China, 3 Guangdong Key Laboratory of Urban Informatics, Shenzhen University, Shenzhen, P.R. China, 4 College of Computer Science and Software Engineering, Shenzhen University, Shenzhen, P. R. China, 5 Department of Geosciences, University of Arkansas, Fayetteville, AR, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * liqq@szu.edu.cn (QQL); yueyang@szu.edu.cn (YY) Editor: Matthew Kahn, Johns Hopkins University, UNITED STATES UNITED STATES Received: February 8, 2019 Accepted: September 13, 2019 Published: October 17, 2019 Copyright: © 2019 Gao 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: February 8, 2019 Accepted: September 13, 2019 Published: October 17, 2019 Copyright: © 2019 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The dataset is obtained through academic cooperation with the Traffic Operation Command Center, Shenzhen Transport Committee in China. The dataset can be accessed at the Shenzhen Transport Committee and Shenzhen University for academic purposes. Anyone who wants to access raw data can contact the Traffic Operation Command Center, Shenzhen Transport Committee directly (http://jtys.sz.gov.cn/ ; email jtzx12328@jtys.sz.gov.cn) or Shenzhen Key Laboratory of Spatial Smart Sensing and Services, Shenzhen University (https://spatial.szu.edu.cn/; email spatial@szu.edu.cn). OPEN ACCESS OPEN ACCESS Citation: Gao Q-L, Li Q-Q, Zhuang Y, Yue Y, Liu Z- Z, Li S-Q, et al. (2019) Urban commuting dynamics in response to public transit upgrades: A big data approach. PLoS ONE 14(10): e0223650. https:// doi.org/10.1371/journal.pone.0223650 Citation: Gao Q-L, Li Q-Q, Zhuang Y, Yue Y, Liu Z- Z, Li S-Q, et al. (2019) Urban commuting dynamics in response to public transit upgrades: A big data approach. PLoS ONE 14(10): e0223650. https:// doi.org/10.1371/journal.pone.0223650 Editor: Matthew Kahn, Johns Hopkins University, UNITED STATES Urban commuting dynamics in response to public transit upgrades: A big data approach Qi-Li Gao1, Qing-Quan LiID1,2,3, Yan Zhuang1, Yang Yue2,3, Zhen-Zhen Liu4, Shui- Quan Li4, Daniel Sui5 Abstract Public transit, especially urban rail systems, plays a vital role in shaping commuting pat- terns. Compared with census data and survey data, large-scale and real-time big data can track the impacts of urban policy implementations at finer spatial and temporal scales. Therefore, this study proposed a multi-level analytical framework using transit smartcard data to examine urban commuting dynamics in response to rail transit upgrades. The study area was Shenzhen, one of the most highly urbanized and densely populated cities in China, which provides the opportunity to examine the effects of rail transit upgrades on com- muting patterns in a rapidly developing urban context. Changes in commuting patterns were examined at three levels: city, region, and individual. At the city level, we considered the average commuting time, commuting speed, and commuting distance across the whole city. At the region level, we analyzed changes in the job accessibility of residential zones. Finally, this study evaluated the potential effects of rail transit upgrades on the jobs-housing relationship at the individual level. Difference-in-difference models were used for causal inference between rail transit upgrades and commuting patterns. In the very short term, the opening of new rail transit lines resulted in no significant changes in overall commuting pat- terns across the whole city; however, two effects of rail transit upgrades on commuting pat- terns were identified. First, rail transit upgrades enhanced regional connectivity between residential zones and employment centers, thus improving job accessibility. Second, rail transit improvement increased the commuting distances of individuals and contributed to the separation of workplaces and residences. This study provides meaningful insights into the effects of rail transit upgrades on commuting patterns. Urban commuting dynamics in response to public transit upgrades Kong, public transit is the travel mode most frequently used for daily commuting. For instance, more than half of the people in New York use the public transportation system [3]; in Tokyo, more than 70% of the population travels on public transit [4], and in Hong Kong, over 90% of people use transit [5]. In particular, the high capacity and efficiency of rail transit (e.g. metro and light rail) has led it to enjoy priority in the transportation and land use development strategies of high-density or compact cities [6, 7]. Worldwide, many cities have made massive investments in improving their urban rail transit systems, especially in fast-growing develop- ing countries [8–10]. Funding: This research was supported by the National Natural Science Foundation of China (Grant no. 41671387) and the National Natural Science Foundation of China-Joint Programming Initiative Urban Europe (Grant no. 7181101150). 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. It is widely believed that efficient public transit can improve job accessibility and address mobility problems [11, 12]. A growing quantity of literature has investigated the connection between rail system improvement and commuting behavior, particularly changes in public transit usage [2, 13]. In the U.S. context, transit ridership has been declining rapidly despite significant improvements in public infrastructure [8]. However, cities like Paris, London, and Tokyo have highly efficient urban rail transit systems that provide effective connections between city center and outer suburbs [14]. Public transit improvements also affect commut- ing patterns by changing residence and jobs locations [15]. Specifically, improvements in urban transportation infrastructure can affect the physical layout of cities by changing the dis- tributions of residences and employment opportunities. Some evidence indicates that expan- sion of a transport network contributes to decentralization of both the urban population and employment, which produces changes in commuting patterns [16]. In recent decades, China has witnessed rapid urban growth and construction of transporta- tion infrastructure. This has resulted in great changes in the spatial organization of residences and employment opportunities, leading to changes in commuting patterns [17–19]. Unlike in Western cities, a large proportion of low-income residents in Chinese cities live in the subur- ban areas and outskirts, and employment decentralization is not as pronounced. Introduction Public transit systems play significant roles in urban development and in shaping commuting behaviors [1, 2]. In high-density populated cities such as New York, Paris, Tokyo, and Hong 1 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Most job opportunities remain concentrated in the inner city, especially for low-skilled workers [20]. As a result, significant commuting inequality appeared in large Chinese cities, and many obstacles prevent people from moving closer to employment centers, particularly limited affordable housing resources in the city center [21–23]. As a result, large Chinese cities have developed significant commuting inequality. In response to these issues, local governments have been actively building rail transit systems, endeavoring to strengthen the geographical connection of suburban residents with employment centers in the inner city [9]. These projects provide opportunities to examine the effects of rail transit upgrades on commuting patterns. Surveys and censuses are two data sources commonly used in traditional commuting stud- ies. However, individual demographics and survey data are usually difficult to access, and sam- ple sizes are always limited. Additionally, it is impossible to estimate effects on individual commuting behavior from aggregated census data; thus, census data is usually used to examine changing commuting patterns at either national or regional levels. In recent years, the develop- ment of information and communication technology has enabled the continuous generation of various large-sample and fine-scale movement datasets, which can effectively capture human behaviors in daily life [24]. One such source for urban big data is public transit smart- card data, which records users’ travel behaviors. Personal commuting information (e.g. resi- dence, workplace and commuting time) can be extracted according to daily travel regularity [25, 26]. More importantly, smartcard records are automatically generated when travel occurs, making it possible to examine commuting dynamics and the jobs-housing relationship at finer temporal resolutions than is feasible with slowly upgraded conventional data. In this sense, real-time movement data enable us to evaluate the short-term and long-term impacts of urban policy implementations in an effective way. real-time movement data enable us to evaluate the short-term and long-term impacts of urban policy implementations in an effective way. 2 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades This study aims to supply evidence regarding the influence of new rail transit lines on com- muting patterns through a case study of Shenzhen, China. Due to the interaction of various factors influencing urban commuting dynamics, the effects of rail transit cannot be intuitively reflected in the overall commuting patterns. Hence, this study introduces a multi-level and individual-based analytical framework to explore commuting dynamics from different per- spectives. The proposed methodology can examine how and to what extent rail transit upgrades affect urban commuting patterns. Specifically, this study aims to address issues at three levels. 1) The city level: what was the average commuting time, commuting speed, and commuting distance across the whole city before and after rail transit upgrades? 2) The region level: what was the change in accessibility of residential zones to employment centers with rail transit upgrades? 3) The individual level: how did the newly opened rail transit lines reshape individual jobs-housing relationships? Difference-in-difference models were used to explore the associations between rail transit upgrades and manifestations of commuting behaviors. This study contributes to existing research in two ways. Firstly, this study proposes an analyti- cal framework for using big data to identify commuting dynamics in response to urban rail transit upgrades. Secondly, the findings in this study illustrate how improvements in rail tran- sit reshape urban commuting patterns in a rapidly- developing country and provide some evi- dence for urban policies and planning strategies. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Data and methodology Public transit smartcard data from two periods were used to explore dynamic changes in the com- muting patterns and jobs-housing relationships of public transit commuters. The first period is 5–11 Jan. 2015, before the opening of three new metro lines (i.e. metro lines 7, 9 and 11), and the second period is 22–28 Nov. 2016, after their opening. When a rider uses a smartcard to pay for a public transit trip, the unique card number, boarding time, boarding vehicle number (bus trip), and boarding/get-off station (metro trip) are automatically recorded. For bus trips, the boarding station and get-off station were inferred from bus GPS data and bus line data. For multimodal transfers, bus trips and metro trips were merged based on the unique card numbers. Fig 2 shows the analytical framework of this study. Typical public transit riders often com- mute between relatively stable places (i.e. residence and workplace) at relatively fixed times; therefore, residences and workplaces can be identified based on specific commuting regularity and spatial-temporal repeat patterns. This study defines as residence the most frequent board- ing station (visited at least three times in a week) of a smartcard user during morning peak hours (6:00~10:00 am). Similarly, a rider’s workplace is defined as the most frequently visited place where the rider stayed for six hours or more. Commuting time is the duration of trips between one’s residence and workplace. For a bus rider, the travel distance is defined as the actual network distance based on bus lines, and for a subway rider as the length of the shortest path based on metro lines. According to the above definitions, approximately 0.63 million commuters were identified out of a total ridership of 2.48 million during the first period, and over 0.96 million were iden- tified from a ridership of 2.57 million during the second period. Given that our analysis is based on comparisons between two identified groups over the two periods, the two groups should have similar demographics. Evidence shows that 2% sampling rate is sufficient in modeling intra-city human mobility patterns (e.g., travel distance and travel time) using public transit smart card data [27]. Therefore, we consider the identified two groups with more than 20% sampling rates are demographically and statistically similar in this study. Urban commuting dynamics are jointly determined by co-location of residences and jobs and transport systems. Study area The study area, Shenzhen, covers 1997.47 km2, consisting of ten districts and 491 traffic analy- sis zones (TAZ) (Fig 1). As one of the most highly urbanized and densely populated cities in China, Shenzhen housed 11.4 million permanent residents in 2015, which increased to 11.9 million the following year (Shenzhen Statistical Yearbook 2016, 2017). The city center is con- stituted of three central districts, Nanshan, Futian and Luohu. Due to urban sprawl, the city’s public transit systems are highly developed. While Shenzhen’s bus system and road networks are relatively stable, its rail transit system has expanded rapidly. At present, it has eight rail transit lines, covering a total of 285 kilometers. Five of the eight metro lines were opened before 2015; the other three (i.e. metro lines 7, 9, and 11) were opened in 2016, increasing the number of metro stations from 131 to 199. Specifically, metro lines 7 and 9 opened on October 28 and mainly serve the central areas. Metro line 11, opened on June 28 and also known as the “airport line”, connects the inner city and suburbs. In addition, more than twenty additional metro lines are under construction or being planned for construction in the coming ten years; this is due to the ‘70/70’ transport plan that calls for 70% of daily travel to be made by public Fig 1. Study area and metro lines. https://doi.org/10.1371/journal.pone.0223650.g001 Fig 1. Study area and metro lines. Fig 1. Study area and metro lines. https://doi.org/10.1371/journal.pone.0223650.g001 3 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades transit, and for 70% of those trips to be made by rail transit. This rapid and ongoing develop- ment of rail infrastructure may continuously change the commuting patterns and spatial lay- out of residences and employment opportunities in Shenzhen over the next ten years, which provides us with excellent opportunities to examine how rail transit upgrades affect urban commuting dynamics in a high-density urban context. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Fig 2. Analytical framework of this study. Data and methodology The development of rail transit affects both commuting efficiency and individual jobs-housing locations. However, individual relocations are also influenced by other factors, such housing factors and individual socioeconomic levels. Due to the interac- tions of various influencing factors, the effects of rail transit on the commuting dynamics can- not be intuitively reflected in the overall patterns. Based on this concern, this study designs a multi-level and individual-based analytical framework to explore commuting dynamics to examine how and to what extent rail transit upgrades affect urban commuting patterns from different perspectives. At the city level, we focus on overall commuting patterns across the whole city, including average commuting time, commuting distance, and commuting speed. At the region level, we concentrate on the changes in commuting efficiency and job accessibil- ity of residential zones. We further detect which areas enjoyed prominent reductions in com- muting time. At the individual level, we use commuting distance as a proxy for jobs-housing separation to explore how rail transit upgrades have influenced the spatial relationships of individuals’ residences and workplaces. 4 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades Fig 2. Analytical framework of this study. https://doi.org/10.1371/journal.pone.0223650.g002 Analysis and findings Overall commuting dynamics. This study first analyzed overall commuting dynamics across the whole city. Fig 3 shows the probability density distribution of one-way commuting time, commuting distance, and commuting speed, along with the relationship between com- muting time and distance. Commuting patterns were derived using all commuting trips, including both bus trips and metro trips. In general, similar distribution patterns were observed in commuting distance and time for both study periods. The largest proportion of commuters traveled 3–4 km from home to work in about 10 minutes. After the highest value, the probability density presents a decay pattern that it decreases as commuting time and dis- tance increase. Commuting speed (commuting distance/commuting time) generally follows a normal distribution, with values ranging from 0 to 60 km/h. Table 1 shows the average commuting time, distance, and speed before and after rail transit upgrades. On the whole, the average commuting distance for bus trips is considerably shorter than that for metro trips. In 2015, the average commuting distances were about 8 km (bus) and 12 km (metro). The average commuting speed of rail transit was 25 km/h. This is much lower than the running speed of rail because commuting time includes not only in-vehicle 5 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades Fig 3. Overall commuting dynamics: (a) 2015, (b) 2016. https://doi.org/10.1371/journal.pone.0223650.g003 Fig 3. Overall commuting dynamics: (a) 2015, (b) 2016. Fig 3. Overall commuting dynamics: (a) 2015, (b) 2016 https://doi.org/10.1371/journal.pone.0223650.g003 https://doi.org/10.1371/journal.pone.0223650.g003 travel time but also the waiting time at stations, which is noticeable due to the traffic delay and crowding, especially during peak hours. The average commuting speed of the bus system was around 20 km/h. Because the bus speed is significantly lower than that of rail transit, the bus system mainly serves short- and middle-distance travel while rail transit is developed to serve long-distance trips. However, the two travel modes are competitive in some areas that are cov- ered by both systems. In the short term, no significant changes were found in average commuting time or dis- tance across the whole city. However, the proportion of people traveling by rail transit increased from 33.62% to 42.59% after the opening of new metro lines. This increase is consis- tent with existent evidence that improvements in rail transit have positive effects on promoting travel modal shifts. Urban commuting dynamics in response to public transit upgrades Table 2. Percentage and cumulative percentage of commuters in different transit time intervals. Time Interval (minutes) 2015 2016 Percentage (%) Cumulative Percentage (%) Percentage (%) Cumulative Percentage (%) 0–15 28.23 28.23 28.38 28.38 15–30 40.52 68.75 39.39 67.77 30–45 20.66 89.41 21.03 88.80 45–60 7.82 97.23 8.14 96.94 60–75 2.19 99.42 2.39 99.33 75–90 0.47 99.89 0.54 99.87 >90 0.11 100 0.13 100 Total 100 - 100 - Table 2. Percentage and cumulative percentage of commuters in different transit time intervals. In summary, in the very short term after the opening of new metro lines, no considerable changes are observed in average commuting distance and time across the whole city. Does it mean that the opening of new rail transit lines has no impact on urban commuting patterns? If not, how and to what extent rail transit upgrades contribute to commuting dynamics? To answer this question, we further examine the potential effects of rail transit upgrades on the commuting patterns at the region level. Regional changes in accessibility to employment centers. At the region level, we con- cern about the changes in commuting efficiency brought by rail transit upgrades. The increase in commuting efficiency can lead to better job accessibility. Therefore, we evaluated the effect by examining the changes in job accessibility of residential zones to employment centers. To carry out this analysis, this study first identified the typical employment centers of Shenzhen, areas with high job densities. Fig 4 presents the analytical framework for identifying employ- ment centers. First, the kernel density method was used to generate the employment density surface of the whole city based on workplace locations derived from smartcard data. To extract high-density employment centers from this surface, we generated a contour map and then Fig 4. Identification of employment centers: (a) distribution of jobs, (b) kernel density map of employment, (c) contour map of employment, (d) employment centers. tion of employment centers: (a) distribution of jobs, (b) kernel density map of employment, (c) contour map of employment, centers Fig 4. Identification of employment centers: (a) distribution of jobs, (b) kernel density map of employment, (c) contour map of employment, (d) employment centers. Fig 4. Identification of employment centers: (a) distribution of jobs, (b) kernel density map of employment, (c) contour map of employment, (d) employment centers. Analysis and findings However, the average travel speed of bus trips was lower in 2016 than that in the preceding year. This study further examined changes in the population commuting for different time inter- vals, i.e. 0–15 minutes, 15–30 minutes, 30–45 minutes, 45–60 minutes, 60–75 minutes, and 75–90 minutes (Table 2). Only a small proportion of public transit commuters travel very long distances; most public transit users are short- and middle-distance commuters. The highest proportion ride for an interval of 15–30 minutes, followed by 0–15 minutes. Approximately 70% of public transit commuters live within 30 minutes travel of their workplaces, and over 90% of commuting trips are less than 45 minutes. This finding suggests that most commuters reside in areas where they can travel to their workplaces by public transit within a reasonable commuting time. There was no obvious change in the proportion of very short-distance (within 15 minutes) commutes after the opening of new rail transit lines. The proportion of middle-distance (between 15 and 30 minutes) commutes decreased slightly, while long-dis- tance (more than 30 minutes) commuting trips increased slightly. Table 1. Average commuting distance/time/speed across the whole city. Commuting indicators 2015 2016 Bus Metro Bus & Metro Bus Metro Bus & Metro 66.38%: 33.62% 57.41%: 42.59% Commuting distance (km) 7.98 12.48 9.53 7.34 12.14 9.33 Commuting time (minutes) 23.49 28.57 25.25 23. 72 28.15 26.40 Commuting speed (km/h) 21.21 25.27 22.61 19.41 25.22 21.81 https://doi.org/10.1371/journal.pone.0223650.t001 Table 1. Average commuting distance/time/speed across the whole city. Commuting indicators 2015 2016 Bus Metro Bus & Metro Bus Metro Bus & Metro 66.38%: 33.62% 57.41%: 42.59% Commuting distance (km) 7.98 12.48 9.53 7.34 12.14 9.33 Commuting time (minutes) 23.49 28.57 25.25 23. 72 28.15 26.40 Commuting speed (km/h) 21.21 25.27 22.61 19.41 25.22 21.81 https://doi.org/10.1371/journal.pone.0223650.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 6 / 18 Table 1. Average commuting distance/time/speed across the whole city. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 6 / 18 https://doi.org/10.1371/journal.pone.0223650.g004 https://doi.org/10.1371/journal.pone.0223650.g004 7 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades classified the density values into five grades using the Jenks classification method. Areas belonging to the fifth grade of the contour map were selected as typical employment centers. Four typical employment centers were identified (Fig 4(D)); all were located in the inner city, suggesting a centralized employment structure for Shenzhen. One employment center is the High-tech Park (HTP) in the Nanshan district, at which the majority of high-tech compa- nies and industries are located. Chegongmiao (CGM) is located in the Futian central business district (CBD) and is the most important financial center in Shenzhen. Another typical employment center is the COCO Park and Exhibition Center (CPEC), which is the biggest shopping, business, and entertainment center in the Futian district. Finally, the highest-density employment center is Huaqiang North and the Laojie area (HNL), which is located in the Luohu CBD. HNL is not only the heart of urban economic activity for Shenzhen but also the most heavily populated area and a cultural hotspot. Ultimately, as the majority of job opportu- nities are located in the city center, job accessibility can be represented as proximity to employ- ment centers in the inner city. h d f b b l l l d h This study uses commuting time as a proxy for job accessibility. We calculated the average commuting time from each residential zone to each employment center based on individual commuting time and residence. Suppose that Zij is the collection of all the commuters who live in TAZ i and work in employment center j. AveTimeij represents the commuting time from residential zone TAZ i to employment center j, and is calculated as follows: AveTimeij ¼ P k2Zijtk Nij ð1Þ ð1Þ where tk is the commuting time of commuter k, and Nij is the total number of commuters who live in TAZ i and work in employment center j; i =1,. . .,491; j = HTP,CGM,CPEC,HNL. We evaluate average effects of rail transit upgrades on job accessibility of residential zones by using a difference-in-difference (DID) estimator. In this model, we controlled for the dis- trict fixed effect and the year fixed effect. The treatment group consisted of residential zones having centroids within 2 km of a station on a newly-opened metro line. Urban commuting dynamics in response to public transit upgrades Table 3. Results of DID model for regional commuting time to typical employment centers. Employment center Commuting time (min) Coef. Std.Err. t P>\|t\| [95% Conf. Interval] HTP (constant) 43.371 0.245 176.76 0.000 42.887 43.854 treatedperiod -2.168 0.887 -2.44 0.015 -3.916 -0.421 (period) after rail transit upgrades 4.350 0.666 6.53 0.000 3.038 5.662 R2 0.240 CGM (constant) 39.194 0.335 116.85 0.000 38.533 39.855 treatedperiod -4.314 1.273 -3.39 0.001 -6.822 -1.806 (period) after rail transit upgrades 5.843 1.128 5.18 0.000 3.620 8.066 R2 0.209 CPEC (constant) 39.705 0.392 101.32 0.000 38.933 40.477 treatedperiod -5.358 1.398 -3.83 0.000 -8.112 -2.604 (period) after rail transit upgrades 3.367 1.092 3.08 0.002 1.216 5.517 R2 0.078 HNL (constant) 40.932 0.225 182.14 0.000 40.490 41.375 treatedperiod -3.250 0.802 -4.05 0.000 -4.828 -1.671 (period) after rail transit upgrades 4.578 0.676 6.77 0.000 3.248 5.909 R2 0.247 https://doi.org/10.1371/journal.pone.0223650.t003 Table 3. Results of DID model for regional commuting time to typical employment centers. commuting efficiency and reducing the travel time of residents who use public transport for commuting [28, 29]. To explore which parts of city enjoyed the greatest reductions in commuting time, we cal- culated the distances from each residential zone to each employment center and the corre- sponding changes in commuting time. Residential zones with reduced commuting times were used to generate a kernel density trend map, shown in Fig 5. The horizontal and vertical coor- dinates are the reduced time, expressed as Δtime (minutes), and distance to employment cen- ter, represented as distance (km), respectively. The effect of new metro lines on residential zones varies depending on the associated employment center. For the HTP employment center, the residential zones that benefited most are those 7–22 km away (Fig 5(A)). In general, the commuting times for these residential zones were reduced by 0–4 minutes. For the CGM and CPEC employment centers, most of the residential zones that benefited are located within 15 km and enjoyed commute improve- ments of 0–6 minutes (Fig 5(B) and 5(C)). Meanwhile, commuting times from residential zones distributed within 20 km of the HNL employment center were only reduced by 0–2 min- utes (Fig 5(D)). Fig 5(E) illustrates the density surface of all benefiting residential zones associ- ated with the four employment centers. The control group consisted of residential zones that are more than 2 km away from the new rail stations. The model is expressed as follows: AveTimeit ¼ b0 þ b1 treatedi periodt þ mi þ lt þ εit ð2Þ ð2Þ treatedi ¼ 1; if minðdistancehtazi; stopsiÞ 2kmÞ 0; if minðdistancehtazi; stopsiÞ > 2kmÞ ð3Þ ( ð3Þ periodt ¼ 0; if t ¼ before rail transit upgrades 1; if t ¼ after rail transit upgrades ð4Þ ( ð4Þ where μi and λt are the district fixed effect and the year fixed effect, respectively; εit represents the error term; and β1 is the average treatment effect of rail transit upgrades on the job accessi- bility of residential zones. The results of the DID model are shown in Table 3. For each employment center, all variables were significantly associated with commuting time. Notably, the coefficient of the average treatment effect is negative and significant at the 0.01 or 0.001 level; this indicates that rail transit upgrades can reduce commuting time from residential zones to employment centers and thereby increase job accessibility. Generally, the average commuting time decreased by 2–5 minutes in the treated area. This result is consistent with existing evidence that improvements in public transport have positive effects, improving PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 8 / 18 The majority of residential zones that benefited are located within 20 km of the employment centers and enjoyed 0–6 minute’s reduction in com- muting times. To determine which residential zones enjoyed the greatest improvement, we selected those whose commuting times were reduced by more than 2 minutes, illustrated in Fig 5(F). Most of these residential zones are very close to the new metro stations, which is con- sistent with the abovementioned finding that rail transit upgrades can strengthen the connec- tions between nearby residential zones and employment centers as well as improve personal mobility. 9 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Fig 5. Changes in commuting time to employment centers: (a) HTP, (b) CGM, (c) CPEC, (d) HNL, (e) all typical employment centers, (f) spatial distribution of residential zones that benefited. Urban commuting dynamics in response to public transit upgrades Urban commuting dynamics in response to public transit upgrades Fig 5. Changes in commuting time to employment centers: (a) HTP, (b) CGM, (c) CPEC, (d) HNL, (e) all typical employment centers, (f) spatial distribution of residential zones that benefited. Fig 5. Changes in commuting time to employment centers: (a) HTP, (b) CGM, (c) CPEC, (d) HNL, (e) all typical employmen spatial distribution of residential zones that benefited. https://doi.org/10.1371/journal.pone.0223650.g005 https://doi.org/10.1371/journal.pone.0223650.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades Table 4. Distribution of commuters in the inner city and suburbs. Working Population (Percentage %) Residential Population (Percentage %) 2015 2016 inner city suburb total inner city suburb total inner city 46.16 24.86 71.02 37.23 25.38 62.61 suburb 3.63 25.35 28.98 4.39 33.00 37.39 total 49.79 50.21 100 41.62 58.38 100 https://doi.org/10.1371/journal.pone.0223650.t004 Table 4. Distribution of commuters in the inner city and suburbs. https://doi.org/10.1371/journal.pone.0223650.t004 in the inner city decreased from 49.79% to 41.62%, based on residence locations derived from smartcard data. This indicates that more commuters resided in suburban areas in 2016. Along- side this suburbanization of the population is a trend of shifting employment from the inner city to the suburbs as well; and this employment decentralization is as significant as population suburbanization. In 2015, more than 70% of job opportunities were concentrated in the inner city; but by 2016, the proportion of the population working in the inner city decreased rapidly, from 71.02% to 62.61%. This means that fewer people in the suburbs commute to the city cen- ter for work; instead more seek jobs in the suburbs. The percentages of suburb-to-center and suburb-to-suburb commuting trips increased while the within-center commutes decreased greatly. In particular, the proportion of suburb-to-suburb commuters increased significantly, from 25.35% to 33.00%. Taken as a whole, this jobs-housing redistribution presents another manifestation of changing commuting pattern. in the inner city decreased from 49.79% to 41.62%, based on residence locations derived from smartcard data. This indicates that more commuters resided in suburban areas in 2016. Along- side this suburbanization of the population is a trend of shifting employment from the inner city to the suburbs as well; and this employment decentralization is as significant as population suburbanization. In 2015, more than 70% of job opportunities were concentrated in the inner city; but by 2016, the proportion of the population working in the inner city decreased rapidly, from 71.02% to 62.61%. This means that fewer people in the suburbs commute to the city cen- ter for work; instead more seek jobs in the suburbs. The percentages of suburb-to-center and suburb-to-suburb commuting trips increased while the within-center commutes decreased greatly. In particular, the proportion of suburb-to-suburb commuters increased significantly, from 25.35% to 33.00%. Taken as a whole, this jobs-housing redistribution presents another manifestation of changing commuting pattern. Although rapid suburbanization has affected commuting patterns, e.g. Relocation of individual residences and workplaces In addition to positive effects on improving job accessibility, rail transit can also affect com- muting patterns through changing the distribution of residential and workplace locations. Since people prefer to live and work near rail transit services [1], the improvements of urban rail systems may encourage people to relocate or look for job opportunities in the areas served by new rail transit lines [30]. Therefore, to identify the potential impact of rail transit improve- ment on commuting patterns, changes in commuters’ residence and workplace locations should be considered. Table 4 shows overall trends in jobs-housing redistribution across the whole city. Gener- ally, the city evidences a rapid suburbanization trend. The proportion of commuters who lived PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 10 / 18 center-to-center commutes changed into suburb-to-suburb commutes, the effects of suburbanization on com- muting distance and time are complex. Relocation to suburban districts does not necessarily lead to increases in commuting distance or time. For example, although some commuters resettled in the suburbs, they may find jobs around their new homes instead of remaining with their prior employers. To understand the changes in commuting pattern associated with indi- vidual relocation, we extracted commuters who moved or changed their jobs during the study period based on changes in residences or workplaces. Following the study in [31], relocated commuters were classified into three categories: 1) home movers, transit commuters who moved with job unchanged; 2) job hoppers, commuters who changed workplace without changing residence; and 3) job and residence switchers, commuters who changed both work- place and home during the study period. Changes in the distance between home and work- place were calculated for each of these three subgroups. As depicted in Fig 6, for each subgroup, the proportion of relocated commuters decreased rapidly with increasing Δdistance. This suggests that most commuters tend to maintain or slightly change the distance between their homes and workplaces, which is consistent with existing evidence that when workers change locations, they prefer commuting zones (i.e., time and distance) similar to their previous commuting zones. These behaviors lead to stable aver- age city-wide commuting time and distance, regardless of high residence and workplace mobility [32]. The symmetrical pattern observed for Δdistance indicates a dynamic balance of jobs-housing redistribution across the whole city. This result is consistent with the findings related to overall dynamics in Table 1 that there is no considerable change in average commut- ing distance. Although the city has witnessed a lot of reallocations over the two years, the large-scale relo- cations to suburban areas are not only driven by rail transit upgrades. Individual’s relocations are motivated by various factors. In addition to transport systems, people are also influenced and constrained by land use patterns, housing factors, individual socioeconomic attributes, and personal preferences. An empirical study of Shenzhen has found that a large number of PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 11 / 18 Urban commuting dynamics in response to public transit upgrades Fig 6. Changes in distances for (a) home movers, (b) job hoppers, and (c) job and residence switchers. https://doi.org/10.1371/journal.pone.0223650.g006 Fig 6. Changes in distances for (a) home movers, (b) job hoppers, and (c) job and residence switchers. https://doi.org/10.1371/journal.pone.0223650.g006 Fig 6. Changes in distances for (a) home movers, (b) job hoppers, and (c) job and residence switchers. https://doi.org/10.1371/journal.pone.0223650.g006 https://doi.org/10.1371/journal.pone.0223650.g006 public transit commuters moved to suburban areas and urban periphery due to surging hous- ing prices in 2015 [33]. In this study, we are interested in how and to what extent rail transit development contrib- utes to changes in individual commuting patterns. To evaluate this effect, we extracted all relo- cated individuals (i.e. home movers, job hoppers, job and residence switchers) and applied another DID model. In this model, the commuting distance is considered as the dependent variable, which measures the extent of home-work separation. We defined transit users who commuted by new metro lines (7, 9, and 11) in 2016 as the treatment group. The model is expressed as follows: distanceit ¼ b0 þ b1 treatedi periodt þ mi þ lt þ εit ð5Þ ð5Þ treatedi ¼ 1; if individuali 2 treatment group 0; if individuali 2 control group ð6Þ ( ð6Þ periodt ¼ 0; if t ¼ before rail transit upgrades 1; if t ¼ after rail transit upgrades ð7Þ ( periodt ¼ 0; if t ¼ before rail transit upgrades 1; if t ¼ after rail transit upgrades ð7Þ ( ð7Þ Where distanceit is the commuting distance of individual i in year t. μi and λt are the indi- vidual fixed effect and the year fixed effect, respectively; εit is the error term. The results of the DID model for relocated individual commuting distances are given in Table 5. The average treatment effect is statistically significant at the 0.001 level and presents a positive association with commuting distance. On average, the commuting distance of relocated individuals increased by 8.6 km due to the opening of new rail transit lines. This suggests that the Table 5. Results of DID model for relocated individual commuting distance. Distance (km) Coef. Robust Std.Err. t P>\|t\| [95% Conf. Interval] (constant) 11.249 0.019 580.80 0.000 11.211 11.287 treatedperiod 8.568 0.399 21.470 0.000 7.786 9.350 (period) after rail transit upgrades -0.624 0.039 -16.05 0.000 -0.700 -0.548 R2 0.013 https://doi.org/10.1371/journal.pone.0223650.t005 Table 5. Results of DID model for relocated individual commuting distance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 12 / 18 Urban commuting dynamics in response to public transit upgrades improvement of rail transit can motivate residents to live farther from their jobs or seek job opportunities farther away from home, exacerbating the separation of residence and workplace. These effects can be explained by two potential factors. On factor is the high commuting efficiency that results from rail transit upgrades, which reduces the cost of commuting to employment centers and other destinations, in turn, increases the attractiveness of new station areas, causing population relocation to zones near the new rail transit lines. Therefore, the opening of suburban metro lines would incite people to relocate into less central areas, leading to the increase in their commuting distances. Moreover, rail transit upgrades also influence commuting patterns through effects on the real estate values of neighboring zones. According to the theory of urban spatial equilibrium based on the trade-off between accessibility and cost of space, reduced travel costs and rising demand to live close to new transit stations are expected to increase housing costs in the bene- ficiary areas [34, 35]. To illustrate these effects, we applied a DID model to the average housing unit prices of residential zones before and after rail transit upgrades derived from a real estate rental and sales service platform in China, Anjuke (https://shenzhen.anjuke.com/). The DID model and results are provided in S1 Appendix. The results demonstrate that the rail transit upgrades considerably increased housing prices in treatment areas close to the new rail transit lines. Consequently, people who cannot afford the extremely high housing costs in central areas will reduce rents by changing locations, moving to the suburban zones along new rail transit lines. As a result, rail transit expansion increases population density in the suburbs and commuting distances of relocated individuals. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Discussion In this study, we conducted the analysis at three levels (i.e. city-region-individual) and drawn the following findings: 1) The average commuting distance and time did not change signifi- cantly across the whole city in the study period. 2) The opening of new rail transit lines reduces the commuting time of new station zones to typical employment centers. 3) Rail transit upgrades increase individual commuting distances by influencing residence and job relocations. Why does the overall commuting time remain stable while new rail transit lines reduce the com- muting time in the treatment zones? Why does the overall commuting distance have no obvious change while new rail transit lines increase individual commuting distances? The overall com- muting dynamics are the outcomes of various influencing factors, the impact of rail transit can- not be intuitively reflected in the overall commuting patterns. In order to examine the changes in commuting patterns brought by only rail transit upgrades, we conducted the analysis at the region level and individual level. Therefore, the influencing results of rail transit upgrades on regions and individuals may not be consistent with the overall commuting dynamics. Moreover, as we aim to study the impacts on commuting dynamics from different perspec- tives (i.e. commuting efficiency and jobs-housing separation), the region level analysis and individual level analysis focus on different groups. The target group for region level analysis can be considered as the commuters who live in the treated zones and work in the employment centers. New rail transit lines increased their commuting efficiency and reduce their commut- ing time with constant commuting distance. The target group for individual level analysis is the commuters who originally lived in the control area and relocated to the treated area. After relocations, their commuting distance increased. For these commuters, their commuting time may increase as well. The two effects will jointly affect the overall commuting dynamics. Therefore, the respective result of region level analysis or individual level analysis may be inconsistent with the overall commuting patterns. 13 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades Fig 7. Relationship between individual, region and urban commuting indicators. https://doi.org/10.1371/journal.pone.0223650.g007 Fig 7. Relationship between individual, region and urban commuting indicators. Discussion https://doi.org/10.1371/journal.pone.0223650.g007 In addition, the analysis at the region level and individual level are based on different analy- sis units, the increase (decrease) in individual commuting indicators may not result in the increase (decrease) in regional commuting indicators. We illustrate the relationships between the commuting indicators of three levels, as displayed in Fig 7. The commuting time and dis- tance of individual i are expressed as Ti and Di, then the regional average commuting time and distance are Tregion ¼ P i2regionTi nregion and Dregion ¼ P i2regionDi nregion , where nregion is the number of commut- ers in the region. The average commuting time and distance at the city level are defined as Tcity ¼ Pn i¼1 Ti n and Dcity ¼ Pn i¼1 Di n , where n is the number of commuters in the city. Firstly, changes in individual’s commuting time/distance can generate a variety of possible outcomes on the regional average commuting time/distance. Increase in individual’s commut- ing time/distance may not lead to an increase in average commuting time/distance in the origi- nal residential area or resettled region. It depends on the numerical relationship (i.e. larger, smaller and equal) between individual’s commuting time/distance and regional average com- smaller and equal) between individual’s commuting time/distance and regional average com- muting time/distance. Fig 8 illustrates three examples that describe different situations. Although all the highlighted commuters moved from one region to another and the individu- al’s commuting time/distance increased, the effects of the relocations on regional commuting patterns vary. In Fig 8(A), the commuter’s previous and present commuting time/distance are respectively larger than the average commuting time/distance in the original residential region and relocated region, therefore the relocation would lead to the reduction in average commut- ing time/distance in the original region while increase in the resettled region. For the com- muter in Fig 8(B), he used to live close to where he works and then moved to a farther place, which would lead to an increase in the average commuting time/distance in both his original and resettled regions. Inversely, the commuter’s relocation behavior in Fig 8(C) would lead to the reduction in commuting time/distance in both regions. The increase in individual’s commuting time/distance is positively associated with the increase in overall commuting time/distance. Regardless of other unknown factors influencing commuting dynamics, the stable overall commuting distance may be attributed to the rela- tively short study period. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Discussion The data used in this study were collected over a period of only five months after the opening of one new metro line (No. 11) and one month for the other two (No. 7 and 9). Although new rail transit can motivate people to relocate, only a very small number of commuters were affected over a period of a few months because residential mobil- ity is time-consuming and complicated. Compared with the total millions of transit users, the PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 14 / 18 Urban commuting dynamics in response to public transit upgrades Fig 8. Illustrations of the effect of individual’s relocation on regional commuting indicators. https://doi.org/10.1371/journal.pone.0223650.g008 Fig 8. Illustrations of the effect of individual’s relocation on regional commuting indicators. https://doi.org/10.1371/journal.pone.0223650.g008 population of relocated commuters is not large enough to make a noticeable difference in overall commuting distance across the whole city. Two factors may contribute to the stable average commuting time. The first is the decrease in commuting speed of bus trips alongside the increased speed of new metro systems (see Table 1). The second possible reason is the “rebound effect”, in which increased speed due to rail transit upgrades reduces commuting cost to make people commute farther, which has been demonstrated in this study. Although improvement of rail transit reduces the travel time of some commuters who live in the treatment area, the greater distance has an opposing effect on the overall commuting time. This shed light on the importance of examining commuting dynamics in response to rail transit upgrades at multiple levels. In the long term, an increase in average commuting distance is expected across the whole city. General demand to live close to efficient public transit will increase local demand for living in the new rail transit zones, thereby attracting more people to relocate into the outer metro zones. This assumption is supported by existing evidence that the expansion of urban rail transit causes suburbanization [8]. Furthermore, rail transit upgrades affect commuting patterns by increasing real estate values; namely, high housing price and rents in the central zones motivate people to relocate to suburban metro zones, leading to increases in their commuting distances. However, the change in commuting time is unpredictable. In addition to the “rebound effect”, population suburbanization and employment decentralization also play vital roles in changing commuting time. Discussion Therefore, changes in commuting time and distance might be not consistent between different development contexts and land use patterns. For instance, exist- ing research shows that commuting time remains relatively stable despite an increase in aver- age commuting distance [36]. Conclusions This study utilized individual-based public transit smartcard data to analyze urban commuting dynamics in response to rail transit upgrades. Using personal commuting information (i.e. 15 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223650 October 17, 2019 Urban commuting dynamics in response to public transit upgrades residence, workplace, commuting time, and commuting distance) derived from smartcard data before and after the opening of new rail transit lines, this study proposed a multi-level analytical framework to explore changes in commuting patterns, including city level, region level and individual level. Difference-in-difference models were adopted to evaluate the effects of the rail transit system on commuting patterns. This study has demonstrated that improve- ment of rail transit had positive effects on improving the job accessibility of residential zones and reducing commuting time to employment centers. Meanwhile, new rail transit also pro- moted individual relocation, which increased the distances between residences and workplaces. Although this study examined only the short-term effects of rail transit improvement, the proposed analytical framework is meaningful in response to the illustration of dynamic changes and exploration of the trend. In the future, we will examine the long-term effects of rail transit upgrades on urban commuting behaviors by collecting data across multiple years. Furthermore, rail transit improvement not only influences the jobs-housing relationships of public transit commuters but also has an impact on the commuting behaviors of all workers, especially in promoting modal shift; this is another subject for future research. In general, the improvement of rail transit will have a positive effect on job accessibility. These improvements enhance personal mobility and regional connectivity, which are helpful in promoting rail tran- sit use and reducing automobile dependence [9]; thus, the jobs-housing relationships of all workers and the urban spatial arrangement can be changed by transit development. However, these impacts cannot be identified from transit users’ travel behaviors alone; therefore, the effect of rail transit improvement on all workers was not analyzed in this study. To determine the overall effect on jobs-housing mismatch for the city, a longitudinal panel of household travel survey data is needed. 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https://openalex.org/W3082175155	https://zenodo.org/record/2228386/files/36_IJRG18_A10_1777.pdf	English	null	ASSESSMENTS OF THE DETERMINANTS OF INVESTMENT ACTIVITIES IN NEKEMTE TOWN	International journal of research - granthaalayah	2,018	cc-by	9,469	ASSESSMENTS OF THE DETERMINANTS OF INVESTMENT ACTIVITIES IN NEKEMTE TOWN Tesfaye Eresso Gofe 1 1 Lecturer, Department of Accounting and Finance; College of Business and Economics; Wollega University; Ethiopia Cite This Article: Tesfaye Eresso Gofe. (2018). “ASSESSMENTS OF THE DETERMINANTS OF INVESTMENT ACTIVITIES IN NEKEMTE TOWN.” International Journal of Research - Granthaalayah, 6(11), 366-383. https://doi.org/10.5281/zenodo.2228386. Cite This Article: Tesfaye Eresso Gofe. (2018). “ASSESSMENTS OF THE DETERMINANTS OF INVESTMENT ACTIVITIES IN NEKEMTE TOWN.” International Journal of Research - Granthaalayah, 6(11), 366-383. https://doi.org/10.5281/zenodo.2228386. ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] ISSN- 2350-0530(O), ISSN- 2394-3629(P) (Received: September 22, 2018 - Accepted: November 01, 2018) DOI: 10.5281/zenodo.2228386 Management Abstract Investment activity plays a crucial role in the economic growth of a country. Economic literatures show that investment is, both empirically and theoretically, the key determinant to economic growth. Investment can increase a country’s productive capacity, provided that investment expenditure regards durable goods that have comparatively long useful lives and embody the latest technological advances. Economic growth refers to an increase in a country’s production or income per capita. The study assessed the determinants of investment activities in Nekemte town which is found in Oromia region with 328km away from Addis Ababa the capital city of Ethiopia. The research was investigated the major determinants of investment activities in the town and indicates the direction for investment how to deal with those determinants. For this study both primary and secondary data were used. Because of the heterogeneity of the activities the researcher was used stratified sampling techniques and the sample size was computed using solvin’s formula. In this research the descriptive analysis was employed. Some of the main findings of the study were difficulties of finance and lack of credits when they started their business, and low encouragement from the investment offices are the major findings of this study. Finally, investment related problem is not the responsibility of the government alone, but also all investors, the local community, the private business man of the town has to work together with the government so as to attain the desired objectives. Keywords: Investment; Investment Activity; Determinants. 1. Introduction Economic literatures show that investment is, both empirically and theoretically, the key determinant to economic growth. Economic growth refers to an increase in a country’s production or income per capita. It is usually measured by gross national product or gross national income, used interchangeably, an economy’s total output of goods and services. Investment is the source of manufactured goods that will be used to produce other goods. It is the major foundation of Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [366] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) (Received: September 22, 2018 - Accepted: November 01, 2018) enhancement in the level of literacy, improvement in technology and increase in the capital stock (Hashmi et al 2012). enhancement in the level of literacy, improvement in technology and increase in the capital stock (Hashmi et al 2012). A rate of investment is one of the key factors that differentiate developed countries from developing countries. In high-growth countries investment is high, where as it is low in low growth countries. The implication of low investment is that the productive capacity of the economy fails to increase. This in turn leads to lower rates of growth and job creation, and fewer opportunities for the poor to improve their livelihoods (White, 2005). According to Sackey 2007, countries with high standards of living are those who have shifted the economic structure from traditional and less diversified to a more diversified one. Commitment to investment is the central issue in the process of structural diversification. Economic growth has a long been one of primary goals in virtually every country of the world weather developed or developing. To this end capital formation, proper mobilization and allocation of resources and reduction unutilized resources to the productive sector of the economy is very crucial. Growth requires investment of both domestic and foreign, both public and private calls for increased domestic mobilization. (Todaro, 2005). Investment activity plays a crucial role in the economic growth of a country. Investment can increase a country’s productive capacity, provided that investment expenditure regards durable goods that have comparatively long useful lives and embody the latest technological advances. In addition, changes in investment expenditure can potentially result in shifts in the level of employment and personal income by affecting the demand for capital goods. 1. Introduction Although gross fixed capital formation usually represents a substantially smaller fraction of an economy’s total expenditure compared to consumption expenditure, it is a highly volatile component that causes strong fluctuations to a country’s economic activity (Olga Kosma, 2015). According to Keynes (1964) cited in Olga Kosma, (2015), investment is volatile because it is determined by the “animal spirits” of investors (optimism and pessimism). Furthermore, investment creates new capital goods so it is a very important determinant of an economy’s long- run productive capacity, in the sense that a higher investment rate suggests that capital stock is growing rapidly. According to Bahar Bayraktar Economic growth and development depend essentially on a country’s ability to invest and make efficient and productive use of its resources. In this regard, the role of the private sector is important both in terms of its contribution to the quantity of Gross domestic investment and its ability to allocate and employ resources efficiently. (Bahar Bayraktar, 2003). Briefly, there cannot be growth without investment of sufficient amount and quality. In fact, investment is both a result and cause of economic growth. Therefore, it is important to examine the main determinants of investment activities. Based on the literature review, this study has identified both dependent and independent variables. It is expected that dependent variable is affected by multiple factors like economic, social, political, institutional, cultural, geographical and etc. This study, however, focuses on macroeconomic factors affecting private investment activities. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [367] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) The role of investment to the development of a nation is very large. Investment increases the productive capacity of an economy. It is a very important to utilize untapped resource to create job opportunity for citizen, to increase foreign currency through import substitution and export promotion of a nation and facilitate the communication and cultural exchange of society. Investment is critical determinant of a long run economics performance. Investment involves the formation of capital, fixed capital, such as reputation or technical knowledge; human capital such as: skill or education (Bond and Jan king 1996). The investment sector is the main engine of growth in market economy. 1. Introduction It thrives and delivers sustained growth when number of factor combines to product conductive environment for the investment sector to develop (Blejer & khan, 2004). Investment played a great role in country’s development especially in developing country whose capital is scarce and their government lack of enough capacity to cover all constrain and bring economic change for development (Asmelash, 2007). Thus, investment sector involved in investment the out and growth domestic product (GDP) country increase (Hernadeze-cata, 2000). One of which could be promoting demand countries have shown that growth the economic have come through increased investment. Thus, investment play a vital role for economic growth and development and for improving the welfare of the society recent studies (collier gunning, 1999; dikumana, 2000; Hernadeze-cata, 2000) conducted has established the critical linkage between investment and the rate of economic growth. Ethiopia is known with ancient civilization, historical culture, proud of tradition and on the top of all the land with immense potential for economic development of a country .The country is rich in natural resource and human resource. The climate and fertile soil are suitable for the cultivation of wide ranges of crops and live stocks. But this land of plenty is also suffering from poverty, lack of infrastructure, under development health center and fundamental ills of the economy system and reacts up on to keep the country under development. Nekemte town’s people almost reflect the real situations of a country in Ethiopia. The town has abundant natural and human resources. It is the habitual of various location of best kept secret of tourism site. It is natural resource such as mineral, forest, energy and diversified of climatic conditions, geographical area of the town hold the interest of the investors. For developing countries like Ethiopia the basic question in their economy is increase the production and hence improve the standard of living of their people so that there will be dramatic change in their economic, political and social conditions. For this purpose different alternatives are on the table. Investment promotion is one key instrument and primary engine of economic growth (Mustefa, 2014). As a result due attention has been given to development of private sector in developing countries to help improve economic growth (Ouattara, 2004). 1. Introduction Understanding the status and determinants of private investment activities is essential for successful and effective implementation of sustainable development goals [368] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) As investment plays a great role in economic development, the government of Ethiopia has given emphasis concerning this activity. Its purpose was to develop the investment activity by giving major incentives for investors and promotion of investment opportunities both for domestic and foreign investor so as to increase the number of participants in this activity. But due to social, political & economic constraints this activity has not shown a remarkable development in the town. Some research findings on promotion of the determinants of investment activities is concluded that, investment is constrained by the interest rate and individual demand determination, human capital base weaker and less diversified economy. Different researches were conducted on the area of determinants of investment activity. The study done by Belay focuses on bureaucracy related problem, power supplies as factors that determine investment activity (Belay Kucchi, 2005). Similarly the research under taken by Gatberuach (2015) finds factors that determine the investment activity are investment incentive, and interest rate. Though there are many factors that determine the investment activities this study would be conducted by taking some factors such as economic factors, socio cultural factors, political factors, corruption, institutional problem like land holding policy, lack of advisory. Thus, this study is conducted to identify determinants of investment activity particularly in Nekemte town. The study attempts to address the question of what really motivate individuals or organizations to invest in Nekemte. According to Edwin, et al. (2006), it is when individuals and organizations anticipate future cash needs, and expect that their earnings in the future will not meet those needs, that they take a decision to invest and make their money grow. However, before they decide as to where they think they will put their money to grow, they take into account numerous factors and these are the factors that this study attempts to identify. The objective of the study is to identify the determinants of investment activities in the Nekemte town. 2. Literature Review Many scholars and academicians have defined the term of investment differently. According to Mankiew investment is defined as spending today for future benefits and it’s the component of national income that links with future”. (Mankiew, 2002) Investment has been viewed and defined by different ways. It has different meaning in finance and economics. In economics investment is related to saving and deferring consumption it involved in many areas of the economy, such as business management and finance whether for households, firms or government. In finance investment is putting money into something with the expectation of gain, usually over a longer term. The term investment refers to a sum of funds committed on the physical and human cavity by both profit and no profit oriented individuals and institutions. It is applied to production of goods not meant for immediate consumption but further production of goods such goods are called, investment goods. The investment of business firms usually comprises of capital goods and inventories (Baddely, 2005). According to Dr. Mohammad, Investment is the flow of capital which is used for productive purposes. There is a great emphasis on investment for being the primary instrument of economic growth and development for a country. Investment means an increase in capital spending and it helps in creating a robust economy (Dr. Mohammad Shafi, 2014). In economics, investment can be defined as the purchase of plant, equipment or inventory. In lay terms investment is the acquisition of an asset such as a stock or a bond. Once an individual receives income, there are two alternatives to spend or to save it. Regardless of how you use your income, investment can be defined as postponed consumption. Individuals may postpone their current consumption to accumulate for the sake of accumulating. For any or all of these reasons individuals save part of their income rather than spend all of their income. The above analysis shows that investment has a strong relationship with saves income. But the extent of investment also depends on the level of consumption. As Mayra observes “there are no other road of economic development than a compulsory rise in the share of the nation’s income which is withheld from consumption. In an economy where living standard of the masses are too low, to curb consumption, it is difficult to mobilize and allocate resource in to investment activity. (mayrad,2003). 1. Introduction The specific objective of the study is focused on the following: The objective of the study is to identify the determinants of investment activities in the Nekemte town. The specific objective of the study is focused on the following: town. The specific objective of the study is focused on the following: • To identify Investments activities mostly involved in the town. • To identify Investments activities mostly involved in the town. • To identify the major factors of investment activities in the town and examine how this factors affect the investment. • To identify the major factors of investment activities in the town and examine how this factors affect the investment. To identify the major investment activities obstacles and opportunities in the town With the above objectives, this research paper answers the following general questions, which are useful in identifying the determinants and opportunities of investment activities in Nekemte town: To identify the major investment activities obstacles and opportunities in the town With the above objectives, this research paper answers the following general questions, which are useful in identifying the determinants and opportunities of investment activities in Nekemte town: • What types of investment activities involved mostly in the town and why • What are the major factors that determine investment activities? • What are the major investment activities obstacles and opportunities in the t This research paper may enrich the knowledge of the reader on the determinants of investment activities. It may add to the existing literature and serve as an additional source of reference. This paper also has the significance in initiating researchers to study the problem in depth, so that the researchers and readers understand the determinants and opportunities of investment in the town. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [369] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] 2. Literature Review As investment activity is affected by social, political and economic condition, there are some criteria have to be considered before starting investment activity. It is obvious that before starting investment activity, one investor must know and analyze in what area he is going to invest, the available funds he/she has for investment, the economic and political situations and profitability. In addition, an investor should know the advantage for the society. That is the quality and quantity of goods and service to be produced, employment, export and utilization of natural resources. In Ethiopia foreign and direct investment has been steadily growth the federal and regional government encourage investment: they provide land incentive such as; tax holding, an improved bureaucracy at the federal and regional investment office. The Ethiopian government special focus on investment and private investors both Ethiopian and non-Ethiopian national undertaken [370] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) investment activities in the agriculture, construction and manufacturing sectors, flower farm, cement factory, steel melting and rolling mills are becomes more and more common in Ethiopia. Investment is the current commitment of dollar (birr) for period of time in order to drive further payments that will compensate the investors: the time the funds are committed, the expected rate of inflation and the uncertainty of the future payment investment activities in the agriculture, construction and manufacturing sectors, flower farm, cement factory, steel melting and rolling mills are becomes more and more common in Ethiopia. Investment is the current commitment of dollar (birr) for period of time in order to drive further payments that will compensate the investors: the time the funds are committed, the expected rate of inflation and the uncertainty of the future payment From this we can answer the question about why people invest and what they want from their investments. They invest to earn a return either income or capital appreciation from saving due to their deferred consumption. Investment emphasis to the capital to be used for investment as well as the risk associated with investment in two ways. The first one is Investment is the commitment of funds with the view to minimize risk and safeguarding capital while earning return (investment constructed with speculation). 2. Literature Review The other one is investment is the commitment of something other than money to a long or term interest or project (Caves 1993). In modern changing economic nature and scope it is defined by the agreement of the association of south. East Asian nations (ASENS) given due emphasis to its scope as the term investment every find of asset and in particulate shall include through of exclusively. In other words investment can be showed as: Movable and immovable property and any other property right such as mortgage liens and pledges; Shares, stocks and debentures of companies or interest in the property in such companies; Claim to money or any performance under contract having a financial value and intellectual property right and good will; Business concession conferred by law or under contract including concession to reach for, cultivate, extract or exploit natural resources (UNCTAD, Vol.11. 1999. P. 19) Element of Investment Element of investment are those which are common in investment activity. These are return, risk and time. Return refers to reward from investment which includes currently income, capital gain or losses and profits. Risk is the change when the expected or prospective again or profit less they expected outcomes is the degree of happiness of losses. Time is the important factor in investment is the time which offers different courses of action. As time move on analysis believe that conditions changes and investors revaluate expected return for each investment (Garagdhia and Babuks, 2003). 3. Conceptual Framework Independent Variables  Economic factors  Political factors  Institutional factors  Socio cultural factors  Geographical factors  others Dependent Variables  Investment activities Source: literatures (conceptual framework) Independent Variables  Economic factors  Political factors  Institutional factors  Socio cultural factors  Geographical factors  others Dependent Variables  Investment activities Source: literatures (conceptual framework) Independent Variables  Economic factors  Political factors  Institutional factors  Socio cultural factors  Geographical factors  others Dependent Variables  Investment activities Dependent Variables Investment activities Source: literatures (conceptual framework) Private investment is a major driver of economic growth. In LDCs, increasing the growth rate of private investment would be a desirable target to achieve development agenda of the country For this purpose policymakers need to find the right balance between creating a climate conducive to investment, removing barriers to investment and they need to find mechanisms to attract private investors. Private investment is a major driver of economic growth. In LDCs, increasing the growth rate of private investment would be a desirable target to achieve development agenda of the country For this purpose policymakers need to find the right balance between creating a climate conducive to investment, removing barriers to investment and they need to find mechanisms to attract private investors. It is important to examine the main determinants of private investment. Based on the literature review, this study has identified both dependent and independent variables. It is expected that dependent variable is affected by multiple factors like economic, social, political, institutional, cultural, geographical and etc. This study, however, focuses on macroeconomic factors affecting private investment ( Muhdin M. Batu, 2016). Investment, as it has seen earlier, is an engine for economic growth and is one of the most important weapons in poverty alleviation. It improves the productive capacity of the nation and also creates job opportunity for many people. One of the most important components of investment is private investment in which business institutions engage in the production of goods and services with the twin objectives of profit maximization and improving national economy. That is why due attention has been given to private investment activities. Different factors are expected to affect the performance of private investment activities, especially in the long run. 3. Conceptual Framework What determine the level of investment are highly continuous and emphasized topics in economics. Investment spending depends on the economic, social and political situation of a country that affect the return, but such of favorable conditions is often lost for most developing countries in realizing this, there is a growing interest in the countries on the factors that influence the pattern of investment activity and some factors identified (Samuelson, 2004). Investors invest more that will enable them to earn profit. According to him there are three main determinates. These are revenue that is an investment will bring more product which leads to encourage investors less and otherwise it discourage. The second one is cost which is interest rate and government taxes. As it is obvious investors invest by borrowing of funds and if the interest [371] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] Gofe , Vol.6 (Iss.11): November 2018] Received: September 22, 2018 - Accepted: November 01, 2018) (Received: September 22, 2018 - Accepted: November 01, 2018) rate is higher it discourage investment. The third one is execration were the investors predict about the future return of investment and if its prediction is negative on its return this leads to discourage. (Samuel son, 2002) rate is higher it discourage investment. The third one is execration were the investors predict about the future return of investment and if its prediction is negative on its return this leads to discourage. (Samuel son, 2002) Source: literatures (conceptual framework) Private investment is a major driver of economic growth. In LDCs, increasing the growth rate of private investment would be a desirable target to achieve development agenda of the country For this purpose policymakers need to find the right balance between creating a climate conducive to investment, removing barriers to investment and they need to find mechanisms to attract private investors. 3. Conceptual Framework Muhidn M.Batu (2016) in his study determinants of private investment in Ethiopia concludes that investment is one of the key elements in economic growth and hence improving living standards of nations. A rate of investment is one of the key factors that differentiate developed countries from developing countries. Low investment leads to lower rates of growth and job creation, and fewer opportunities for the poor to improve their livelihoods. The primary objective of this study was to systematically review factors affecting private investment. For this purpose eleven studies were collected from nine African countries. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [372] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) The result shows that output/national income, public investment and exchange rate are the critical variables affecting the performance of private investment. The higher real GDP per capita is assumed increase effective demands for goods and services and thereby inspire private investors. Public investment, in majority of the case, is seen a complement to private investment and hence promote private sector expansion and development. Exchange rate, like GDP and public investment, is important in the promotion of private investment. However, countries should take care in its management as its implication is in both directions: positive and negative. The others variables: interest rate, credit, inflation rate, international trade, and money supply are also slightly important in explaining the performance of private investment. In this regard, reasonable interest rate, broad money expansion and trade liberalization positively contributes to private investment. On the other hand, inflation and miss-targeted credit reduces private investment. Because of poverty, people, sometimes, would borrow to finance other matters like education, healthcare and basic necessities. The study finally recommends that countries should seriously work in creating enabling environment for private investment. To promote private sector investment, countries needs to improve real income of people; maintain macroeconomic stability and make public investment in basic infrastructures and institutions that are fundamental to promote private investment. 4. Empirical Literature The empirical research on determinants of investment activity has in variably been limited in the developed country. However many studies have been made in developing country recently and have been conducted about the determinants of investment activity in different countries. Corruption can be affect investment activity and there by lower the economic development (Mauro, 1995). Corruption is a specific measure of illegal activities with in the political system. The opportunity for corruption occurs when public officials are the gate keeper of public goods and services. Shaum’s (1999) has set the process for evaluation and selecting long term investment in long term asset such as property, plant and equipment or resource commitments in the form of new product development, market research, refunding and replacement decisions such as replacement of existing facilities with new facilities Daniel (2004) has tried to model the determinant of investment activity in Ethiopia following the argument that investment is externally constrained in developing countries and it is affected by risk and unattained variables. The model tries to determining the investment activity, effects of external financial constraints domestic and international risk variables, public investment of infrastructure and out on the private investment. In this research we are concluded the determinants of investment activity by availability of finance, the real exchange rate, investment policy, debt service payment and the debt over hanging. [373] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) It is important to examine the main determinants of private investment. Based on the literature review, this study has identified both dependent and independent variables. It is expected that dependent variable is affected by multiple factors like economic, social, political, institutional, cultural, geographical and etc. This study, however, focuses on macroeconomic factors affecting private investment. Understanding the status and determinants of private investment is essential for successful and effective implementation of sustainable development goals (M. Muhammed hussen Batu, 2016). 5. Research Methodology 𝑛= 𝑁 1 + 𝑁(𝑒)² 𝑛= 𝑁 1 + 𝑁(𝑒)² 𝑛= 1 + 𝑁(𝑒)² Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [374] [Gofe , Vol.6 (Iss.11): November 2018] ISSN- 2350-0530(O), ISSN- 2394-3629(P) (Received: September 22, 2018 - Accepted: November 01, 2018) DOI: 10.5281/zenodo.2228386 𝑛= 273 1 + 273(0.1)² 𝒏= 𝟕𝟑 I November 01, 2018) 𝑛= 273 1 + 273(0.1)² 𝒏= 𝟕𝟑 Therefore 73 investors were selected Therefore 73 investors were selected Therefore 73 investors were selected RR= ratio sample size to total population RR= ratio sample size to total population RR= ratio sample size to total population Therefore sample size from each sector can be determined proportionally as follow RR= 𝑛 𝑁 𝑁 RR= 73 273=0.27= 27%; Agriculture = 10* 27 100= 3 Education = 19* 27 100= 5; Health = 13* 27 100= 3 Trade = 172* 27 100= 46; Hotel and tourism = 59* 27 100= 16 Total = 73 Types of investment activities No of population Sample size Agriculture 10 3 Education 19 5 Health 13 3 Trade 172 46 Hotel and tourism 59 10 Total 273 73 The designed questionnaires were distributed to the computed sample size in order to gather the needed information and since investors of the town are not homogeneous, stratified sampling techniques were used for the selection of typical sample out of the total population of investors. After collected necessary data the researcher was classified, analyzed and summarized the data in order to give meaningful results. The simple descriptive analysis was used because of its simplicity and clarity for drawing inferences. 5. Research Methodology This study was conducted in one of the urban center, Nekemte town, which is located in East Wollega. The town is established long years ago. But still the investment activity is in its infancy stage. Nekemte town has vast natural resources and good climatic condition which indicates the relative advantage for the potential investment opportunities. The town was considered to be the middle route to almost every districts, town and Regional Ethiopia. But due to economic, social and political constraints, the investment activity has not shown a considerable development. Now a day the town has its own investment offices to encourage private inventors and foreign investors through giving major incentives to potential investors and promotional activities about investment opportunity in the town. The investment office is also responsible to facilitate infra structural facility for investment activities, about marketability of project, technical and advisory service to investors. But due to problems both on activities investors and government side, the investment activity in the town is very limited. The study was descriptive and carried out using both primary and secondary data. Primary data was collected through structural questionnaires distributed to individuals who engaged in investment activity in the town and oral questions for the managers of Nekemte investment office. Secondary data was gathered from documents, reports books, magazines, internet and manuals. The target population for this study was individual investor’s and government organization of the town who give important information about the investment. The town has 273 investors who are investing permanently in the town and out of these investors the researcher was selected 73 investor who represent the other else and to fill the questionnaires for data collection purpose by using Slovin’s Formula, which is written as n = N / (1 + Ne2) where n = Number of samples, N = Total population and e = Error tolerance Th f h Sl i ’ f l l d fi d h l i p , p p Therefore, the Slovin’s formula was employed to find the sample size. 6. Results and Discussions This section deals with the analysis and discussion of the results from individual investors in the town. Regarding questionnaires, the researcher designed a questionnaire and distributed them to the total samples that are found at different age, education level and marital status and the researcher collected all of the questionnaires distributed to the respondents and all questionnaires were properly filled as well as give the necessary information for the researcher. The personal characteristics and basic information of the study reveals that the women participation in investment activities are very low because of culture and traditional beloved particular in the town and generally in the country as a whole. [375] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Gofe , Vol.6 (Iss.11): November 2018] ISSN- 2350-0530(O), ISSN- 2394-3629(P) (Received: September 22, 2018 - Accepted: November 01, 2018) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepte 6.1. Present Investments and Their Problems 6.1. Present Investments and Their Problems Types of investment currently engaged Question raised Alternative offered No of respondents Percentage In which type of investment are you engaged currently? Hotel trade Agricultural Health Education Total 16 45 4 3 5 73 21.92% 61.64% 5.48% 4.11% 6.85% 100% The finding shows that 21.92% engaged in Hotel, 61.64% investors were engaged in trade, 5.48% investors were engaged agriculture, 4.11% investors engaged in health6.85 % investors were engaged education. From the types of investment currently engaged in the town most of the investors engaged in service sector. Investment response to the problem they faced What types of economic factor you faced during your engagement? Lack of capital Rate of taxation Infrastructural facilities(water, Road, Electricity and Communication services) Total 26 14 33 73 35.62% 19.18% 45.20% 100% What types of social awareness & initiative problems you faced during you engagement? Lack of educational facilities Lack of health facilities Lack of social stabilities Lack of social attitude towards work Expenditures behaviors Total 20 15 25 10 3 73 27.39% 20.55% 34.25% 13.69% 4.11% 100% What types of political factor you faced during your engagement? Political participation in decision making Lack of women empowerment Lack of good administrative services Corruption& related issue Land administration & management problem Investment policy &administration problem Total 10 12 18 15 10 8 73 13.69% 16.44% 24.66% 20.55% 13.69% 10.96% 100% What types of Environmental factor you faced during your engagement? 6. Results and Discussions Pollution Seasonality Land degradation Waste management Total 8 42 5 18 73 10.96% 57.53% 6.85% 24.66% 100% [376] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) (Received: September 22, 2018 - Accepted: November 01, 2018) The above reveal that 27.39% of the respondents are replied lack of capital during their engagement in investment activities, 19.18 % of the respondents are answered that rate of taxation during their engagement in investment and 45.20 % of the respondents are answered lack of infrastructural facilities. The finding shows that the majority of investors face the problem of infrastructural facilities during their engagement of investment activities. Out of 73 respondents, 27.39% said lack of educational facilities, 20.55% said lack of health facilities, 34.25% said lack of social stability, 13.69% said lack of social attitude towards to work and 4.11% said expenditures behaviors face the investors during their engagement in the investment activities. From the above information the researchers under stood that majority of investors face the problem of social stability during their engagement in the investment activities and relatively lack of educational facilities. Out of 73 respondents,13.69% said political participation in decision making ,16.44% said lack of women empowerment,24.66% said lack of good administrative services, 20.55% said corruption and related issue 13.69% said land administration and management problem and 10.69% said investment policy and administration problem. From the above information the researcher under stood that the majority of investors face the problem of corruption and related issue and land administration and management problem during their engagement in the investment activities. From the total 73 respondents, 10.96% of the respondents said there is pollution problem, 57.53% of the respondents said there was seasonality problem, 6.85% of the respondents said there was the problem of land degradation and 24.66%of the respondents said there was waste management problem faced during their engagement in the investment activities. From the above information the researchers conclude that the majority of problem that faced the investors are the seasonality problem and waste management problem that faced during their engagement in the investment activities. In addition, investments significantly affect the intensity of overall economic activity and growth in general. Changes in size, structure and purpose of investment may indicate forthcoming conjuncture changes, but also longer-term developmental characteristics of the economy. Therefore, investment decisions are of special interest not only to policy makers and researchers, but also to firm managers and owners (M. Peric, J. Ðurkin, 2015) [377] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepte 6.2. (Received: September 22, 2018 - Accepted: November 01, 2018) It also shows that 27.39% of the respondents are to get information about the areas of investment activities from the newspaper, 20.55% of the respondents are get information from the magazines, 35.66% of the respondents are get information from media and 16.44% of the respondents are get information from the documents [378] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Gofe , Vol.6 (Iss.11): November 2018] ISSN- 2350-0530(O), ISSN- 2394-3629(P) (Received: September 22, 2018 - Accepted: November 01, 2018) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 p p Investor’s future review Question raised Alternative offered No of respondents Percentage Dou you have any plan to start the investment activities? Yes No Total 50 23 73 68.49% 31.51% 100% Is there any training program related to the investment activities? Yes No Total 40 33 73 54.79% 45.21% 100% Have you got any skill training about investment since you have started it? Yes No Total 40 33 73 54.79% 45.21% 100% As the table of investors future review above shows 68.49% of the respondents have been plan to start in investment activities of the town. While only 31.51% of the respondents have no plan to start the investment activities of the town. This implies that most of the investors have plan to start to their business which seems to have positive relationship with capacity The table shows 54.79 % of the respondents have got training program related to investment activities. But 45.21 % of the respondents have not any training program related to the investment activities. This implies that most of the investors have got training program to enhance the investment activities. According to many investors ideas, most of them 54.79% skill development training about investment movement and how to manage their resources and how to minimize risk i.e. risk diversification in order to achieve their objective goals and 45.21% of the respondents did not get skill development training about investment before they had started the investment activities in the town because they haven’t enough time to get skill development and nobody initiate them to participate on skill development training program. As the view of the investors for encouraging nature of current investment policy, they argued that the policy had made efforts to improve the operating environments through serious problems of implementation limits the benefits of the policy. (Received: September 22, 2018 - Accepted: November 01, 2018) Sources of Finance and Investors Prospect Sources of data and accessibility of financial source Question raised Alternative offered No of respondents Percentage What are your sources of fund to invest? Government bank Private banks Credit and loan association Personal saving Total 20 19 25 9 73 27.39% 26.03% 34.25% 12.33% 100% How do you rank the accessibility of their financial soundness to investors? Easy Difficult Medium Total 18 32 23 73 24.66% 43.84% 31.50% 100% What is your attitude towards the current investment strategy of government in the study town? Encourage Discourage Total 49 24 73 67.12% 32.88% 100% What means to get information about the areas of investment activities in the investment offices? News paper Magazines Media Documents Total 20 15 26 12 73 27.39% 20.55% 35.62% 16.44% 100% 6.2. Sources of Finance and Investors Prospect As the above table shows that most of respondents 34.25% used credit and loan association, 27.39% used government bank, 26.03% used private banks and personal saving15.33% used their own capital and borrowed from their families and friends. With regarding to accessibility condition of financial sources to investors from the financial institution, 24.66% of the respondents as easy, 43.84% of investors rank as Difficulties and 31.50% of respondents rank as Medium to get financial sources (funds) from financial institutions. According to the views of the investors responded to the difficulties in using financial sources, the un-ability to meet collateral requirements, high interest rate, lack of long term and absence of financial intermediary are the most frequent reason for not obtain financial sources. According to many investors ideas the current investment policy of the town is encouraging 69.12% of the respondents said that current investment policy of the town is encouraging if they compare with the previous investment policy of the town and 32.88% of the respondents said that the current investment policy of the town is discourage because of there is a discrimination and corruption is more involved in the investment activities of the town. 6.3. Summary of the Types of Investment Activity in the Nekemte Town There are different types of investment activity; those are agriculture, Education, health, trade and hotel and tourism sectors. This classification is presented in the following table with their respective number of members and job created by them. Types of profile investment activities in Nekemte town Investment type Sub-city Percentage (%) Chalalki Darge Kaso Baknisa kase Burka Jato Bake jama Total Agriculture 4 1 - 2 1 - 8 2.93 education 6 2 4 1 4 2 19 6.96 health 5 1 3 2 1 1 13 4.76 trade 56 22 32 18 12 34 174 63.74 Hotel and tourism 20 5 10 11 4 9 59 21.61 Total 91 30 49 34 20 46 273 100 Types of profile investment activities in Nekemte town ent Sub-city Types of profile investment activities in Nekemte town Investment type Sub-city Percentage (%) Chalalki Darge Kaso Baknisa kase Burka Jato Bake jama Total Agriculture 4 1 - 2 1 - 8 2.93 education 6 2 4 1 4 2 19 6.96 health 5 1 3 2 1 1 13 4.76 trade 56 22 32 18 12 34 174 63.74 Hotel and tourism 20 5 10 11 4 9 59 21.61 Total 91 30 49 34 20 46 273 100 According the table in Nekemte town there are five investment activities, those are classified in to two major investment activities. Thus sectors are agriculture and service sectors. The agriculture which contributes to 2.93 % of the total investment activity and it is contributed lowest when compared the other sector. The second sector is service sector which is contributed the largest total investment when compared with the rest one which is 33.33 %. In generally in Chalalaki sub-city there are 91 investment activities, in Darge 30 types of investment activities, Kaso 49 types of investment activity, in Bakenisa kase sub city there are 34 investment activities, in Burkajato there are 20 investment activities and in Bake jama there are 46 investment activities. According the table in Nekemte town there are five investment activities, those are classified in to two major investment activities. Thus sectors are agriculture and service sectors. The agriculture which contributes to 2.93 % of the total investment activity and it is contributed lowest when compared the other sector. The second sector is service sector which is contributed the largest total investment when compared with the rest one which is 33.33 %. 6.3. Summary of the Types of Investment Activity in the Nekemte Town In generally in Chalalaki sub-city there are 91 investment activities, in Darge 30 types of investment activities, Kaso 49 types of investment activity, in Bakenisa kase sub city there are 34 investment activities, in Burkajato there are 20 investment activities and in Bake jama there are 46 investment activities. (Received: September 22, 2018 - Accepted: November 01, 2018) However, others considered if as discover aging because of enormous practical problems of the policy rather than the theoretical attractiveness of the policy. One can see from the study that most investors have good attitude to the theoretical design of the policy than what practically have been done in implementing it. In other words the current investment policy have got theoretical acceptance by most of investors, though it has serious problems of implementation that discouraged investors. This leads us to the conclusion that the current investment policy would be more import and through giving emphasis is to the implementation of the policy from work. As investors suggested the government and investors should express their interest too jointly work together to identify constrains and examines solution for lack of properly implementing policy. To sum up, most investors have many problems regarding source of funds to invest, political instability, corruption, inaccessibility of financial resources, economic, lack of infrastructure and tax levied on the investors of the town. [379] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] , (Received: September 22, 2018 - Accepted: November 01, 2018) 7. Conclusion and Recommendations Investment is one of the key elements in economic growth and hence improving living standards of nations. A rate of investment is one of the key factors that differentiate developed countries from developing countries. Low investment leads to lower rates of growth and job creation, and fewer opportunities for the poor to improve their livelihoods. The primary objective of this study was to systematically review factors affecting investment activities. In this area different studies were conducted over the world and also even particularly in Ethiopia. The result shows that output/national income, public investment and exchange rate are the critical variables affecting the performance of private investment. The higher real GDP per capita is assumed increase effective demands for goods and services and thereby inspire private investors. Public investment, in majority of the case, is seen a complement to private investment and hence promote private sector expansion and development. Exchange rate, like GDP and public investment, is important in the promotion of private investment. However, countries should take care in its management as its implication is in both directions: positive and negative. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [380] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018 (Received: September 22, 2018 - Accepted: November 01, 2018) Investment, as it has seen earlier, is an engine for economic growth and is one of the most important weapons in poverty alleviation. It improves the productive capacity of the nation and also creates job opportunity for many people. One of the most important components of investment is private investment in which business institutions engage in the production of goods and services with the twin objectives of profit maximization and improving national economy. That is why due attention has been given to investment activities. Different factors are expected to affect the performance of investment activities, especially in the long run. For developing countries like Ethiopia the basic question in their economy is increase the production and hence improve the standard of living of their people so that there will be dramatic change in their economic, political and social conditions. Investment promotion is one key instrument and primary engine of economic growth (Mustefa, 2014). Recommendations Based on the finding the following suggestion is forwarded to improve the factors that determine the investment activities in the town and also to reduce or eliminate some problem faced by the investors. To enhance the investment activity in the town in the future, the following recommendations are suggested by the researcher. • The main constraint for business activity in the town is the difficulty to easily obtain land for new investment. In this regard the land lease policy which is being used by the local authorities for implementation in the town should be handled in a manner that will contribute to the investment activities so that the investment may be enhanced which indirectly contribute to the economic growth of the country. • Development of infrastructure facilities plays a major role for the development of investment activities. In order to facilitate transportation service, deteriorated roads should be maintained, gravel surface roads should be asphalted and the construction of additional inner roads which can help for fast movement of goods and service within the town. • It is recommendable to diversify the business activities. To diversify the business activities, it is essential to provide sufficient information to private investors about alternative investment opportunities. In this regard, the institution capacity of the town should strengthen to give advisory and consultation service for potential investors before they start their investment activities by seeing only others investments so that they can easily identify the new investment activity in the town. recommendable to prepare land for investment with infrastructural facilities. • It is recommendable to prepare land for investment with infrastructural facilities. • The other thing is it is advisable to Expand loan institutions to increase the financial capacity of the investors and enhancing promotions for investment • In addition it is advisable to provide different training, group discussions, seminars on various issues so as to avoid or minimize the corruption behavior of individual investors and the concerned offices. Generally the researcher recommended that the government body should facilitate infrastructure and also to promote essential information to investors and should establish efficient advisory and consolation service. Generally the researcher recommended that the government body should facilitate infrastructure and also to promote essential information to investors and should establish efficient advisory and consolation service. 7. Conclusion and Recommendations Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [381] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2228386 [Gofe , Vol.6 (Iss.11): November 2018] (Received: September 22, 2018 - Accepted: November 01, 2018) In general, the town has strategic location as a middle route with favorable climatic condition can be seen as an opportunity for development of new investment activity and due to economical, infrastructures and political hindrances this activity in its low of stage in the town. 7. Conclusion and Recommendations As a result due attention has been given to development of private sector in developing countries to help improve economic growth (Ouattara, 2004). Reliable and continuous increase in domestic private investment also helps in reduction of poverty. Understanding the status and determinants of private investment is essential for successful and effective implementation of sustainable development goals. Allocation of capital on different sectors of economic activity is essential to move the available resources in to more productive. To increase the development, allocation of human and natural resources in effective and efficient manner on investment activity is very crucial aspect. Without investment it is impossible to have any development rather it remains in poverty. This is mainly seen in developing countries like Ethiopia. If there is unemployment, there will be low productivity which results in low level of income, there is poverty. This research is conducted in Nekemte town to investigate the factors that determine the determinants of investment activity. Data was collected through primary and secondary method. The data obtained was analyzed through descriptive method. According to data obtained shows that the investment activity was found in its low level to activity due to various a constraints. However, the town has good opportunity to become an important distribution center in East wollega zone. Owing to its strategic location due to its proximity to Addis Ababa and as a Junction point for roads that head to various directions. Yet the level of investment activity in the town is rather small particularly when compared with the level of the investment in other small towns that recently gained a higher administrative status. Bureaucratic system of land lease policy has a great impact on investment activity. Due to high competitions among investors, a small unit of land has high price in the town and it is difficult to get land easily for construction purpose. Low level of institutional capacity due to absence of well qualified workers to study investment opportunities in the town and to promote it to potential investors, to give advisory and incentives for investors would have its own limitation on this investment activity. The town’s low level of infrastructure facilities like deteriorated and overcrowded roads, unbalance educational and health services with the town’s population have also negative influence on investment activity. References [1] Agu, O. C. (2015). Determinants of Private Investment in Nigeria an Econometric Analysis. International Journal of Economics, Commerce and Management United Kingdom Vol. III, Issue 4. 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https://openalex.org/W4312038074	https://www.journal.aleftrust.org/index.php/cstp/article/download/35/51	English	null	transpersonal exploration of a leader’s transformative journey, and the effects on the organization	Consciousness, spirituality & transpersonal psychology	2,022	cc-by	6,524	Correspondence concerning this article should be addressed to Laís Cossermelli. Email: consultoria@laiscossermelli.com.br Recommended citation: Cossermelli, L. (2022). A transpersonal exploration of a leader’s transformative journey, and the effects on the organization: A case study. Consciousness, Spirituality & Transpersonal Psychology, 3, 144-157. https://doi.org/10.53074/cstp.2022.35 A Transpersonal Exploration of a Leader’s Transformative Journey, and the Effects on the Organization: A Case Study Laís Cossermelli Alef Trust and Liverpool John Moores University This paper explores how the transpersonal approach might bring an actualized view to business and leadership, enabling a collective transformation, giving rise to more engaged, healthier and happier people, reaching high performance and positive business results. This case study examines a real-life experience, investigating the inner transformative journey of a senior business leader, followed by a collective transformation of the company he worked for. An in-depth understanding was formulated through multiple data sources: interviews, associate surveys, reports, office images and meeting protocols. Six themes emerged: preparation, the leaders’ journey of dissolution, the organization’s soul encounter, metamorphosis, enactment and actualization of the senior leader. These themes were explored in relation to the experience of other global business leaders, followed by an exploration of a calling for leadership transformation which aimed to cope with uncertainty and rapid change, and finally, the impact on the organization, when leadership matured from silo and individual-thinking to a common-purpose direction. A description was written to explore the essence of each step of the transformative journey, starting from the senior leader and followed by a collective shift of the organization’s identity. This lived experience shows what can be achieved in an organization when people feel deeply connected with each other and part of a greater purpose, beyond organizational structures. Keywords: leadership, organizational transformation, transpersonal, transformative journey Correspondence concerning this article should be addressed to Laís Cossermelli. Email: consultoria@laiscossermelli.com.br Recommended citation: Cossermelli, L. (2022). A transpersonal exploration of a leader’s transformative journey, and the effects on the organization: A case study. Consciousness, Spirituality & Transpersonal Psychology, 3, 144-157. https://doi.org/10.53074/cstp.2022.35 144 144 Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 Two topics have led my attention in the last twenty years. One has been business organizations, leadership and performance, and the other, contemplative practices, intuitive intelligence and the science of attention. In my viewpoint these are not two worlds, as both encompass human experiences relating to self and others. We are dialogical selves who relate to the various dimensions in our private world, and in relationship with others (Gergen, 2013). These relational experiences can occur either in the work environment or through inner quietude in search for self-knowledge. The lived experience referred to in this study is especially valuable to me, as at the time of this transformative journey I was part of the leadership team as a human resources (HR) manager. During this journey, I experienced my own process of strengthening my connection to what might be referred to as Source, Divine or Self. According to Jaworski (2012), this is “the underlying reality out of which all forms” (p. 58). This investigation is based on the case study of a senior business leader who chose to open a space for an in-depth exploration, which further led to a collective transformation embracing the whole organization. He acknowledged that he was in a place of “not knowing” and invited his team to journey together towards the future. Discovering new knowledge (Jaworski, 2012) means being rooted in surrender and a sense of service, implying the need to detach from current knowledge in order to access new emerging wisdom and insight. Through inviting these new ways of knowing, he stepped out of the continuum of the past, allowing for a vantage point that reviewed multidimensional possibilities. When the senior leader detached from classical models of management based on Taylorism, with the purpose of economic efficiency and labour productivity (Ehiobuche & Tu, 2012), he invited himself and the leadership team towards new ways of seeing and sensing reality, which included and transcended the cognitive and logical dimension. An organisational transformation is a process that happens from the inside out. No external power can generate personal transformation, as it is a personal choice (Rooke & Torbert, 1998). Some leaders can successfully tailor their actions to create greater transformational capacity (Cook-Greuter, 2004). In its life journey, an organization will go through a range of transformational changes (Taborga, 2012) in order to shape, adjust and co-create its reality. Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 When leadership sees and senses the organization as part of one living system, if the observer is the observed (Bohm & Nichol, 1996), a new paradigm might be revealed. An organization is an interconnected system which produces a pattern of behaviour that serves a purpose, and the quality of the relationship between its parts will affect the behaviour of the system (Meadows, 2008). As a living organism, no change should be imposed from the outside, as such ecosystems have the capacity to sense changes and adapt (Loloux, 2014). The underlying question of this research was: What was the experience of a senior leader of a global technological organization during a transformative journey, and how was the organization affected? This research aimed to uncover the fundamental steps of the transformative journey and to clarify how this senior leader influenced and further nurtured collective change. It also explored the transformation of worldviews of other leaders as they abandoned past assumptions and assumed a beyond-mind-and-ego perspective. Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation 145 Method The case study methodology was well suited to answer the research question, as my objective was to narrate a meaningful, contemporary and real-life experience within a defined context and time frame (Yin, 2009). It is a type of qualitative methodological design in which the investigator explores a system (a case) “over time, through detailed in-depth data collection involving multiple sources of information” (Creswell & Poth, 2018, p. 96). The company in this study is part of a German multinational technological conglomerate, with about 400,000 associates worldwide and sales of 70 billion Euros in 2020. This original case study research took place in the Latin American subsidiary, which currently employs 800 associates in 18 countries. The case study affected all hierarchical levels and business areas of the organization: Research and Development, Production, Quality, Purchase, Logistics, Marketing & Sales, Controlling & Finance, and supporting departments such as Human Resources and Project Management. It included all 18 countries and four Business Units. The parameters of the case study were that both the senior leader and organization being led were to undergo a transformation, and the timeframe was to start from the beginning of the transformative journey until the completion of data gathering. Sampling Three semi-structured interviews were conducted, one with the senior leader and two others with managers (Table 1), who held leadership positions at the organisation since at least 2015 – when the organizational transformation began. In order to reach the other interviewees, I requested the HR department to provide interested people to perform the interviews and due to time availability the two interviewees were chosen. Table 1 Table 1 Participant Information Pseudonym Bruce John Louise 59 48 50 White German White Brazilian White Brazilian Regional President Department Manager Department Manager 30 22 16 Age Declared ethnicity Organizational position Years in the company Male Male Female Gender Participant Information Participant Information Procedure Three interviews were conducted, and the following six documents were analysed and integrated: Two associate surveys showing the improvement of main indicators of performance, providing directions, driving change, managing people and systems, and engagement; a corporate presentation summarizing the main activities of the transformation; a set of pictures taken from the offices before and after the transformation; and finally, two business reports showing positive development in terms of market share and profitability. The documents were gathered through the organization managers and HR. Through a holistic analysis of the entire case, a detailed description emerged. The three interviews were analysed, and 13 codes emerged. In the second round of data analysis, codes were separated, considering (a) aspects that were only addressed by the senior leader informing when/how and why the transformation was ignited; (b) aspects that were experienced only by the two interviewees; and (c) the common codes for all. In the third round, codes were merged, which finally delivered six major themes. At this point, the hard data was integrated into the coding process, shaping and reinforcing the emergence of six final themes and 13 sub-themes. The hard data reflected the changes perceived in the organization regarding people’s engagement, the leadership culture and business results. At this point of the research process, I realized the revelation of the major steps of a journey, lived in a sequence of time and within specific conditions. There was a correspondence with the canyon image (Plotkin, 2021), a psychospiritual expedition into the depths of psyche, encountering our unique niche (soul) and reemerging radically transformed by the encounter, which offered a resonant and logical container for the major themes and subthemes. Data Interview Protocol The interview questions were developed from the research question and attempted to cover the main aspects of the experience, both of the individual (senior leader) and the collective (leadership team and organization). They allowed the participants to describe their individual experience as well as the effects on the organization. Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 146 Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 146 A semi-structured interview protocol was designed with consistent open-ended questions which enabled participants to express perceptions, insights, and experiences. Questions to all participants were approached with the following three aspects: the shift in individual and collective consciousness; the impact on selves, others and the business; and finally the integration process. The design included questions that had the potential to bring the transpersonal aspects (Hartelius et al., 2007) to the experience, allowing participants to uncover deeper meaning and aspects beyond the professional dimension. Questions were divided into three themes of beyond-ego psychology, integrative/holistic psychology, and transformative psychology. The interviews were held via Zoom and were around 60 to 80 minutes long. Bracketing My Experience At the time when the journey started and gained momentum, I was in the role of HR manager, and recognize that it may have influenced my understanding of the transformative journey. I am keen to interpret the experience of the participants from transpersonal lenses, which may impact the interpretation and analysis of my interviews. Considering this, I endeavoured to remain objective when conducting the research and attempting to understand participants’ experiences. Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation 147 Results The following results derive from the revelation delivered by the interviews and hard data, delineating a narrative that encompasses the six themes: preparation, the leaders’ journey of dissolution, the organization’s soul encounter, metamorphosis, enactment and actualization of the senior leader (Table 2). It follows a logical sequence which reveals a transformative journey that began with the senior leader, and further developed to a collective cultural change. Table 2 Table 2 Themes and Subthemes that Emerged from Data Analysis Theme Subtheme Preparation The leaders’ journey of dissolution The organization’s soul encounter Metamorphosis Enactment Actualized senior leader 1. 2. 3. 4. 5. 6. 1.1 1.2 2.1 2.2 2.3 2.4 3.1 3.2 3.3 3.4 4.1 5.1 5.2 Sensing the emerging future Strong need to change The spark of the senior leader: A place of not knowing Experiencing the journey What needs to dissolve Transforming self An actualized leadership emerges Temporary purpose team emergence The interconnectedness: We are one When not in flow An integrated organizational culture Contemplating the present moment Business results flourish Table 2 Themes and Subthemes that Emerged from Data Analysis Actualized senior leader 6. Preparation: Sensing the emerging future/Strong need to change The case study narrative begins when the senior leader started sensing and envisioning the future of his organization, connecting with a feeling of new possibilities, the image of which was fundamentally different from a continuum of the past. Bruce expressed: “I felt separated from the organization”, and at the same time he was sensing new ways of relating with self and others, feeling deep empathy towards people. Bruce noticed that he “was feeling really powerless to solve this within the classic management model” and believed leaders and Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 148 associates should nurture a deeper level of connection, where associates felt empowered to lead with mastery in an environment with less hierarchy, more flow and autonomy. A sense of unease and discomfort were present, manifesting an inner calling for transformation. The leaders’ journey of dissolution: A place of not knowing/ Experiencing the journey/What needs to dissolve/Transforming Self At a leadership team event Bruce openly shared that they would carry out a journey without a clear idea of the destination: “I was very confident in the organization … not so much in me, and it was highly disturbing, disruptive for me”. Bruce did not know how it would unfold, but affirmed that together, the team could co-create its future. As noted by Louise: “When he recognized in front of all of us: I do not know. I just know that we need. I do not know how and we find out together. This was something that … as I said, the impact was really fantastic, amazing because we understood that we were together.” She added: “It was also a mark for me because as a leader we always thought that we had to have all the answers”. This step into the unknown and the expression of vulnerability moved the team to a new place, as an invitation to novel ways of knowing and accessing the future. A sequence of four leadership events followed in the course of one year and a half, where leaders experienced an inner discovery process through new ways of relating with the self and others. Louise shared: “The third [Leadership Day] was … I think it totally changed the scenario that people just opened up, showed vulnerability and started to connect. Forgot their role in the company”. Feelings of discomfort in the beginning have transmuted to barriers dissolution, when they were able to relate as vulnerable human beings rather than only professionals. John added: “The bonds came from the personal. I am connecting with you as a person, because before the professional there is a person.” They felt the need to let go of old ways of being, mainly involving power and control as well as attachment to the ego, meaning ideas, roles, statuses or positions. As mentioned by John: “There was a very old-fashioned style, a leadership style, that was really based on fear, on the authority, on hierarchy”, and noted the need of “letting go of the ego and understanding and fighting against the fear. Or transmuting the fear.” They assumed facing a shift in their belief system on how to experience the leadership role, expressing the pain that originated from the transformation process: letting go of a learned way of being, and accessing a novel, actualized self. The organization’s soul encounter: An actualized leadership emerges/ Temporary purpose teams/The interconnectedness/When not in flow When more connected as a community, the company leaders strengthened bonds beyond functions, roles or positions, relating as human beings serving a common purpose. They learned that openness and vulnerability were key attributes to leadership and realized that their main task was the development of their teams. While the leaders journeyed from dissolution to renaissance, multifunctional, autonomous teams gained stage while working towards common purpose projects, experiencing the assumption of responsibility and becoming the protagonists of relevant projects. Louise pointed out: “As I started providing Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation 149 a high level of autonomy for the group, it was so nice because they didn’t want to disappoint me. It’s a matter of giving and receiving back. And this was a very successful cycle”. The actualized leaders and the autonomous teams embraced a new type of relationship with their selves, others and the needs of the organization. John explains: “In a lot of situations I let my team lead me. In some issues that they have much more experience than me.” A healthy container was created and nurtured by people who relate from a place of being, serving the needs of the whole organization. Bruce says: “It’s a kind of radical collaboration that puts collaboration above everything … above the interests of the departments, above obviously the private interests of leaders”. This phenomenon is described by the expression, “We are one”, which was repeated 14 times by the interviewees. It can be perceived in the statements of Bruce: “The connection of working with an open heart too … without fake, with real connections”. “Obviously has a lot to do with consciousness … and for me the most important thing! It has to do with self-knowledge, it has to do with connection itself, it has to do with connection with others”. It was well described by John: “So, there is something different that we are doing. And I think that the most different thing is that ‘we are one’. He adds: “There is a huge, huge force that moves everybody when we are connected.” Ultimately, a natural consequence of a newly built cultural system was formed. However, some leaders did not fit in the new paradigm, and, for different reasons, they sooner or later left the enterprise. Bruce expresses: “There were also a lot of leaders who left, because couldn’t identify with that way.” Metamorphosis The next step was the integration of the experiences, manifested in a newly created collective being. The challenge of this phase was to sustain the high-quality level of the relationships in order to nurture a healthy living system. Bruce states: “[It] is collective intelligence and mindfulness itself. And these kinds of things started to become usual to the organization. That is, we live it more and more, organically”. As a collective transformation, it required the manifestation of the incorporated individual attributes, along with a sustaining force to maintain the confidence of the collective. The teams seem to have found their own way of leading projects and deciding things. Bruce mentions: “When the topic is getting very serious, they know that they can come to me and say: Bruce, now I need you because it’s getting critical”. The new way of leading the business showed to be not in a defined, process-based manner, but by creatively discovering ways to offer solutions to the problems arising in the organization. “The organization took more and more courage to act in this way”, says Bruce. Enactment: Contemplating the present moment/Business results flourish The organization felt and lived its growth potential towards a life-enhancing stage. The word “growth” embraces three aspects: a) Individuals experiencing a place of higher self-expression; p g p g p b) A deeper level of connection between individuals along and across the organization, for instance, between colleagues of the same area as well as across departments, Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 150 business units, countries and hierarchical levels. This is well expressed by John: “We are marching. We are walking. We are flying. We are doing. We are working. We are living towards a common objective”; c) The growth of the business itself. “Since we … ah ... since the second year when we implemented this, the result is constantly, despite all the crises, it has improved significantly”, says Bruce. At this stage, the statements from participants echo a feeling of bliss and an attempt to comprehend how it was possible to reach the place they currently are in. In the words of Bruce: “Happiness, satisfaction, that all we dreamed has worked out. I think for many executives it is a dream. Actualized Senior Leader A fundamental aspect to being integrated in this narrative, which encompasses the journey as a whole, is the role of the senior leader. Bruce intuitively envisioned and ignited a shape-shifting process of the organization’s collective ego, being led to actively co-create this cultural regeneration. As mentioned above, he had not delineated any plan to reach a defined objective, but rather, let the organization give birth to what was willing to be born. In his following statement we can observe his self-perception: “I can define things in the way I think it is right and paying the price for that … the price of disconnecting people, of doing something that is against their will, or against their conviction, and everything. And if you open up more space for the organization, it’s the other way around … but you pay the price of losing your power, your ego”. According to John: “He’s much closer (to people) and he brings this bond to the business, so connecting people through connecting aspects of different business units, different interests, targets. And then everything he connects to the whole business in a holistic way”. Bruce finally summarizes: “The collective intelligence, collective perception is greater or better than the individual, in this case mine.” Metamorphosis It is a huge relief … it takes away … I think it is, maybe something I didn’t mention so much, but the weight of the responsibility. It is huge when you carry all that responsibility alone. When you don’t share.” The positive business outcome that followed was mentioned 10 times. This was when the transformation reached momentum, when the level of trust allowed for and sustained the fruitful initiatives co-created by teams. It was only possible with the mindful presence and care of the leadership team. John could well summarize: “But we generated the collective consciousness that drives almost alone the organization.” Discussion The transformative steps mirror Plotkin’s (2021) framework, following the metaphor of a canyon. The preparation phase which included the discovery process of the senior leader “as solitary intimations of a problem to be discovered” (Jaworski, 2012, p. 143) was followed by the journey of other company leaders through the dissolution phase where old beliefs required reconsideration. The next major step was the high Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation 151 sense of interconnectedness and wholeness among leaders and associates, which led to a metamorphosis. No longer from a top-down perspective, but through creative collaboration for mutual benefit (Montuori & Donnelly, 2017), leaders and associates experienced a novel participatory process with more autonomy (Pink, 2009), psychological safety (Edmondson, 1999) and collective intelligence (Woolley et al., 2015), leading to a learning organization (Senge, 1990). People felt motivated to move beyond self-interest leading to a sense of purpose (Givens, 2008). What emerged was an actualized collective being in the final enactment phase. If through transformation we become more uniquely who we are (Hart, 2000), in this case, the organization became able to experience its uniqueness, through the sense of unity and interconnectedness. The transpersonal aspects, beyond ego, including the whole person and a catalyst leading to transformation (Hartelius et al., 2007) could be observed. When realizing that old patterns would no longer fit the needs of the future, and feeling powerless to solve the problem within the classical management tools, a strong need to change was perceived. According to Plotkin (2021), that is when one arrives at the rim of the canyon “from where you can gaze down toward the intimidating and alluring mysteries that await below” (p. 22). It can be interpreted as a threshold to new possibilities that want to emerge. As posited by Scharmer (2009), such doorways appear when the conventional ways in which we operate no longer produce the expected results. When the space was opened for new ways of knowing and a safe container for the leadership team was nurtured, an underlying intelligence of the universe was accessed (Jaworski, 2011). This is the ability to go beyond the cognitive and emotional intelligences, reaching multiple levels of consciousness, meaning the manifestation of a spiritual intelligence (Amram, 2009). Discussion A future with more connection, empathy, autonomy and flow with less hierarchy had been imagined by the senior leader, showing that his attention was on how people were relating with each other and the need to nurture stronger relationships, not only based on roles or functions. This is sustained by Meadows (2008), when she affirms that the behaviour of the system will be affected by the relationship quality of its parts. Leaders operating from a novel perspective were able to see and sense the organization as one living system (Bohm & Nichol, 1996). From this viewpoint, the senior leader did not impose any change, but opened the space for the organization to co-design their future. Following Plotkin’s (2021) framework, in the phase of dissolution, one enters a kind of identity indeterminateness, a suspension of whom you believed you were. In this case, it was a dismemberment of what leaders understood as leadership. The findings describe leaders tapping into old ways of being, manifested in terms of distancing from people, having the last word, carrying the heavy burden Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 152 Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 152 of decision-making and being encapsulated in the armour of hierarchy. This approach applies to the mechanistic view of reality opposed to the system view of life to management (Capra & Luisi, 2014), which implies a living organism in the totality of its mutual interactions. As further described by Cook-Greuter (2004), the human development model describes human potential moving towards deep understanding and effectiveness in the world, from an ego-centric to a world-centric view. As development unfolds, tolerance for ambiguity, flexibility and self-awareness increase while defences decrease. Results show the dissolution of patterns such as power and control and the attachment to the ego. A newly-born leadership culture met an empowered team, giving rise to the unfoldment of an actualized organizational culture. Individuals felt connected with each other and collaborated for the sake of the whole business, moving towards a common purpose beyond functions. Leaders felt connected with each other beyond functions, meaning a relationship nurtured by their beingness instead of the roles they represent. While fostering this kind of relationship, teams tend to operate smoothly, capitalizing on the creativity of the individuals. When leaders operate from this novel place, allowing openness and vulnerability to be intrinsic values of leadership, the organization consequently mirrors their behaviour. Cossermelli: A transpersonal view of leadership transformation Discussion One of the outcomes is that teams start feeling safe to assume responsibility and act autonomously. Edmondson (1999) suggests that when team members feel confident that colleagues will not hold the errors against them, they tend to speak up and assume responsibility. When this is constantly perceived and lived by the collective, it starts facilitating a learning behaviour in the organization, meaning the ability to cope sustainably and creatively with new challenges (Senge et al., 2004). The perception of interconnectedness was translated into the statement repeatedly spoken, “we are one”. As expressed by John: “There is a huge, huge force that moves everybody when we are connected”, which led me to assume the perception of a deeper level of interconnectedness encompassing what Tibetan Buddhism calls “wisdom awareness” (Senge et al., 2004). This study reveals the possibility to bring the transpersonal view to the transformative journey of leaders, bridging the perception of the nondual with the dual world (Sharma, 2017). The experience led to heightened levels of interconnectedness, a non-dual sense, where inner wisdom resides (Sharma, 2017). When leaders and team members start relating from a place of non-boundaries, it seems they access a beyond “analytic knowing”, allowing collectively a “primary knowing” to emerge. According to Rosch in Senge et al. (2004), primary knowing arises by means of interconnected wholes and a sense of unconditional value, leading to compassionate actions based on wholes larger than the self. Cossermelli: A transpersonal view of leadership transformation 153 153 Results showed that going up the canyon (Plotkin, 2021) and crystallizing an integrated organizational culture requires specific forces: collective intelligence and a collaborative decision making process. According to Woolley et al. (2015), “collectively intelligent groups communicate more and participate more equally than other groups” (p. 422), and they also suggest that it is derived from bottom-up processes, combined with top-down interactional processes. The second attribute is the way the decision- making process is performed in the integrated culture. The results of the current study illustrate the affirmation of Montuori and Donnelly (2017) who define transformative leadership as a participatory process of collaboration and mutual benefit. The basic premise from the authors is a relational view of leadership, actualizing the idea of someone holding a specific position in the system. They sustain that everyone can lead in a specific moment, which is particularly aligned with the emerging networked culture of collaboration (Montuori & Donnelly, 2017). Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 Conclusion The research suggested that the senior leader’s main role was to move to a place of vulnerability, accessing a space of not knowing. While stepping out of the patterns from the past, the organization was free to pursue its own path. It was only possible when the leadership team experienced an ego-shifting process, abandoning their old belief system and co-creating a new collective self. It required courage and willingness to dig in our darkest places until we found our unique, authentic and heartfelt selves. It was also suggested that a core competence of leaders is the ability to co-create and hold a safe space for people, sustaining a position of caring and mindful presence, allowing the living organism to accomplish its evolutionary purpose. This allowed associates to assume projects beyond their functional roles, meeting the needs of the organization and serving a common purpose, which all together formed the organizational culture based on vulnerability, human development, autonomy, purpose, collaboration and empathy. Finally, the case study has demonstrated the effects of interconnectedness, revealed by the emergence of “We are one”. It was manifested when silos were dissolved and people were invited to live their fullest potential beyond the boundaries of roles, countries, business units or departments. This can be seen and felt in people’s contentment, engagement and, consequently, in the high performance revealed by the business results. Limitations and suggestions for future research I believe that observations of the individuals’ interactions in meetings would provide a fruitful amount of qualitative data for analysis and integration into the findings. According to Edgar Schein in Senge et al. (2004), we can learn about organizational culture through careful observation and reflective participation during interactions. Due to the COVID-19 restrictions, it was not possible to participate in a live event. Further research could be conducted with a group of case studies, to explore how transpersonal approaches have contributed to organizational transformative journeys. The researcher could explore how senior leaders’ level of awareness impacts the development of organizations. This will allow a focus on different perspectives of the issue, bearing in mind that the contexts of cases differ and generalization should be avoided. Discussion Formal leaders and associates assume business decisions collaboratively and interchangeably. The last phase is enactment, when one learns to embody his or her identity through acts of service to the community (Plotkin, 2021). When individuals feel free to manifest their identity without fear and through new eyes, the collective operates from a new level of awareness. According to Cook-Greuter (2004), such development allows for an expansion of what we are aware of, and consequently what can be influenced and integrated. The present findings show that people were happier and business results improved in many aspects. There was no master plan leading to this outcome, only an impetus “to do things differently”. This self-made path led to the enactment of a better place to work, where results flourish while people feel empowered to do business, and where the leader’s main role was to remove barriers and help people develop. Laloux (2014) affirms that each new state of consciousness allows for more of our human potential and talent to unfold. As far as this case study is concerned, it is possible to claim that the transformative journey has led to a place of higher performance, with more content people, as expressed by Bruce: “Happiness, satisfaction. All we dreamed has worked out, right? I think for many executives it is a dream. It is a huge relief.” An approach to leadership development reinforces that the interior condition of leaders makes the ultimate difference in terms of a team’s performance and their ability to face change. According to Scharmer (2009), the interior condition is “the inner place from which they operate” (p. 7). This inner place, also known as the inner source, will originate the actions performed by leaders –the what – and the processes leaders use to make their actions come to life – the how. This study highlights how the senior leader’s inner condition influenced the actions (what) and the processes (how) experienced by the organization. Consciousness, Spirituality & Transpersonal Psychology, Vol. 3, 2022 154 154 Acknowledgements This article is based on the final dissertation research project undertaken by the author and supervised by Dr. David Lipschitz. It was submitted in partial fulfillment of the requirements for the MSc programme in Consciousness, Spirituality and Transpersonal Psychology. The programme was delivered by Alef Trust in partnership with Liverpool John Moores University, UK. Cossermelli: A transpersonal view of leadership transformation Cossermelli: A transpersonal view of leadership transformation 155 155 References Amram, J. Y. (2009). 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Current Directions in Psychological Science, 24(6), 420–424. https://doi.org/10.1177/0963721415599543 Woolley, A. W., Aggarwal, I., & Malone, T. W. (2015). Collective intelligence and group performance. Current Directions in Psychological Science, 24(6), 420–424. https://doi.org/10.1177/0963721415599543 Yin, R. K. (2009). Case study research: Design and method (4th ed.). Thousand Oaks. Yin, R. K. (2009). Case study research: Design and method (4th ed.). Thousand Oaks. Yin, R. K. (2009). Case study research: Design and method (4th ed.). Thousand Oaks. About the Author Laís Cossermelli has 20 years experience working with multinational organizations, primarily in the field of leadership, organizational development and cultural transformation. She holds a Bachelor in Law, an MBA and is specialized in the field of Anthroposophy. Cossermelli is deeply passionate about individual and collective transformation and this drives all of her personal and professional endeavours. She has been a committed mindfulness practitioner for more than 20 years and holds an MSc in Consciousness, Spirituality and Transpersonal Psychology. Currently, Cossermelli is a MBA professor in the area of change management and cultural transformation. In addition, she is an organizational consultant in the company she cofounded, Flowing 3, which specifically focuses on corporate cultural change. She is a transpersonal psychologist, coach and mentor working with leaders/professionals who are called to integrally work with mind, body, emotions and spirit. Copyright © 2022 Cossermelli. This is an open-access article licensed and distributed under the terms of the Creative Commons Attribution 4.0 International License. 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 © 2022 Cossermelli. This is an open-access article licensed and distributed under the terms of the Creative Commons Attribution 4.0 International License. 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 © 2022 Cossermelli. This is an open-access article licensed and distributed under the terms of the Creative Commons Attribution 4.0 International License. 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. Cossermelli: A transpersonal view of leadership transformation 157 157
https://openalex.org/W2034527479	https://storage.googleapis.com/jnl-sljo-j-sljch-files/journals/1/articles/31/submission/proof/31-1-131-1-10-20080923.pdf	English	null	<i>Mycoplasma pneumoniae</i>: An unrecognized cause of pyrexia of unknown origin	Sri Lanka Journal of Child Health	2,008	cc-by	1,213	Abstract of onset. Causes of continuous fever include viral infections, bacterial infections, parasitic infections (such as malaria and toxoplasmosis), collagen vascular diseases and malignant conditions (such as leukaemia and lymphoma). Objective To find out the incidence of Mycoplasma pneumoniae infection in patients with pyrexia of unknown origin. Design Prospective study. Mycoplasma pneumoniae is a common aetiological agent in community acquired pneumonia, accounting for about 18% of cases1. It is also known to produce infections in extra- pulmonary sites including central nervous system, pericardium or myocardium, hepatobiliary system and genitourinary system2,3,4,5. Children below 4 years of age are rarely infected with this organism. According to the literature mycoplasma pneumoniae is not considered a cause of pyrexia of unknown origin (PUO). Since respiratory tract infections and infections in other body systems due to mycoplasma pneumoniae are not uncommon, it was considered that looking for mycoplasma pneumoniae infection would be a worthwhile exercise in patients with PUO. Setting University Paediatric Unit, Teaching Hospital, Karapitiya. Setting University Paediatric Unit, Teaching Hospital, Karapitiya. Method Patients presenting with fever of more than 7 days with no obvious reason for its occurrence (PUO) to University Paediatric Unit, Teaching Hospital, Karapitiya from January to November 2003, were included. Patients with features of lower or upper respiratory tract infections, urinary tract infections, hepatitis, meningitis, myositis and arthritis were excluded. Routine tests for continuous fever viz. full blood count, test for malaria parasites, ESR, urine full report, urine culture, blood picture, SAT, chest x-ray, Paul-Bunnel test, hepatic transaminases and blood cultures were done in all patients. Mycoplasma antibody titre was done in each patient using the particle agglutination test. Method A prospective study was carried out in the university paediatric unit, Teaching Hospital, Karapitiya from January to November 2003 to find out the incidence of mycoplasma pneumoniae infection among patients with PUO. Patients presenting with fever of more than 7 days with no obvious reason for its occurrence were considered to have PUO and were included in the study. Patients with upper and lower respiratory symptoms, urinary tract symptoms, features of hepatitis, meningitis, myositis and arthritis were excluded. Results There were 40 patients. Age distribution was 2-12 years. 10 patients had mycoplasma pneumoniae infection, mycoplasma antibody titres ranging from 640-20,480. Conclusion 10 out of 40 (25%) children with PUO were due to mycoplasma pneumoniae infection. Sri Lanka Journal of Child Health, 2006; 35: 125-127 Sri Lanka Journal of Child Health, 2006; 35: 125-127 (Key words: Mycoplasma pneumoniae, pyrexia of unknown origin, particle agglutination test) Original Articles Original Articles Introduction The commonest cause of fever in childhood is viral infection and this usually resolves within a week Routine tests for continuous fever such as full blood count, test for malaria parasites, urine full report, urine culture, blood picture, SAT, chest x-ray, Paul- Bunnel test, hepatic transaminases and blood cultures were done in each patient. Mycoplasma antibody test was done by particle agglutination method at the Department of Microbiology, Faculty of Medicine, Karapitiya, Galle. This is a highly sensitive and 1Senior Lecturer in Paediatrics, Faculty of Medicine, Galle (Received on 25 November 2005. Accepted after revision on 22 June 2006). Table 2 Patients with positive Mycoplasma antibody test Pt. Index Age yrs. Sex Clin. features Mycopl. antibody titre 1 13 M Fever 8 days, Headache, Mild hepato- splenomegaly 640 2 8 M Fever 10 days 640 3 8 M Fever 10 days, Mild splenomegaly 640 4 5 F Fever 14 days, Headache 640 5 8 F Fever 8 days, Headache 640 6 8 M Fever 12 days, Mild hepato- splenomegaly 640 7 7 F Fever 14 days, Headache 1240 8 4 F Fever 13 days, Mild splenomegaly 5120 9 8 M Fever 8 days 20480 10 7 F Fever 10 days, Headache, Mild splenomegaly 640 specific test to diagnose acute mycoplasma infection. A titre of 1:80 or more establishes the diagnosis. Table 2 Written consent was obtained from the parents. Ethical approval was obtained from the Ethics committee of the Faculty of Medicine, Karapitiya, Galle. Results 40 patients were included in the study. 24 patients were boys. Age distribution was 2-13 years. The aetiology of continuous fever is shown in Table 1. Mycoplasma pneumoniae infection accounted for 10 patients. In 18 patients the cause of fever was not evident with the conventional laboratory tests. Table 1 Aetiology of continuous fever Cause of fever No. of patients Mycoplasma pneumoniae infection 10 Typhoid fever 5 Infectious mononucleosis 3 Urinary tract infection 3 Juvenile chronic arthritis 1 Unknown 18 Table 1 Aetiology of continuous fever The description of patients with positive mycoplasma antibody test is given in Table 2. The male: female ratio was 1:1. None of the patients were below 4 years of age. Mycoplasma antibody titres ranged from 640-20,480. Discussion When fever has been present for over a week and no cause has been found, hospital admission and adequately investigating for the cause of fever is a common practice. Mycoplasma pneumoniae is commonly considered as a cause of respiratory tract infection. It can cause multiple body system infections including meningitis, encephalitis, pericarditis, myocarditis, hepatitis, nephritis or arthritis. Mycoplasma pneumoniae is not listed under the causes of PUO. In this series of patients 10 out of 40 (25%) presenting with PUO had mycoplasma pneumoniae infection. All of them were above 4 2. Balaguer A, Boronat M, Carrascosa A. Successful treatment of pericarditis associated with mycoplasma pneumoniae infection. Paediatric Infectious Disease Journal 1990; 9(2): 141-3. years of age. Headache was a common presenting feature among these patients and there were hardly any symptoms or signs except mild hapatomegaly or hepatosplenomegaly, in a few of them. Mycoplasma pneumoniae antibody titres were very high, ranging from 640 – 20,480. Micro-particle agglutination test has been used for the above antibody detection and this has 100% sensitivity and 90% specificity6. All these patients responded rapidly to macrolide antibiotics without developing complications. 3. Yamamoto K, Takayanagi M, Yoshihara Y, Murata Y, Kato S, Otake M, et al. Acute disseminated encephalomyelitis associated with mycoplasma pneumoniae infection. Acta Paediatrica Japonica 1996; 38(1): 46-51. Conclusions and recommendations 4. Horii Y, Sugimoto T, Sakamoto I, Sawada T. Acute acalculous cholecystitis complicating mycoplasma pneumoniae infection. Clinical Pediatrics Philadelphia 1992; 31(6): 376-8. Twenty five percent children with PUO were due to mycoplasma pneumoniae infection. It is recommended that mycoplasma pneumoniae infection be considered as a cause of PUO in children. Since micro-particle agglutination test has very high specificity and sensitivity presence of antibody titre more than 320 in a single sample could be considered as an acute infection. 5. Shibasaki T, Gomi H, Otino I, Ishimoto F, Sakai O. A case of chromic renal failure following cold agglutinin due to mycoplasma pneumoniae infection. Nephron 1991; 57(2): 249-50. 6. Aubert G, Pozzetto B, Gaudin OG, Hafid J, Mbida AD, Ros A. Evaluation of five commercial tests: complement fixation, microparticle agglutination, indirect immuno- fluorescence, Enzyme linked immuno sorbent assay and Latex agglutination in comparison to immuno-blotting for mycoplasma pneumoniae serology. Annales de biologie clinique Paris 1992; 50(8): 593-7. Acknowledgements Author thanks Dr. Nelun de Silva and the staff of the Department of Microbiology for doing mycoplasma antibody assay using micro particle agglutination test. References 1. Karalus NC, Cursons RT, Leng RA, Mahood CB, Rothwell RP, Hancock B, et al. Community acquired pneumonia: aetiology and prognostic index evaluation. Thorax 1991; 46(6): 413-8.
https://openalex.org/W4281570264	https://ijbnpa.biomedcentral.com/counter/pdf/10.1186/s12966-022-01298-w	English	null	Associations of ultra-processed food intake with maternal weight change and cardiometabolic health and infant growth	The international journal of behavioural nutrition and physical activity	2,022	cc-by	7,863	Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 https://doi.org/10.1186/s12966-022-01298-w Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 https://doi.org/10.1186/s12966-022-01298-w Open Access © 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Excessive intake of ultra-processed foods, formulated from substances extracted from foods or derived from food constituents, may be a modifiable behavioral risk factor for adverse maternal and infant health outcomes. Prior work has predominately examined health correlates of maternal ultra-processed food intake in populations with substantially lower ultra-processed food intake compared to the US population. This longitudinal study investigated relations of ultra-processed food intake with maternal weight change and cardiometabolic health and infant growth in a US cohort. Methods: Mothers in the Pregnancy Eating Attributes Study were enrolled at ≤12 weeks gestation and completed multiple 24-Hour Dietary Recalls within six visit windows through one-year postpartum (458 mothers enrolled, 321 retained at one-year postpartum). The NOVA (not an acronym) system categorized food and underlying ingredient codes based on processing level. Maternal anthropometrics were measured throughout pregnancy and postpartum, and infant anthropometrics were measured at birth and ages 2 months, 6 months, and 1 year. Maternal cardiometa- bolic markers were analyzed from blood samples obtained during the second and third trimesters. Results: Holding covariates and total energy intake constant, a 1-SD greater percent energy intake from ultra- processed foods during pregnancy was associated with 31% higher odds of excessive gestational weight gain (p = .045, 95% CI [1.01, 1.70]), 0.68±0.29 mg/L higher c-reactive protein during pregnancy (p = .021, 95% CI [0.10, 1.26]), 6.7±3.4% greater gestational weight gain retained (p = .049, 95% CI [0.03, 13.30]), and 1.09±0.36 kg greater postpar- tum weight retention (p = .003, 95% CI [0.38, 1.80]). No other significant associations emerged. Conclusions: Ultra-processed food intake during pregnancy may be a modifiable behavioral risk factor for adverse maternal weight outcomes and inflammation. Randomized controlled trials are needed to test whether targeting ultra-processed food intake during pregnancy may support optimal maternal health. Trial registration: Clinicaltrials.gov. Registration ID – NCT02217462. Date of registration – August 13, 2014. Keywords: Ultra-processed food, Pregnancy, Postpartum, Maternal weight change, Cardiometabolic health, Infant weight-for-length Background Excessive intake of ultra-processed foods, including snacks, drinks, ready meals, and other products formu- lated from substances extracted from foods or derived from food constituents, is widespread [1] and may com- promise maternal and infant health. Ultra-processed Correspondence: nanselt@mail.nih.gov 1 Social and Behavioral Sciences Branch, Division of Population Health Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 6710B Rockledge Drive, Bethesda, MD 20817, USA Full list of author information is available at the end of the article Dietary intake assessment The current longitudinal study examined relations of ultra-processed food intake during pregnancy and postpartum with maternal weight change and cardio- metabolic health and infant growth in a US cohort. We hypothesized that greater ultra-processed food intake during pregnancy and postpartum would be associated with excessive maternal weight gain and retention, worse maternal cardiometabolic health, and greater infant growth. Mothers were asked to complete the well-validated Auto- mated Self-Administered 24-Hour (ASA24) dietary recall [14] once within each visit window, indicating all foods consumed in the past 24 hours, including details on food preparation, brands, portion size, and additions. The ASA24 program assigns food codes from the US Depart- ment of Agriculture Food and Nutrient Database for Die- tary Studies (FNDDS) and provides nutrition information including kilocalories [14]. Research staff at the Univer- sity of North Carolina Nutrition and Obesity Research Core corrected implausible and missing food codes and nutrition information. Records (1.9%) with implausi- ble daily energy intake (< 600 kcal/day), based on estab- lished cutoffs adjusted for increased energy requirements of pregnancy, were excluded from analysis resulting in © 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Page 2 of 11 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act early pregnancy BMI (kg/m2) ≥18.5 and absence of pre- existing diabetes, any medical condition contraindicating study participation, participant-reported eating disorder, and use of medication that could affect diet or weight, were previously described in detail [12]. Mothers pro- vided informed consent before enrollment, and visits occurred at <12 weeks (baseline/first trimester), 16-22 weeks (second trimester), and 28-32 weeks (third tri- mester) gestation and at 4-14 weeks (~2 months), 23-31 weeks (~6 months), and 50-58 weeks (~1 year) post- partum. At baseline, mothers were on average 30.5±4.7 years of age; 71.6% attained at least a bachelor’s degree; 71.4% were white; 47.8% were with normal weight (BMI ≥18.5, <25), 25.1% were with overweight (BMI ≥25, <30), and 27.1% were with obesity (BMI ≥30) (see Nansel et al., 2020 for other sociodemographic characteristics of the sample). foods are formulations resulting from a series of indus- trial processes including whole food fractioning, chemical modification (e.g., hydrolysis), assembly (e.g., pre-frying), additions for palatability (e.g., colors, flavors, emulsifiers), and packaging with synthetic materials [1]. These foods comprise more than half of the total dietary energy con- sumed in the US and other high-income countries [1]. A systematic review indicated that, in non-pregnant adults, greater ultra-processed food intake was associated with higher risk of overweight and obesity, all-cause mortal- ity, overall cardiovascular disease, and overall cancer in cross-sectional and longitudinal studies [2]. Given that excessive maternal weight change [3] and infant weight [4] increase risk of adverse maternal and infant health outcomes, investigating their associations with ultra-pro- cessed food intake may elucidate a modifiable behavioral risk factor. Procedures were approved by the University of North Carolina Institutional Review Board (study #18-2030) and were in accordance with the ethical standards of the Helsinki Declaration of 1975 as revised in 1983. Recruitment began in November 2014 and completed in December 2016; data collection completed in June 2018. Figure 1 presents a flow diagram of the number of moth- ers at each study stage. Of 458 mothers enrolled, 367 and 321 were retained through delivery and one-year post- partum, respectively [13]. The predeclared endpoint of PEAS was to examine the role of reward-related eating in maternal diet and weight change during pregnancy and postpartum (https://clinicaltrials.gov/ct2/show/NCT02 217462). The present study was a secondary data analy- sis using all available data for variables of interest; since these analyses were not predeclared, they are considered exploratory. Ultra-processed food intake during pregnancy and postpartum may increase maternal weight gain and car- diometabolic disease risk, and increase infant weight through the prenatal environment and breastfeeding [5]. In Brazilian women, greater ultra-processed food intake during pregnancy was associated with greater gestational weight gain [6, 7], diabetes risk [6], and pro-inflammatory potential of diet [8]. Greater postpartum intake of ultra- processed foods was associated with lower concentra- tions of essential vitamins in breastmilk [9] and higher incidence of infant overweight [10]. Although the aver- age energy intake from ultra-processed foods in the US is nearly double that of Brazil [1], only one small study (n = 45) has examined ultra-processed food intake during pregnancy in American women, finding associations with greater gestational weight gain and offspring adiposity at birth [11]. Further research is needed to understand rela- tions of maternal ultra-processed food intake with mater- nal and infant weight and health outcomes in populations with high ultra-processed food intake. Methods Design, setting, participants, and procedures Th b d ( g , g, p p , p The Pregnancy Eating Attributes Study (PEAS) examined dietary intake and weight change during pregnancy and postpartum in women from a metropolitan area in North Carolina, United States [12]. Eligibility criteria, including Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act Page 3 of 11 Fig. 1 Flow Diagram of the number of mothers at each study stage Fig. 1 Flow Diagram of the number of mothers at each study stage or in the form of steaks, fillets, and other cuts; fresh or pasteurized milk; pasta, couscous, and polenta made with flours, flakes, or grits and water), exclusion of one participant during pregnancy and one participant during postpartum [15, 16]. To assess maternal ultra-processed food intake, food codes were categorized according to the NOVA (not an acronym) classification system, which is a set of descrip- tive criteria developed by an academic group at the University of São Paulo [1]. Standardized Stata (College Station, TX) code for applying NOVA was used [17], which is available upon request from the University of São Paulo group. Ultra-processed food categorization according to NOVA has demonstrated convergent valid- ity with calculations of added sugars and macro- and micro-nutrients [1]. Methods There are four categories: ll (b) group 2 – processed culinary ingredients, which are substances obtained directly from group 1 foods or from nature by industrial processes such as press- ing, centrifuging, refining, extracting, or mining (e.g., vegetable oils crushed from seeds, nuts, or fruits; butter and lard obtained from milk and pork; sugar and molasses obtained from cane or beet), (c) group 3 – processed foods, which are products made by adding group 2 ingredients to group 1 foods, using preservation methods such as canning or bottling and industrial processes such as non- alcoholic fermentation (e.g., freshly made unpack- aged breads and cheeses; fruit preserves; salted or sugared nuts and seeds), (a) group 1 – unprocessed or minimally processed foods, which are foods in their natural form or altered by industrial processes such as removal of inedible or unwanted parts, drying, crushing, grinding, fractioning, roasting, boiling, pasteuriza- tion, refrigeration, freezing, placing in containers, or vacuum packaging (e.g., fresh, squeezed, chilled, frozen, or dried fruits and vegetables; fresh, chilled, and frozen meat, poultry, fish, and seafood, whole (d) group 4 – ultra-processed foods (e.g., ‘instant’ foods; ready-to-heat pre-prepared pies, pasta, and pizza dishes; mass-produced packaged breads; reconstituted meats; sweet or savory packaged snacks; confectionery desserts; sweetened drinks). (d) group 4 – ultra-processed foods (e.g., ‘instant’ foods; ready-to-heat pre-prepared pies, pasta, and pizza dishes; mass-produced packaged breads; reconstituted meats; sweet or savory packaged snacks; confectionery desserts; sweetened drinks). Page 4 of 11 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act For food codes indicating a homemade recipe, underly- ing ingredient codes and correspondent nutrition infor- mation were obtained from the FNDDS and categorized according to NOVA [17]. See Additional file 1 for the 15 most-frequently reported foods from the ultra-processed food category in the current study sample and see Mon- teiro et al. [1] for full definitions and lists of examples of foods assigned to each NOVA category. calculated by multiplying 100 by the difference in weight from last prenatal medical visit to 1-year postpartum divided by gestational weight gain; percent of gestational weight gain retained (but no other maternal anthropo- metrics) was coded as missing for participants who had ≤2 kg gestational weight gain. Infant anthropometrics Patient medical records indicated infant weight (kg) and length (head to foot; cm) at birth. At 2 months, 6 months, and 1 year, infant weight was measured to the nearest 0.01 kg on an infant scale and length was measured to the nearest 0.1 cm using a recumbent infant board with a stadiometer. Each measure was obtained twice; if the two measurements varied by more than 0.2 kg (weight) or 1 cm (length), a third measure was taken. The mean of the two closest measurements was calculated. Infant weight- for-length z-scores were calculated based on US Cent- ers for Disease Control and Prevention reference growth curves for infant age and sex [28]. Methods Postpartum weight change (kg) was calculated by subtracting baseline weight from weight at 1-year postpartum. Given that there is little change in dietary intake across pregnancy trimesters [18–20], total daily energy intake (kcal/day) during pregnancy and postpartum were cal- culated by averaging total daily energy intake across all ASA24 dietary recalls collected during pregnancy and across all ASA24 dietary recalls collected during post- partum, respectively. Percent daily energy intake from ultra-processed foods during pregnancy and postpar- tum were calculated by dividing the average total daily energy from ultra-processed foods by the average total daily energy intake [17]. The intent of using percent daily energy intake from ultra-processed foods was to reduce bias introduced by non-differential calorie misreporting from all foods [21]. Maternal anthropometrics At baseline, maternal height was measured to the nearest 0.1 cm using a wall-mounted stadiometer. At each study visit, weight was measured to the nearest 0.1 kg using a standing scale, and skinfolds thickness was measured to the nearest 0.1 mm using skinfold calipers. Each anthro- pometric measurement was obtained twice; if the two measurements varied by more than 1 cm (height), 0.2 kg (weight), or 2 mm (skin folds), a third measure was taken. The mean of the two closest measurements was calculated. Early pregnancy BMI was calculated from baseline height and weight. Patient medical records indi- cated maternal weight at prenatal medical visits. Con- sistent with clinical practice and prior research [22, 23], gestational weight gain was calculated as the difference between baseline weight and the last prenatal medical visit weight [M(SD) = 0.35(0.75) weeks prior to deliv- ery]. Gestational weight gain was categorized as inad- equate, adequate, or excessive using 2009 Institute of Medicine guidelines [24], which indicate optimal range of weight gain according to pre-pregnancy BMI (here, early pregnancy BMI). Gestational weight gain was cat- egorized because the direction of association between absolute gestational weight gain and health outcomes differs depending on these categories [24]. Gestational fat gain (kg) was calculated from weight and thigh skin- fold change using an equation validated for pregnancy: 0.77weight change + 0.07*thigh skinfold change – 6.13 [25]. Percent of gestational weight gain retained was Cardiometabolic markers At the second trimester visit, mothers were instructed to fast for at least 8 hours before blood samples (40 ml) were collected; HDL (mg/dL), LDL (mg/dL), triglycer- ides (mg/dL), glucose (mg/dL), insulin (pmol/L), and c-peptide (nmol/L) were analyzed from the fasting sec- ond-trimester samples. At the third trimester visit, non- fasting blood samples (30 ml) were collected; Interleukin 6 (IL-6; pg/mL), tumor necrosis factor alpha (TNF-a; pg/ mL), and c-reactive protein (CRP; mg/L) were analyzed from the non-fasting third-trimester samples. While natural changes in cardiometabolic markers occur during pregnancy, excessive elevations of cardiometabolic mark- ers, particularly during the second and third trimesters, have been associated with adverse maternal and child outcomes [26, 27]. Samples were processed within 30-60 minutes after collection including ensuring proper dis- tribution of anticoagulant, transferring to cryovials, and affixing barcode labels. Samples were stored in a freezer at -20°C for up to 5 days and then transferred on dry ice to a freezer at -80°C. Lipids and CRP were measured using standardized methods on a Roche COBAS 6000 (Roche Diagnostics, Indianapolis, MN) and IL-6 and TNF-a were measured using standard sandwich ELISAs (R&D Systems, Minneapolis, MN). Statistical analysis E f h β1i = γ10+ γ11(%Energy Intake from Ultra-Pro- cessed Foods During Pregnancy1i) + γ12(%Energy Intake from Ultra-Processed Foods During Postpartum1i) + u1i β1i = γ10+ γ11(%Energy Intake from Ultra-Pro- cessed Foods During Pregnancy1i) + γ12(%Energy Intake from Ultra-Processed Foods During Postpartum1i) + u1i Except for the cardiometabolic markers and weight change variables, which have clinically interpretable units, continuous variables were z-scored for ease of interpretation. Linear regressions estimated relations of maternal ultra-processed food intake during pregnancy and postpartum with maternal weight change and car- diometabolic markers. Logistic regression examined relations with gestational weight gain (adequate used as reference category). Model 1 included percent energy intake from ultra-processed foods as the only independ- ent variable; Model 2 added covariates and total energy intake. In Model 2, intercept was additionally predicted by maternal total energy intake during pregnancy, and slope was predicted by maternal total energy intake during pregnancy and postpartum (entered at Level 2). Analyses were conducted in SAS 9.4 (Cary, NC) using maximum likelihood estimation/restricted maximum likelihood estimation (REML) to account for missing data. Signifi- cance was set at p < .05. To determine which covariates to include in Model 2, bivariate associations of potential covariates with inde- pendent and dependent variables (uncorrected for mul- tiple comparisons) were examined (see Additional files 2 and 3). Variables that were significantly associated (p < .05) with both independent and dependent variables were included as covariates. While total energy intake was only modestly associated with ultra-processed food intake during pregnancy and postpartum, it was included as a covariate to examine the independent associations of total energy intake and ultra-processed food intake with dependent variables. In addition, early pregnancy BMI was included as a covariate (a) in the gestational fat gain analysis because gestational fat gain was inversely corre- lated with early pregnancy BMI and adverse cardiometa- bolic health during pregnancy and (b) in the CRP analysis because prior work indicates adiposity (as proxied by Covariatesh The system of equations was as follows, wherein t = time and i = individual: Level 1: Infant Weight-for-lengthti = β0i + β1i(Timeti)+ ɛti Level 2: β0i = γ00+ γ01(%Energy Intake from Ultra- Processed Foods During Pregnancy0i) + u0i Level 2: β0i = γ00+ γ01(%Energy Intake from Ultra- Processed Foods During Pregnancy0i) + u0i Covariatesh The following covariates were considered because of their potential causal influence on both ultra-processed food intake and dependent variables: maternal age, Page 5 of 11 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 BMI) is a major determinant of CRP independent of die- tary intake [32]. low-intensity physical activity, moderate- and vigorous- intensity physical activity, and income-poverty ratio at baseline and smoking status and alcohol use during preg- nancy. Patient medical records indicated maternal age (in years) at baseline and smoking status (1 = “Smoker During Pregnancy,” 2 = “Former Smoker,” 3 = “Never Smoker”) and alcohol use during pregnancy (0 = “No,” 1 = “Yes”). At baseline, mothers reported how often they typically engage in multiple physical activities (e.g., bicy- cling, dancing, tennis) via questions adapted for pregnant women in the National Health and Nutrition Examina- tion Survey [29]; the intensity of the activities was coded as low, moderate, or vigorous based on standard MET intensities [30], and low-intensity and moderate- and vigorous-intensity physical activity variables were calcu- lated by summing the number of times per week mothers engaged in respective activities. Mothers reported their total annual household income, and income-poverty ratio was calculated by dividing this by the US Census Bureau 2016 poverty thresholds accounting for household size and number of children [31]. To examine relations of maternal ultra-processed food intake during pregnancy and postpartum with infant weight-for-length, multilevel growth modeling nesting repeated weight-for-length measures within infants was conducted with time (coded as 0, 1/6, 1/2, and 1 year) entered at Level 1. Time was centered such that the mod- el’s intercept represented infant weight-for-length at birth and the model’s slope represented infant weight-for- length trajectory from birth to 1 year. Random effects for intercept and slope were included because they improved model fit as indicated by a significantly smaller -2LL (log-likelihood). In Model 1, intercept was predicted by maternal ultra-processed food intake during pregnancy only and slope was predicted by maternal ultra-processed food intake during pregnancy and postpartum (entered at Level 2). Results Univariate statistics for variables of interest are pre- sented in Table 1. On average, participants consumed 52.6±15.1% and 50.6±16.6% of energy intake from ultra- processed foods during pregnancy and postpartum, respectively. Estimates for associations of maternal ultra-pro- cessed food intake with maternal weight change and cardiometabolic markers are presented in Tables 2 and 3, respectively. Holding covariates and total energy intake constant, a 1-SD greater percent daily energy intake from ultra-processed foods during pregnancy was associated with 31% higher odds of excessive ges- tational weight gain and 0.68±0.29 mg/L higher CRP during pregnancy. Holding covariates and total energy intake constant, a 1-SD greater percent daily energy Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Page 6 of 11 Cummings et al. Int J Behav Nutr Phys Act Table 1 Univariate statistics for variables of interest n Notes: Untransformed data are presented. CRP C-Reactive Protein, IL-6 Interleukin 6, TNF-a Tumor Necrosis Factor Alpha Maternal Age at Baseline, years (M, SD) 458 30.46 (4.74) Week of Gestation at Baseline (M, SD) 458 9.82 (1.75) Smoking Status (n, %) 291 Smoker During Pregnancy 7 (2.4%) Former Smoker 51 (17.5%) Never Smoker 233 (80.1%) Alcohol Use During Pregnancy (n, %) 290 No 164 (56.6%) Yes 126 (43.5%) Low-Intensity Physical Activity, times/week (M, SD) 294 3.81 (4.19) Moderate- and Vigorous-Intensity Physical Activity, times/week (M, SD) 294 1.07 (2.00) Income-Poverty Ratio (M, SD) 364 3.84 (1.97) Total Energy Intake During Pregnancy, kcal/day (M, SD) 365 2017.46 (678.74) %Energy Intake from Ultra-Processed Food During Pregnancy (M, SD) 365 52.58 (15.12) Total Energy Intake During Postpartum, kcal/day (M, SD) 266 1980.22 (655.07) %Energy Intake from Ultra-Processed Food During Postpartum (M, SD) 266 50.57 (16.62) Early Pregnancy BMI, kg/m2 (M, SD) 458 27.19 (6.94) Gestational Weight Gain (n, %) 367 Inadequate 70 (19.1%) Adequate 124 (33.8%) Excessive 173 (47.1%) Gestational Fat Gain, kg (M, SD) 355 0.48 (3.46) %Gestational Weight Gain Retained (M, SD) 285 3.56 (51.82) Postpartum Weight Change, kg (M, SD) 293 0.78 (5.37) HDL During Second Trimester, mg/dL (M, SD) 360 73.43 (14.55) LDL During Second Trimester, mg/dL (M, SD) 360 125.33 (34.65) Triglycerides During Second Trimester, mg/dL (M, SD) 360 133.64 (64.04) Glucose During Second Trimester, mg/dL (M, SD) 360 79.49 (10.05) Insulin During Second Trimester, pmol/L (M, SD) 359 79.41 (108.29) C-peptide During Second Trimester, nmol/L (M, SD) 360 0.66 (0.43) IL-6 During Third Trimester, pg/mL (M, SD) 350 0.59 (1.02) TNF-a During Third Trimester, pg/mL (M, SD) 350 5.81 (1.84) CRP During Third Trimester, mg/L (M, SD) 350 5.11 (4.30) Infant Weight-for-length (M, SD) At birth 331 -0.44 (1.51) At 2 months 331 -0.40 (1.52) At 6 months 293 -0.06 (1.15) At 1 year 298 0.30 (0.97) Infant maternal weight change and cardiometabolic marker variables were non-significant. intake from ultra-processed foods during pregnancy was associated with 6.7±3.4% greater gestational weight gain retained and 1.09±0.36 kg greater post- partum weight retention; however, ultra-processed food intake during postpartum was not significantly associated with these weight outcomes. Associations of maternal ultra-processed food intake with other i Estimates for associations of maternal ultra-processed food intake with infant weight-for-length are presented in Table 4. On average, infant weight-for-length z-score at birth (intercept) was -0.50±0.07 (p < .001, 95% CI [-0.64, -0.35]) and infant weight-for-length z-score Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Page 7 of 11 Table 2 Estimates for associations of maternal ultra-processed food intake with maternal weight change Table 2 Estimates for associations of maternal ultra-processed food intake with maternal weight change Notes: In pregnancy and postpartum models, adjusted for atotal energy intake. In pregnancy models, adjusted for bearly pregnancy body mass index, cage, dincome- poverty ratio %Energy Intake from Ultra-Processed Foods (z-scored) During Pregnancy During Postpartum OR p 95% CI OR p 95% CI Gestational Weight Gain Model 1 Inadequate > Adequate 0.88 .461 0.64, 1.23 -- -- -- Excessive > Adequate 1.33 .033 1.02, 1.73 -- -- -- Model 2a Inadequate > Adequate 0.88 .451 0.63, 1.23 -- -- -- Excessive > Adequate 1.31 .045 1.01, 1.70 -- -- -- B±SE p 95% CI B±SE p 95% CI Gestational Fat Gain (kg) Model 1 0.07±0.20 .712 -0.32, 0.46 -- -- -- Model 2a,b,c,d 0.32±0.19 .094 -0.05, 0.69 -- -- -- %Gestational Weight Gain Retained Model 1 7.68±3.29 .020 1.20, 14.17 -3.81±3.35 .256 -10.41, 2.79 Model 2a,c,d 6.66±3.37 .049 0.03, 13.30 -4.33±3.39 .202 -11.00, 2.34 Postpartum Weight Change (kg) Model 1 1.19±0.35 <.001 0.50, 1.87 -0.36±0.36 .318 -1.06, 0.35 Model 2a,c,d 1.09±0.36 .003 0.38, 1.80 -0.44±0.36 .222 -1.14, 0.27 weight gain corroborates with previous findings [6, 7, 11], while the positive relations with gestational weight gain retained and postpartum weight retention were previously unexplored. Prior work showed a higher pro- inflammatory potential of diet for women who consumed more ultra-processed foods during pregnancy [8], which the present study also built upon by finding a positive association of maternal ultra-processed food intake with CRP during pregnancy. However, the lack of significant relations of pregnancy ultra-processed food intake with pregnancy elevated glucose and infant weight-for-length is inconsistent with prior studies [6, 10, 11]. Infant Prior stud- ies included smaller sample sizes [6, 11], samples with pregestational diabetes [6], dietary assessment using food frequency questionnaires [6, 10, 11], adjustment for different covariates (e.g., number of prenatal consulta- tions) [6, 10], and different infant weight and adiposity measurements (i.e., time to infant overweight, infant per- cent body fat) [10, 11], which may explain inconsistent findings. trajectory from birth to 1 year (slope) was 0.75±0.10 (p < .001, 95% CI [0.56, 0.95]). Positive associations of mater- nal ultra-processed food intake during pregnancy with infant weight-for-length at birth and with the trajectory from birth to 1 year were non-significant. The negative association of maternal ultra-processed food intake dur- ing postpartum with infant weight-for-length trajectory from birth to 1 year was also non-significant. Discussion l Adjusted for atotal energy intake, bincome-poverty ratio, cmoderate- and vigorous-intensity physical activity, dage, elow-intensity physical activity, and fearly pregnancy body mass index %Energy Intake from Ultra-Processed Foods During Pregnancy (z-scored) B±SE p 95% CI Second Trimester HDL (mg/dL) Model 1 -0.95±0.80 .232 -2.52, 0.61 Model 2a,b,c -0.14±0.96 .885 -2.03, 1.76 LDL (mg/dL) Model 1 -0.50±1.90 .791 -4.25, 3.24 Model 2a -0.73±1.92 .705 -4.49, 3.04 Triglycerides (mg/dL) Model 1 4.11±3.59 .254 -2.95, 11.16 Model 2a,b,c,d -0.89±3.18 .779 -7.14, 5.37 Glucose (mg/dL) Model 1 -0.70±0.48 .145 -1.65, 0.24 Model 2a,d -0.39±0.49 .425 -1.35, 0.57 Insulin (pmol/L) Model 1 5.00±4.23 .238 -3.32, 13.31 Model 2a,b,c 2.27±4.92 .645 -7.43, 11.96 C-peptide (nmol/L) Model 1 0.04±0.02 .066 0.00, 0.08 Model 2a,b,c 0.03±0.02 .247 -0.02, 0.08 Third Trimester IL-6 (pg/mL) Model 1 0.07±0.06 .277 -0.05, 0.19 Model 2a,b 0.06±0.07 .346 -0.07, 0.19 TNF-a (pg/mL) Model 1 0.18±0.11 .090 -0.03, 0.39 Model 2a,b 0.10±0.11 .356 -0.12, 0.33 CRP (mg/L) Model 1 0.89±0.25 <.001 0.39, 1.39 Model 2a,b,c,d,e,f 0.68±0.29 .021 0.10, 1.26 Table 3 Estimates for associations of maternal ultra-processed food Intake with cardiometabolic markers obtained during the second and third trimesters low-protein foods to meet protein needs) [33, 35], quicker eating rate [33], exposure to phthalates and bis- phenol [21], and alterations to the gut microbiome [36], is warranted. In the present study, positive associations of ultra-processed food intake with maternal weight gain and retention and pregnancy CRP did not weaken when statistically accounting for total energy intake, sug- gesting that increased total energy may not be a primary mechanism. Strengths and limitations of the present study should be considered when interpreting findings. This second- ary analysis was strengthened by the relatively large sample size wherein over half of the women were with overweight or obesity, enhancing internal validity. Internal validity was further strengthened by the pro- spective study design; repeated measures of maternal diet and anthropometrics throughout pregnancy and postpartum and infant anthropometrics from birth to age 1 year; directly measured maternal and infant weight; multiple maternal cardiometabolic biomarkers; and measurement of and adjustment for several con- founds including maternal physical activity and total energy intake. Ultra-processed food intake was assessed according to the NOVA classification system by using data from mul- tiple 24-hour dietary recalls, the least biased self-report assessment available [37]. Discussion l Greater ultra-processed food intake during pregnancy was associated with greater gestational weight gain and retention, postpartum weight retention, and pregnancy CRP in a US cohort. However, pregnancy intake of ultra- processed foods was not significantly associated with other indicators of maternal cardiometabolic health and infant weight-for-length, and postpartum intake was not significantly associated with maternal or infant out- comes. The magnitude and significance pattern of the results were consistent when controlling for total energy intake. i The present study findings do not elucidate the mechanism(s) by which pregnancy intake of ultra-pro- cessed foods would promote adverse maternal weight outcomes and inflammation. Future research investigat- ing mechanisms, including increased total energy and added sugars intake [17, 33], displacement of nutrient- rich foods [34], protein leverage (i.e., overconsuming Women consumed just over half their daily energy intake from ultra-processed foods during pregnancy and postpartum, consistent with observations in non-preg- nant U.S. populations [1]. The positive relation of ultra- processed food intake during pregnancy with gestational Page 8 of 11 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act Table 3 Estimates for associations of maternal ultra-processed food Intake with cardiometabolic markers obtained during the second and third trimesters Notes:CRP C-Reactive Protein, IL-6 Interleukin 6, TNF-a Tumor Necrosis Factor Alpha. Notes: In pregnancy and postpartum models, adjusted for atotal energy intake Funding g This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Develop- ment for the funding of the study (contracts #HHSN275201300012C and #HHSN275201300026I/HHSN27500002) and support of Jenna R. Cummings and Carolina Schwedhelm. The funding agency had no role in the design of the study; collection, analysis, and interpretation of the data; and writing of the manuscript. Open Access funding provided by the National Institutes of Health (NIH). Availability of data and materials Data described in the manuscript, code book, and analytic code will be made available from the corresponding author upon request pending application and approval. Competing interests The authors declare that they have no competing interests. ASA24: Automated Self-Administered 24-Hour Dietary Recall; BMI: Body Mass Index; CRP: C-Reactive Protein; FNDDS: Food and Nutrient Database for Dietary Studies; IL-6: Interleukin 6; PEAS: Pregnancy Eating Attributes Study; REML: Conclusions d d Findings indicate that maternal pregnancy intake of ultra-processed foods may be a modifiable behavio- ral risk factor for excessive maternal weight change and inflammation for women in the US. Although the present study findings need to be confirmed in other pregnant populations, decreasing maternal ultra-processed food intake may be an important behavioral target in prenatal interventions. Authors’ contributions JRC, LML, & TRN designed research; CS obtained underlying ingredient codes and correspondent nutrition information from the FNDDS; JRC analyzed data; JRC wrote paper and LML, CS, AL, & TRN reviewed and edited; JRC & TRN had primary responsibility for final content. All authors read and approved the final manuscript. Limitations notwithstanding, the current study find- ings support interventions that reduce exposure to ultra-processed foods in pregnant women. Public health proposals include strategies to de-incentivize ultra- processed food intake, such as changing regulations for ultra-processed food production, increasing taxation of ultra-processed foods, and decreasing subsidies for ultra- processed food ingredients [41]. Proposed strategies to incentivize unprocessed and minimally processed food intake include increasing accessibility to and affordability of meal kits and meal sharing [41]. In a recent non-ran- domized trial in Brazilian women, an educational inter- vention during routine prenatal medical visits reduced maternal ultra-processed food intake by 19% [42], dem- onstrating the efficacy of targeting this behavioral risk factor at the individual level. Randomized controlled tri- als in diverse populations are also needed to test whether targeting ultra-processed food intake during pregnancy may support optimal maternal weight change and health. Author details 1 1 Social and Behavioral Sciences Branch, Division of Population Health Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 6710B Rockledge Drive, Bethesda, MD 20817, USA. 2 Molecular Epidemiology Research Group, Max-Delbrueck-Center for Molecu- lar Medicine in the Helmholtz Association, Berlin, Germany. 3 Biostatistics and Bioinformatics Branch, Division of Population Health Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 6710B Rockledge Drive, Bethesda, MD 20817, USA. Received: 18 January 2022 Accepted: 10 May 2022 Received: 18 January 2022 Accepted: 10 May 2022 Consent for publication Not applicable Consent for publication Not applicable Discussion l Although misclassification is possible when applying NOVA [38], the potential for misclassification was mitigated by using standardized statistical code for applying NOVA and by disaggregat- ing foods to underlying ingredients [17]. Limitations of NOVA, however, are that the categories lack quantitative cutoffs (e.g., two foods could both be in group 3 regard- less of differing amounts of added processed culinary ingredients), that food additives may be legally permitted in a food but not present, and that nutritious and sustain- able foods (e.g., soy-based meat and dairy alternatives) can be classified as ultra-processed [39, 40]. Notes:CRP C-Reactive Protein, IL-6 Interleukin 6, TNF-a Tumor Necrosis Factor Alpha. Adjusted for atotal energy intake, bincome-poverty ratio, cmoderate- and vigorous-intensity physical activity, dage, elow-intensity physical activity, and fearly pregnancy body mass index Table 4 Estimates for associations of maternal ultra-processed food intake with infant weight-for-length Notes: In pregnancy and postpartum models, adjusted for atotal energy intake %Energy Intake from Ultra-Processed Foods (z-scored) During Pregnancy During Postpartum γ±SE p 95% CI γ±SE p 95% CI Weight-for-length at birth (Intercept) Model 1 0.02±0.08 .785 -0.14, 0.19 -- -- -- Model 2a 0.02±0.08 .817 -0.15, 0.18 -- -- -- Weight-for-length trajectory from birth to 1 year (Slope) Model 1 0.10±0.12 .405 -0.13, 0.33 -0.04±0.08 .610 -0.19, 0.11 Model 2a 0.11±0.12 .361 -0.12, 0.34 -0.06±0.08 .472 -0.21, 0.10 Table 4 Estimates for associations of maternal ultra-processed food intake with infant weight-for-length Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Page 9 of 11 Restricted Maximum Likelihood Estimation,; TNF-a: Tumor Necrosis Factor Alpha. Given the observational study design, causal infer- ences from the present study are limited. The temporal precedence of ultra-processed food intake was limited in some of the examined associations. Specifically, dietary assessments were averaged across pregnancy to examine associations with cardiometabolic markers obtained at the second and third trimesters, and dietary assessments were averaged across postpartum to exam- ine the association with infant weight-for-length slope from birth to 1 year. However, averaging of multiple dietary recalls provides a more valid estimate of typi- cal intake than does a single recall, and dietary recalls, cardiometabolic markers, and weight are thought to reflect individuals’ typical behaviors and health status. The sociodemographic characteristics were consistent with those from the metropolitan area in North Caro- lina, US, but are not representative of women through- out the US. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12966-022-01298-w. The online version contains supplementary material available at https://doi. org/10.1186/s12966-022-01298-w. Additional file 1. Additional file 2. Additional file 3. Additional file 1. Additional file 2. Additional file 3. Discussion l Restricted Maximum Likelihood Estimation,; TNF-a: Tumor Necrosis Factor Alpha. Received: 18 January 2022 Accepted: 10 May 2022 Ethics approval and consent to participate Procedures were approved by the University of North Carolina Institutional Review Board (study #18-2030) and were in accordance with the ethical stand- ards of the Helsinki Declaration of 1975 as revised in 1983. Mothers provided informed consent. References 6. 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BMJ. 2020;26:369. Page 11 of 11 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 Cummings et al. Int J Behav Nutr Phys Act (2022) 19:61 42. de Barros Gomes C, Malta MB, da Costa Louzada ML, Benício MHDA, Bar- ros AJ, Carvalhaes MA. Ultra-processed food consumption by pregnant women: the effect of an educational intervention with health profession- als. Matern Child Health J 2019;23(5):692-703. Publisher’s Note 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. 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https://openalex.org/W3217802519	https://www2.ifrn.edu.br/ojs/index.php/RBEPT/article/download/12037/pdf	Portuguese	null	Reflexões metodológicas para o ensino de Direito aplicado aos Cursos Técnicos do Instituto Federal de Rondônia, Campus Porto Velho Zona Norte	Revista Brasileira da Educação Profissional e Tecnológica	2,021	cc-by	5,649	Resumo Recebido: 20/02/2021 \| Revisado: 20/05/2021 \| Aceito: 25/05/2021 \| Publicado: 17/11/2021 Cássia Luciana de Melo Lima ORCID: http://orcid.org/0000-0002-7263- 2010 Instituto Federal de Educação Ciência e Tecnologia de Rondônia E-mail: cassia.lima@ifro.edu.br Iza Reis Gomes Ortiz ORCID: http://orcid.org/0000-0001-8668- 1692 Instituto Federal de Educação Ciência e Tecnologia de Rondônia E-mail: iza.reis@ifro.edu.br Como citar: LIMA, C. L. de; ORTIZ, i. r. g. Reflexões metodológicas para o ensino de Direito aplicado aos Cursos Técnicos do Instituto Federal de Rondônia, Campus Porto Velho Zona Norte. Revista Brasileira da Educação Profissional e Tecnológica, [S.l.], v. 2, n. 21, p. e12037, nov. 2021. ISSN 2447-1801. A partir de um contexto específico – o ensino do Direito nas turmas dos cursos técnicos do Instituto Federal de Rondônia (IFRO), Campus Porto Velho Zona Norte – o texto tem por objetivo encontrar soluções didáticas mais adequadas para a construção de conhecimento jurídico. Dessa forma, são apresentadas diferentes concepções metodológicas pautadas nas tendências pedagógicas, dando ênfase aos métodos de ensino participativos. O esforço permite, ainda, uma reflexão sobre o significado e importância do Direito para a formação cidadã e profissional. Para tanto, apoia-se em pesquisa bibliográfica e documental. Como resultado, apresenta possibilidades metodológicas para o ensino de Direito em cursos técnicos, tendo como foco o desenvolvimento de sujeitos autônomos e reflexivos, capazes de resolver questões jurídicas em diferentes âmbitos. Cássia Luciana de Melo Lima ORCID: http://orcid.org/0000-0002-7263- 2010 Instituto Federal de Educação Ciência e Tecnologia de Rondônia E-mail: cassia.lima@ifro.edu.br Como citar: LIMA, C. L. de; ORTIZ, i. r. g. Reflexões metodológicas para o ensino de Direito aplicado aos Cursos Técnicos do Instituto Federal de Rondônia, Campus Porto Velho Zona Norte. Revista Brasileira da Educação Profissional e Tecnológica, [S.l.], v. 2, n. 21, p. e12037, nov. 2021. ISSN 2447-1801. Palavras-chave: Metodologia de Ensino; Formação de professores; Conhecimento Jurídico; Sala de Aula Invertida. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021 CC BY 4.0 \| ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021 CC BY 4.0 \| ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Reflexões metodológicas para o ensino de Direito aplicado aos Cursos Técnicos do Instituto Federal de Rondônia, Campus Porto Velho Zona Norte Methodological reflections for the teaching of law applied to the Technical Courses of the Federal Institute of Rondônia, Campus Porto Velho Zona Norte 1 INTRODUÇÃO A intenção deste texto é apresentar uma proposta que responda a necessidade de se fazer um ensino exequível no âmbito jurídico, oferecendo ao aluno dos cursos técnicos do Instituto Federal de Rondônia, Campus Porto Velho Zona Norte, uma oportunidade de desenvolver habilidades de raciocínio e instrumentalização do conhecimento, de forma que ele possa preparar-se para atuar como profissional qualificado em processos formativos e como cidadão. Apresenta-se, inicialmente, uma definição de conhecimento jurídico, destacando sua necessidade e importância nas diversas esferas da vida social. Traz, ainda, alguns enfoques no sentido de conceituar o termo Direito. Em seguida, recorre-se à análise, através de uma abordagem sucinta, de diferentes concepções metodológicas, utilizando como referência o professor Libâneo, bem como apresenta a ideia de aprendizagem ativa, dando destaque para os métodos de ensino participativo. Nesse propósito, são apresentadas algumas ideias de Moran e Ghirard. O presente trabalho também descreve um contexto específico em que as disciplinas jurídicas são ministradas, ou seja, apresenta as informações relevantes sobre o Instituto Federal de Rondônia, sobre os cursos oferecidos pelo Campus Porto Velho Zona Norte e algumas características do público-alvo. Abstract From a specific context – the teaching of law in the classes of the technical courses of the Federal Institute of Rondônia (IFRO), Porto Velho Zona Norte campus – this work brings a reflection on teaching practice and legal education, based on different methodological conceptions. To this end, it is based on bibliographic and documentary research. The effort allows a reflection on the meaning of law and the importance of legal knowledge. It brings, albeit briefly, some methodological conceptions, taking the opportunity to make notes about participatory teaching methods from a perspective of methodologies and formative processes of teachers. Finally, it presents a methodological proposal for legal education based on the view of the professional who intends to graduate. This work is licensed under a Creative Commons Attribution 4.0 Unported License. This work is licensed under a Creative Commons Attribution 4.0 Unported License. Keywords: Teaching Methodology; Teacher training; Legal Knowledge; Inverted Classroom. Keywords: Teaching Methodology; Teacher training; Legal Knowledge; Inverted Classroom. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 2 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 2 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 2.1 O QUE É DIREITO? Começaremos por trazer uma noção elementar do Direito, utilizando a teoria de Miguel Reale, de forte base para o conhecimento jurídico brasileiro. Para o autor, o Direito corresponde à exigência essencial e indeclinável de uma convivência ordenada, pois nenhuma sociedade poderia existir sem um mínimo de ordem, de direção e solidariedade (REALE, 2001). Citando Santi Romano, Reale observa o direito concebido como realização de convivência ordenada, e pontua que os fatos e fenômenos sociais só podem ser concebidos e existir na sociedade, e não fora dela. A Teoria Tridimensional do Direito, de Miguel Reale, correlaciona “Direito como fato social e o Direito como ciência, a tal ponto que, ainda hoje, a mesma palavra serve para designar a realidade jurídica e a respectiva ordem de conhecimentos”. Complementa ainda o autor ao destacar a dificuldade em separar “[...] a experiência jurídica das estruturas lógicas, isto é, das estruturas normativas nas quais e mediante as quais ela se processa” (2001, p. 3). Isso significa que o homem vive o Direito, mesmo quando não se preocupa com o seu significado jurídico, porque ele é indissociável da vida social. Mas, afinal, o que é Direito? Mesmo aqueles que passam a vida toda trabalhando com Direito, quando é perguntado sobre o seu conceito, muitas vezes não têm uma resposta para dar, justamente porque vivemos diante muitas coisas que são denominadas Direito, e diante de tantas outras às quais atribuímos o caráter de justo ou injusto. Para tentar explicar o que é Direito utilizaremos as lições de Mascaro (2015), que conceitua o Direito como fenômeno histórico, com especificidade quando a sociedade se estrutura no modo de produção capitalista. Em sociedades anteriores ao capitalismo, as formas de interação entre as pessoas eram de dominação e de exploração diretas. Somente o capitalismo estrutura um tipo de articulação social lastreado num instrumento, que é o contrato, ou seja, todos aqueles que vivem numa sociedade capitalista passam a se vincular por meio do contrato. Importante frisar que a dinâmica de todas as coisas do mundo contemporâneo se dá mediante mercadoria. No passado, o Direito tinha uma ligação muito clara com questões morais e religiosas. O que se falava sobre o Direito estava basicamente no plano ético e moral. Já na sociedade capitalista, o Direito se apresenta como um fenômeno distinto, diferenciando-se dos demais fenômenos sociais em qualidade e quantidade. 2 CONHECIMENTO JURÍDICO Quando temos como pauta a prática docente em relação ao ensino jurídico, a construção de uma representação do Direito e sua função na sociedade deve ocupar um plano central, independente do conteúdo a ser ministrado. Nesse sentido, faz-se necessário, como ponto de partida, refletir sobre o conhecimento jurídico, antes mesmo de abordar metodologias de ensino aplicáveis. O início desta abordagem tomando como base o conhecimento se justifica uma vez que “nós, educadores, utilizamos como matéria prima para o nosso trabalho o conhecimento acumulado pela humanidade” (VERMELHO, 2012, p. 3) O termo conhecer possui inúmeras definições. Cretella Júnior o define como uma representação do objeto na mente do sujeito cognoscente, ou seja, aquele que tem a capacidade de conhecer (2004, p.4). Para que isso ocorra é necessária uma apreensão que se configura num ato complexo. Nesse processo, o sujeito circunda o objeto em busca de sua essência, relacionando-o com outros, comparando e tirando conclusões. O conhecimento jurídico, um dos ramos do conhecimento humano, é o que interessa a esse trabalho. Convém, agora, destacar a importância do conhecimento jurídico, não só para os operadores do Direito, mas para todos que querem se aprimorar como pessoas e como profissionais. Isso porque a função do Direito é tutelar o indivíduo onde existe sociedade, protegendo, amparando, defendendo e servindo ao indivíduo. Além disso, o direito tem a função de mostrar quais são os comportamentos apoiados pela lei, bem como os limites que regem os relacionamentos entre as partes. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 3 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Conhecimento jurídico é fundamental para que os indivíduos possam exercer plenamente sua cidadania. 2.1 O QUE É DIREITO? A possibilidade de alguém vincular-se a outro mediante contrato, quando as pessoas não são mais exploradas pela força bruta ou pela escravidão, dá a qualidade do Direito conforme conhecemos na atualidade. “Entender o Direito a partir do movimento mais simples do capitalismo – as trocas mercantis – é captar o ponto que dá a qualificação específica do Direito moderno” (MASCARO, 2015, p. 12). Assim, percebemos que a apreensão do capital pelas mãos de alguns e, eventualmente, circulando para todos, se faz mediante garantia e vínculos que são dados pelo Direito. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 4 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 4 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Contrato, vinculação com autonomia da vontade, garantia de que o capital está respaldado não por aquele que possui, mas por um terceiro - que é o Estado - vão constituindo a qualidade do Direito. Dentro dessa qualidade existem muitas quantidades de arranjos jurídicos. As pessoas, sendo sujeito de direito, encontram diversas modulações para seus vínculos contratuais. 3 DIFERENTES CONCEPÇÕES METODOLÓGICAS Nessa concepção, os objetivos são voltados para o professor, que está no centro e é detentor do conhecimento. O aluno é passivo e considerado uma tábula rasa. As relações são verticalizadas. trazidas por Libâneo. De acordo com o autor, podemos falar de uma Didática Tradicional, cujo conceito está relacionado à instrução, ou seja, transmissão de cultura, das grandes descobertas da humanidade. Tem por objetivo a preparação intelectual e moral dos alunos. Nessa concepção, os objetivos são voltados para o professor, que está no centro e é detentor do conhecimento. O aluno é passivo e considerado uma tábula rasa. As relações são verticalizadas. Na pedagogia tradicional podemos identificar o conceito de educação bancária definido por Paulo Freire, em que o aluno é mero receptor do processo de aprendizagem. Sendo assim, a ênfase está nos conteúdos, na repetição e na memorização. A aprendizagem é mecânica e a avaliação punitiva e classificatória. Quando falamos em Didática Renovada, nos referimos ao ato de experimentar, aprender fazendo. Isso significa aprender a aprender para perpetuar as condições de vida. Está classificada em renovada progressivista e renovada não-diretiva. A tendência liberal renovada progressivista está ligada ao movimento chamado Escola Nova que se manifestou no Brasil no movimento Manifesto dos Pioneiros, instrumento que se impôs a algumas premissas da escola tradicional. É uma concepção baseada na motivação e na estimulação de problemas, e valoriza mais os processos cognitivos que os conteúdos. A tendência liberal renovada não diretiva é ligada ao humanismo, e tem como principal característica o foco que se dá no desenvolvimento pessoal e nas relações interpessoais, sendo voltada para formação de atitudes e centrada no aluno. Por fim, no grupo das liberais, temos a tendência tecnicista, voltada para a preparação de mão de obra. Nessa tendência, o essencial não é o conteúdo, mas as técnicas. A educação é vista como um recurso tecnológico e modelador do comportamento humano. Entramos agora na análise das concepções progressistas, cujo verbo instrumentalizar traz um significado de apropriação dos conhecimentos para emancipação/transformação. Dentre elas, a concepção libertadora, que tem cunho político, e está voltada para uma educação problematizadora. Tem como principal representante Paulo Freire. Nessa concepção, a Educação assume o papel de questionar a transformação da realidade social, política e econômica. As relações são dialógicas, e o trabalho gira em torno de temas geradores. Por sua vez, a tendência progressista libertária está voltada aos princípios da autogestão. 3 DIFERENTES CONCEPÇÕES METODOLÓGICAS Tendo como intenção investigar uma forma para melhorar a práxis para o ensino de Direito nos cursos técnicos do Instituto Federal de Rondônia, faz-se necessário um levantamento teórico de diferentes correntes metodológicas, diferentes didáticas. Tais correntes possuem diferentes concepções para elaboração de plano de ensino, porém todas têm como elementos o conhecimento, o professor e o aluno. Nessa esteira, importante se faz frisar a seguinte lição de Libâneo: A prática escolar, assim, tem atrás de si condicionantes sociopolíticos que configuram diferentes concepções de homem e de sociedade, e consequentemente, diferentes pressupostos sobre o papel da escola, relações professor-aluno, técnicas pedagógicas, etc. Fica claro que o modo como o professor realiza seu trabalho, selecionam e organizam o conteúdo das matérias, ou escolhem técnicas de ensino e avaliação tem a ver com pressupostos teórico-metodológicos, explícita ou implicitamente (2013, p. 19). Na obra Democratização da Escola Pública, Libâneo (1992) classifica e descreve diferentes tendências pedagógicas com o objetivo de servir ao professor como instrumento de análise de sua prática pedagógica. Classifica, assim, as tendências em liberais e progressistas, utilizando como critério os condicionantes sociopolíticos da escola. A pedagogia liberal divide-se em tradicional, renovada e tecnicista, e a pedagogia progressista divide-se em libertadora, libertária e crítico-social dos conteúdos. Diferenciando a concepção liberal da concepção progressista, é importante destacar que as pedagogias progressistas são de base marxista, estando voltada para as questões de transformação social e da criticidade. Em contrapartida, a pedagogia liberal não entra num contraponto crítico ao modelo capitalista vigente. De acordo com Libâneo (2013, p. 6), A doutrina liberal apareceu como justificativa do sistema capitalista que, ao defender a predominância da liberdade e dos interesses individuais na sociedade, estabeleceu uma forma de organização social baseada na sociedade privada dos meios de produção, também denominada sociedade de classes. Passamos a expor, agora, as peculiaridades de cada uma das concepçõe Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 5 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 5 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 trazidas por Libâneo. De acordo com o autor, podemos falar de uma Didática Tradicional, cujo conceito está relacionado à instrução, ou seja, transmissão de cultura, das grandes descobertas da humanidade. Tem por objetivo a preparação intelectual e moral dos alunos. 3.1 METODOLOGIAS ATIVAS DE APRENDIZAGEM Para falar sobre práticas pedagógicas por meio das metodologias ativas, é preciso, antes, refletir sobre o significado da aprendizagem ativa. Nesse sentido, José Moran (2018) observa que a aprendizagem por meio da transmissão é importante, mas a aprendizagem por questionamento e experimentação é mais relevante para a compreensão mais ampla e profunda. Para o autor, a aprendizagem ativa faz parte da vida do homem, desde o nascimento, porque aprendemos em situações concretas, enfrentando desafios cada vez mais complexos. Além disso, aprendemos aquilo que é relevante e faz sentido. Vários autores, entre eles Dewey, Freire, Piaget e Vygotsky, procuram demonstrar em suas teorias como as pessoas aprendem de forma ativa aquilo que lhes é significativo e relevante, e que a aprendizagem deve ser compatível com o nível de competência do aprendiz. As metodologias ativas, segundo Moran (2018, p. 4), dão ênfase ao papel protagonista do aluno, ao seu envolvimento direto, participativo e reflexivo em todas as etapas do processo [...]”. No mesmo sentido de proporcionar uma aprendizagem ativa aos estudantes de Direito da Escola de Direito Fundação Getúlio Vargas, Ghirard e outros educadores investigam e têm como práxis o método de ensino participativo. Referido método é conceituado como aquele nos quais o aluno é o protagonista da construção do seu próprio conhecimento, entendendo que o discente deve deixar de ser mero expectador, e ser colocado como agente de sua aprendizagem. Considera- se que o protagonismo do aluno nas aulas de Direito é bastante raro. Há uma série de metodologias de ensino pautadas na proposta do ensino participativo ou em aprendizagem ativa. O importante é que, ao utilizá-los, nós tenhamos bem claro o que queremos com ele, o que queremos dos nossos alunos. Se bem empregados, eles contribuem enormemente para o ensino de maior qualidade, maior autonomia dos alunos e maior densidade da reflexão jurídica. Uma dessas metodologias, a Sala de Aula Invertida, é uma modalidade de educação em que “o que tradicionalmente é feito em sala de aula agora é feito como trabalho de casa, e o que tradicionalmente é feito como trabalho de casa, agora é realizado em sala de aula” (BERGMNN; SAMS, 2018, p. 11). Ao contrário das aulas em que o professor apresenta os conceitos e propõe atividades de fixação, na Sala de aula Invertida vigora a autoaprendizagem, e a interação do professor com os alunos é para tirar dúvidas e auxiliar na realização das atividades de compreensão. 3 DIFERENTES CONCEPÇÕES METODOLÓGICAS Nessa concepção pedagógica, professores e alunos estão no mesmo patamar decisório, e a aprendizagem ocorre por intermédio de grupos, de maneira informal. Por fim, no grupo das progressistas, temos a tendência crítico-social dos conteúdos, cuja característica marcante é a contextualização curricular, em que os conteúdos são apresentados de forma indissociável da realidade, ou seja, não desvaloriza os conteúdos historicamente acumulados, mas parte-se deles para possibilitar ao estudante uma análise crítica desse conteúdo. O professor é visto como mediador do processo, e está voltado a uma abordagem sociointeracionista. A investigação das diferentes concepções aqui pretendidas tem por objetivo melhorar a prática educativa. Assim considera Zabala (1998, p 13), acrescentando que esse objetivo pode ser alcançado mediante “[...] o conhecimento e a experiência: Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 6 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 6 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 o conhecimento das variáveis que intervém na prática e a experiência para dominá- las”. O autor ainda pontua que o conhecimento provém da investigação, das experiências dos outros e de modelos, exemplos e propostas. o conhecimento das variáveis que intervém na prática e a experiência para dominá- las”. O autor ainda pontua que o conhecimento provém da investigação, das experiências dos outros e de modelos, exemplos e propostas. 1 Disponível em www.ifro.edu.br Acesso em 15 dez de 2020. 3.1 METODOLOGIAS ATIVAS DE APRENDIZAGEM O modelo Sala de Aula Invertida pressupõe a gravação de vídeos expondo conteúdo e a disponibilização deles para que os alunos possam assistir em casa. Essa proposta ajuda a dinamizar o processo de aprendizagem, e os alunos seguem seu próprio ritmo. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 7 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 7 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 4.1 CONTEXTO – INSTITUTO FEDERAL DE RONDÔNIA, CAMPUS PORTO VELHO ZONA NORTE 8 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 8 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 MISSÃO. O Instituto Federal de Educação, Ciência e Tecnologia de Rondônia, tem como Missão, promover educação científica e tecnológica de excelência no Estado de Rondônia voltada à formação de cidadãos comprometidos com o desenvolvimento e a sustentabilidade da sociedade. VISÃO. Tornar-se padrão de excelência no ensino, pesquisa e extensão na área de ciência e tecnologia. VALORES. Nas suas atividades, o IFRO valorizará o compromisso ético com responsabilidade social, o respeito à diversidade, à transparência, à excelência e à determinação em suas ações, em consonância com os preceitos básicos de cidadania e humanismo, com liberdade de expressão e atos consonantes com os preceitos da ética pessoal e profissional, com os sentimentos de solidariedade, com a cultura da inovação e com os ideais de sustentabilidade social e ambiental. (PORTAL IFRO. Disponível em www.ifro.edu.br Acesso em 20 dez 2020). Com base no supracitado, os cursos do campus Porto Velho Zona Norte são pensados e elaborados de forma a atender à comunidade local. Observa-se uma ênfase em cursos de empreendedorismo, e cursos voltados para gestão pública, pois o município de Porto Velho é o centro administrativo do estado, e tem economia baseada nas empresas e serviços. Quase todos os cursos possuem disciplinas jurídicas no currículo, das quais cabe mencionar Direito Administrativo, Direito Constitucional, Direito Tributário, Direito Previdenciário e Direito Comercial. Ressalta-se, ainda, Introdução ao Estudo do Direito, que passou a fazer parte do currículo do curso de graduação Tecnólogo em Gestão Pública. 4.1 CONTEXTO – INSTITUTO FEDERAL DE RONDÔNIA, CAMPUS PORTO VELHO ZONA NORTE O Instituto Federal de Educação, Ciência e Tecnologia de Rondônia – IFRO, parte da rede centenária de educação federal, é uma autarquia vinculada ao Ministério da Educação – MEC. Foi criado através da Lei 11.892, de 2008, que reorganizou a rede federal de educação profissional, científica e tecnológica, transformando as escolas técnicas, agrotécnicas e CEFET’s em Institutos Federais de Educação, Ciência e Tecnologia. O IFRO está em Rondônia desde 2010 e busca aproximar a instituição da comunidade, ofertando cursos que atendam à demanda de cada região do Estado. Ao todo são oito campis localizados estrategicamente em 7 cidades rondonienses. Além dos campis, o IFRO conta com polos de educação a distância em cidades e distritos do norte ao sul de Rondônia e em cidades do Acre, que recebem as aulas via satélite. Atua oferecendo cursos técnicos e tecnológicos, além de licenciaturas, bacharelados, cursos de especialização lato sensu e Mestrado Profissional em Rede Nacional. Entre os campis, damos destaque para o localizado na Zona Norte do município de Porto Velho, capital do estado. Referido campus oferece curso técnico de nível médio nas modalidades concomitante e subsequente. Na modalidade concomitante, a formação profissional é feita no IFRO, no formato semipresencial, em que o aluno participa uma vez por semana de aulas presenciais, e realiza as demais atividades no ambiente virtual, e o Ensino Médio é cursado em outra escola. Essa modalidade é oferecida no turno diurno. Oferecida no período noturno, a modalidade subsequente é destinada aos alunos que já terminaram o Ensino Médio e querem se inserir no mercado de trabalho. O Campus oferece, ainda, cursos superiores de tecnologia, destinados aos alunos que já terminaram o Ensino Médio, e um curso de pós-graduação. Os graduados, chamados de tecnólogos, são “profissionais aptos a utilizar, desenvolver ou adaptar, de forma plena e inovadora, tecnologias para a produção de bens ou serviço em uma área profissional”1 Os cursos técnicos oferecidos pelo Campus Zona Norte são em Finanças, Informática para internet, Administração, Computação gráfica, Cooperativismo e Recursos humanos. Os cursos tecnólogos são em Gestão Pública, Gestão Comercial e Rede de Computadores. O campus oferece ainda uma pós-graduação em Planejamento Estratégico em Gestão Pública. No processo de construção da proposta metodológica, é importante ter em mente a missão, a visão e os valores do IFRO, destacados na citação a seguir. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 4.2 PROPOSTA METODOLÓGICA Normalmente, as aulas de Direito são centradas em exposição do conteúdo pelo professor, de forma monologada, sem intervenção dos alunos, a não ser quando paira dúvida sobre a intelecção daquilo que está sendo ministrado. Vimos que essa prática educativa se molda de acordo com as características das tendências tradicionais de ensino. Considera-se, neste contexto, que tal prática educativa se traduz em verdadeiro obstáculo para promover a educação profissional que o Instituto Federal de Rondônia pretende, qual seja, “formação científica e tecnológica de excelência, [...] com foco na formação de cidadãos comprometidos com o desenvolvimento humano, econômico, cultural, social e ambiental sustentável (BRASIL. IFRO, 2018). Também não caberia, nesta proposta, tendências metodológicas renovadas, pois os conteúdos normativos do Direito não permitem a configuração que elas apresentam. Dessa forma, propõe-se a aplicação da metodologia progressista ao ensino jurídico, de modo a promover uma reflexão crítica sobre o papel do aluno e do professor no processo ensino-aprendizagem. Em especial, propõe-se a adoção, para aprimorar a prática docente no contexto em questão, de metodologias de ensino participativo com suas respectivas técnicas, o que significará uma verdadeira reestruturação do ensino jurídico. Em que pese o desafio, frisa-se que o objetivo é possível de ser alcançado. Os docentes podem encontrar dificuldades na preparação e desenvolvimento d Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 9 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 9 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 aulas participativas, pois precisam estar preparados, entre outros fatores, com a possível oposição por parte dos estudantes, uma vez que estão acostumados com a configuração das tradicionais de Direito. No entanto, acreditamos que os alunos vão percebendo que podem aprender ao se inserirem nesta nova metodologia, como seres pensantes e independentes. Importa destacar que métodos participativos de ensino possibilitam a construção da aprendizagem significativa proposta por Ausbel (apud MOREIRA, 2006), pois valorizam os conhecimentos prévios dos estudantes, e exigem a organização de organizadores prévios quando o assunto a ser aprendido é totalmente novo. Espera- se que os conhecimentos construídos ativamente possam ser significativos na vida do estudante, possibilitando a flexibilidade de uso na prática da vida social e profissional. Além disso, com a adoção das metodologias ativas, centradas nas tendências progressistas, o aluno pode demonstrar aprendizado, crescimento e, sobretudo, capacidade de caminhar sozinho, independente do professor. 4.2 PROPOSTA METODOLÓGICA 10 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 10 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 5 CONSIDERAÇÕES FINAIS O presente trabalho analisou diferentes concepções metodológicas para, ao final, propor qual a mais adequada na construção de um programa de ensino jurídico no contexto dos cursos técnicos do Instituto Federal de Rondônia em Porto Velho, campus Zona Norte. Procurou-se, num primeiro momento, refletir sobre a visão do Direito de acordo com teóricos da área, bem como trazer um conceito do que seria o próprio direito, destacando a importância do conhecimento jurídico em todos os âmbitos sociais. Concebido de acordo com a teoria Tridimensional do Direito, que considera outros fatores além das normas jurídicas, como os políticos, ideológicos, econômicos e psicológicos, o certo é que o Direito deve ser conceituado a partir da ótica do capitalismo, pois somente ele estrutura um tipo de articulação social lastreado no contrato. Sobre a importância do ensino jurídico, frisa-se que a função do Direito é servir ao indivíduo, protegendo, amparando e defendendo, ao mesmo tempo que mostra os comportamentos apoiados pela lei e os limites que regem os relacionamentos entre as partes. Destacou-se a importância de utilizar métodos ativos/participativos de ensino no contexto em questão, pois pretende-se que o aluno deixe de ser mero expectador para atuar como protagonista na construção do seu próprio conhecimento. Tem-se observado que o protagonismo do aluno nas aulas de Direito ainda é bastante raro. As aulas são, geralmente, monologadas, seguindo a tendência metodológica tradicional. Essa situação, portanto, carece de mudança. Desse modo, ao propor uma metodologia que tenha como objeto do processo ensino aprendizagem a participação ativa do aluno, espera-se tornar a aprendizagem desse componente curricular mais envolvente onde os alunos sintam-se construtores e partícipes de seu processo de aprendizagem. 4.2 PROPOSTA METODOLÓGICA Ressalta-se que o uso das metodologias participativas impõe mudanças tanto na forma de ministração das aulas, como no ambiente físico, que se deve adequar à técnica pertinente. Uma das mudanças implica, por exemplo, na alteração das disposições das carteiras, que não mais deverão ser colocadas em fila, pois os semicírculos e os grandes círculos servirão ao propósito. O professor passa a expor ou inquirir algo, dialogando com os alunos, e dessa forma será visto com mais proximidade, como a um amigo. Para a preparação das aulas será necessário selecionar textos ou livros de fácil compreensão. Espera-se, que ao final do período, os alunos estejam acostumados aos termos e raciocínio jurídico porque estiveram lendo, discutindo e refletindo temas vivenciados no dia a dia que foram extraídos desse material. Espera-se contar com a contribuição do aluno em todo tempo, trazendo para a discussão informações e experiências pessoais, de forma a enriquecer o processo de ensino-aprendizagem. Ao dedicar-se efetivamente, o aluno poderá conquistar, ao final, um patrimônio considerável de leitura, pois os métodos de ensino participativo exigem um trabalho agradável e prazeroso de leitura e discussão em sala. No entanto, o mais considerável nesta proposta é a possibilidade de tornar os alunos capazes de entender o Direito de maneira crítica e, acima de tudo, as preocupações em estudar para as provas tendem a desaparecer, uma vez que a cada aula estarão aprendendo, gradativamente. Dentre as metodologias participativas, sugere-se para o contexto dois métodos: a sala de aula invertida que, como já mencionado, o aluno realiza estudos autônomos em casa e durante as aulas realiza atividades práticas, o que auxiliará na otimização do tempo, e a Aprendizagem Baseada em Problemas, ou simplesmente conhecida como ABP (ou até mesmo PBL, sigla oriunda do inglês problem based learning), que é uma metodologia voltada para aquisição de conhecimento por meio de resolução de situação problemas. Esse método tem como base um problema em que é ofertado aos alunos situações reais para possíveis soluções. Pode ser, por exemplo, um conflito jurídico ou religioso. Os alunos devem avaliar qual a melhor solução real para esse conflito, seja ela jurídica ou não jurídica. A habilidade é avaliar o caminho jurídico em relação a outros caminhos possíveis, inclusive pesando a consequência econômica e social dessa decisão. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. BRASIL. IFRO. Ministério da Educação. O Instituto: Missão, Visão, Valores. Atualizado em dez. de 2018. Disponível em: https://portal.ifro.edu.br/apresentacao. Acesso em: 02 out. 2020. REFERÊNCIAS BACICH, Lilian; MORAN, José. Metodologias Ativas para uma Educação Inovadora. Porto Alegra: Penso, 2018. BERGMANN; Jonathan; SAMS, Aaron. Sala de Aula Invertida: Uma metodologia ativa de aprendizagem. Rio de Janeiro: LTC, 2018. BACICH, Lilian; MORAN, José. Metodologias Ativas para uma Educação Inovadora. Porto Alegra: Penso, 2018. BERGMANN; Jonathan; SAMS, Aaron. Sala de Aula Invertida: Uma metodologia ativa de aprendizagem. Rio de Janeiro: LTC, 2018. BRASIL. IFRO. Ministério da Educação. O Instituto: Missão, Visão, Valores. Atualizado em dez. de 2018. Disponível em: https://portal.ifro.edu.br/apresentacao. Acesso em: 02 out. 2020. BACICH, Lilian; MORAN, José. Metodologias Ativas para uma Educação Inovadora. Porto Alegra: Penso, 2018. BERGMANN; Jonathan; SAMS, Aaron. Sala de Aula Invertida: Uma metodologia ativa de aprendizagem. Rio de Janeiro: LTC, 2018. BRASIL. IFRO. Ministério da Educação. O Instituto: Missão, Visão, Valores. Atualizado em dez. de 2018. Disponível em: https://portal.ifro.edu.br/apresentacao. Acesso em: 02 out. 2020. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 11 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 CARVALHO NETO, Silvio; TOLEDO, Noemia Lopes. Sistema de Informação Online para a Disseminação do Ensino e Pesquisa: Ambientes Virtuais de Aprendizagem e MOOCs. São Paulo: Uni-FACEF, 2014. CRETELLA JUNÍOR, José. Introdução à Filosofia do Direito. 10ª ed. - Rio de Janeiro: Forense, 2004. CRETELLA JUNÍOR, José. Introdução à Filosofia do Direito. 10ª ed. - Rio de Janeiro: Forense, 2004. GHIRARD, José Garcez; FEFERBAUN, Marina (Organizadores). Ensino do Direito em Debate: Reflexões a partir do 1 Seminário Ensino Jurídico e Formação Docente. São Paulo: Direito GV, 2013. GHIRARD, José Garcez. O instante do encontro: questões fundamentais para o ensino jurídico. São Paulo: FGV, 2012. KHALIL, Ranan; EBNER, Martin. “How Satisfied Are You With Your MOOC?” A Research Study About Interaction in Huge Online Courses. Journalism and Mass Communication, Vol. 5, nº. 12, p. 629-639. December/2015. INSTITUTO FEDERAL DE RONDÔNIA. Missão, Visão, Valores. Porto Velho, 2017. Disponível em: http://portal.ifro.edu.br/visao-missao-valores INSTITUTO FEDERAL DE RONDÔNIA. Apresentação. Porto Velho, 2017. Disponível em: http://portal.ifro.edu.br/apresentacao LIBÂNEO, José Carlos. Democratização da Escola Pública: A Pedagogia Crítico Social dos Conteúdos. São Paulo: Loyola, 1992. LIBÂNEO, José Carlos. Didática. 2ª ed. São Paulo: Cortez, 2013. MASCARO, Alysson Leandro. Introdução ao estudo do direito. 6ª ed. São Paulo: Atlas, 2018. MASCARO, Alysson Leandro. Introdução ao estudo do direito. 6ª ed. São Paulo: Atlas, 2018. MORÁN, J. Mudando a educação com metodologias ativas. IN: Carlos Alberto de Souza e Ofelia Elisa Torres Morales (orgs.). Coleção Mídias Contemporâneas. Convergências Midiáticas, Educação e Cidadania: aproximações jovens. Vol. II PG: Foca Foto-PROEX/UEPG, 2015. MOREIRA, Marco Antônio. A aprendizagem significativa e sua implementação em sala de aula. Brasília: Editora Universidade de Brasília, 2006. NOGUEIRA, Cláudio Marques Martins; NOGUEIRA, Maria Alice. A Sociologia da Educação de Pierre Bourdieu: Limites e Contribuições. Educação e Sociedade, ano XXIII, nº 78, abril/2002. OLIVEIRA, Eliane Maria Pavan de OLIVEIRA. Docência em Direito e a “Sala de Aula Invertida” como opção metodológica ativa. Evidência, Araxá, v. 12, p. 59- 77, 2016. OLIVEIRA, Eliane Maria Pavan de OLIVEIRA. Docência em Direito e a “Sala de Aula Invertida” como opção metodológica ativa. Evidência, Araxá, v. 12, p. 59- 77, 2016. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 21, e12037, 2021, p. 12 de 12 ISSN 2447-1801 \| DOI: https://doi.org/10.15628/rbept.2021.12037 RANGEL, Tauã Lima Verdan. Ensino Jurídico e Protagonismo Discente: um repensar das aulas ministradas no curso de Direito. Revista Philologus, ano 22, nº 66. Rio de Janeiro, set/dez 2016. RANGEL, Tauã Lima Verdan. Ensino Jurídico e Protagonismo Discente: um repensar das aulas ministradas no curso de Direito. Revista Philologus, ano 22, nº 66. Rio de Janeiro, set/dez 2016. REALE, Miguel. Filosofia do Direito. 19ª ed. São Paulo: Saraiva, 2000 (versão digital). VERMELHO, Sonia Cristina Soares Dias. Práxis no Ensino Superior. Paraná: Editora FAEL, 2012. VERMELHO, Sonia Cristina Soares Dias. Práxis no Ensino Superior. Paraná: Editora FAEL, 2012. ZABALA, Antoni. A Prática Educativa: Como ensinar. Porto Alegre: ArtMed, 1998. ZABALA, Antoni. A Prática Educativa: Como ensinar. Porto Alegre: ArtMed, 1998.
https://openalex.org/W2902207365	https://www.matec-conferences.org/articles/matecconf/pdf/2018/98/matecconf_ts2018_01027.pdf	English	null	Study of the structurally sensitive physical characteristics of materials used to fabricate highly loaded elements of rolling stock	MATEC web of conferences	2,018	cc-by	4,306	, 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 Study of the structurally sensitive physical characteristics of materials used to fabricate highly loaded elements of rolling stock Yury Matyash1,, Yury Sosnovsky1, Evgeniy Kondrikov1 and Dmitriy Muravyov1 1Omsk State Transport University, Karl Marx Ave., 35, Omsk, 644046, Russia Abstract. The paper presents the results of laboratory tests of steel samples. The values of the coercive force and the results of spectral analysis of the samples under study at room temperatures are given. The dependences of the thermoelectric power, the resistivity (specific conductivity), and the Young's modulus on the number of loading cycles at a frequency of 50 Hz are studied. The loading amplitude did not exceed 1% of the elastic limit. 1 Introduction According to the strategic direction of innovative development of the transportation process of JSC “Russian Railways”, the following are the priority directions of development: introduction of heavy cargo trains (up to 18 thousand tons, 780 axles); an increase in the specific load per axle (27 tons/axle and more), and an increase in the speed (up to 120 km/h). At the same time, issues related to the safety and accident-free operation of the transportation process become especially important and relevant. For the timely detection of the occurrence of various types of malfunctions of freight cars, complexes (MCTM-02, HSC, CTM, ACCS, and АSDND) are located on the approaches to maintenance points, which provide diagnostics of the technical condition of the freight rolling stock on the moving train [1]. MCTM-02 is a multifunctional complex of technical means for diagnostics of running parts of railway rolling stock. For the processing of incoming signals from KTSM-02, a hardware and software complex (HSC) is used on the basis of a laptop with special software. CTM - complex of technical measurements. ACCS - automatic coupler control system. АSDND - automatic system for detecting cars with negative dynamics. The table shows the main units of cars, the defects of which are identified by these diagnostic complexes. Despite the fact that the introduction of these complexes is extremely necessary and relevant, they all allow determining only the critical values of the diagnosed parameters and do not allow detecting a number of defects (for example, loss of the elastic properties of the shock absorbing device of the coupler), which significantly affect the safety transportation process. © 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/). Corresponding author: sosnovskyym@mail.ru * Corresponding author: sosnovskyym@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 2018 TransSiberia Table 1. Typical types of defects detected by existing diagnostic complexes. Units of freight cars Areas of application of diagnostic complexes MCTM-02, HSC DDW, CSVDL (NIIAS), WILD CTM ACCS ASDND Axlebox units + Wheel pairs + + Automatic coupler + Car body and frame + Brake equipment Details of car trucks + Table 1. Typical types of defects detected by existing diagnostic complexes. In depot conditions, the most common practical methods for identifying defects are visual inspection, ultrasonic and magnetic particle methods of nondestructive examination. At the same time, the assessment of the mechanical properties of metal at repair facilities of JSC Russian Railways is not carried out at all. This is due to the lack of equipment that makes it possible to detect a change in the structure of metal, the mechanical properties and the ability to predict the behavior of the part during operation. The magnetic particle inspection method is used to detect defects in the surface and subsurface layers of the material. Using this method, it is possible to detect defects of about 0.5 mm in length, 2.5 mm in width and more [2]. The ultrasonic method is based on recording the parameters of elastic waves that arise or are excited in the object of control. This kind of control allows determining defects in the form of cracks, pores, discontinuities, measuring the geometric parameters of the inspection object, slag and graphite inclusions, coarse granulation, as well as determining the depth of defects, coordinates, the orientation of the crack relative to the surface of the introduction of elastic vibrations, and the equivalent size of the defect [3]. Among the domestic works, special attention is paid to the paper [4] in which the authors established about twenty correlation dependences of the physical characteristics of inorganic crystals with their structural weakness: melting temperature, relative and absolute hardness, surface energy, Young's modulus, etc. At the same time, under weakness is meant a certain parameter that indirectly characterizes the energy of the interatomic interaction of inorganic crystals. It should be noted that the issue of the quick registration of the above-mentioned characteristics is still open. * Corresponding author: sosnovskyym@mail.ru In Finland, the control system of vertical forces of interaction of wheels with rails WILD [5] became widely used. This system is designed to check the condition of the wheels and to prevent defects that can affect the safety of traffic: sliders, chips, asymmetrical wheels, etc., which can cause rail and/or car damages. The module of the WILD system consists of a track panel with a length that includes two rails, sixteen sleepers, twenty strain gauges for sleepers, and twenty-two strain gauges for rails. At the end of 2015, Finland has installed and commissioned thirteen such systems. In the USA and other countries, the technology of machine or computer vision is widely used [6, 7], based on the monitoring of car structures and their defects. Such a system makes it possible to control the position of the friction wedges as a criterion for the detach of car due to the uneven load on the side frame of the car truck, the presence or absence of brake pads, the absence of the axlebox elements (bolts, covers), the closing of the spring coils, the difference in the height of the axle beams of the coupler and much more. The development of this technology in combination with additional preventive maintenance systems has a good prospect for ensuring the safety of the railway traffic. On the railways of the Netherlands, the Quo Vadis system for the weighing of rolling stock in motion was introduced and widely used [8]. At the same time, an analysis of the existing methods for diagnosing the residual resource showed that none of them makes it possible to estimate the dynamics of states associated 2 2 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 with a change in the structure of the metal, which leads to the appearance of defects registered by existing methods. with a change in the structure of the metal, which leads to the appearance of defects registered by existing methods. Expansion of information about structural changes in steel products during their operation can significantly increase the accuracy and reliability of existing methods and lead to the emergence of new, most effective ways of diagnosing the residual resource. For this, it is necessary to know the relationship between the available registrable steel parameters (for example, the physical characteristics associated with the metal structure) and its mechanical properties. * Corresponding author: sosnovskyym@mail.ru Analysis of the scientific literature has shown that such dependences are nearly absent or extremely rare and contradictory. The existing methods do not allow making a reliable assessment of the nature of the development of changes in the structure of the metal, which, in turn, lead to the appearance of defects registered by methods of nondestructive examination. One of the reasons for this situation is the lack of reliable methods and appropriate equipment that allows determining and predicting the change in the structure and mechanical properties of metal units and parts during operation [9,10]. That is why the problem of determining the residual resource remains very relevant. 2 Materials and methods The problems associated with the accumulation of fatigue damages in the process of elastic deformations of metals are quite wide and varied. It is established that the process of fatigue failure of metals consists of two periods: the initiation and propagation of fatigue cracks [11]. Taking into account the fact that the crack propagation in the metal has an explosive character [12], the authors suggested that the process of fatigue crack initiation (predefective state) is associated with structural changes in the studied metal objects and, accordingly, with the change of various structurally sensitive physical characteristics [13]. On the other hand, various semi-empirical relationships [4] connecting the energy parameters of metal products with their structural weakness are known. These dependences are characterized by a rather high degree of reliability of approximation and are not inferior to the corresponding data obtained in the framework of other energy approaches. Qualitative and quantitative confirmation of the relationship between the physical and chemical properties of metals and their performance characteristics are other results found in the scientific literature. For example, the authors of [14] established a connection between the eddy-current magneto- optical images of the surface of the main gas pipelines and the structure of the material after many years of operation. The influence of performance characteristics on the hardness of automatic couplers (embrittlement) of freight cars of railway transport has been established [15]. The work [16] shows the effect of temperature on the internal surface of distillation columns after long-term operation. To determine the relationship with structural changes in the metal objects under study and taking into account the materials presented above, methods and tools for experimental studies were determined. To achieve this goal, a unique laboratory facility was designed and created at OmSRTU with which it is possible to establish a connection between the physical and technical characteristics of steel ferromagnetic samples [17]. As the objects under study, steel samples in the form of a rectangular parallelepiped made of steel used for making automatic couplings, rails, etc. were fabricated at the enterprise VRK-1 Omsk (Fig. 1). 3 , 0 (2018) MATEC Web of Conferences 239 1027 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 2018 TransSiberia Fig. 1. Form and designation of the sample. c d b a Fig. 1. Form and designation of the sample. 2 Materials and methods The test sample is rigidly fixed at one end to the fixing device (part d), and the loose end is placed in the region of the variable magnetic field, under which the free end of the sample begins to vibrate at a frequency equal to the frequency of the variable magnetic field. The nature of the load can be variable: pulsed, sinusoidal, meander, asymmetric. Through certain intervals of oscillation, a weight of a different mass is suspended from the free end of the sample. Under the influence of the load weight F, the free end bends by the value Δ (Fig. 2). The deflection is measured by a micrometer. F L ∆ Fi 2 P f d i i h Y ' d l F L ∆ b F a L Fig. 2. Parameters for determining the Young's modulus. Fig. 2. Parameters for determining the Young's modulus. By the size of the deflection and the parameters of the sample, the Young's modulus can be calculated from the formula: 3 3 4FL E ba   (1) (1) The specific electrical resistivity (ρ) or reciprocal to it specific electrical conductivity γ determines the current density (j) in the conductor when an external electric field with intensity (E) is applied to it (Ohm's law). To determine the specific electrical resistivity of the steel samples ρ or the specific conductivity γ, a special hinged module was developed, which provided electrical contact with the sample under study. The circuit for measuring the electrical conductivity is shown in Fig. 3. Fig. 3. Scheme for measuring the electrical conductivity of samples. Fig. 3. Scheme for measuring the electrical conductivity of samples. , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 As a voltage source, the power supply unit VS-24M was used. To calculate the specific electrical conductivity and resistivity, the following expressions are used: (2) (2) Determination of the thermoelectric power coefficient α is based on the Seebeck effect - the appearance of the electromotive force ε in a conductor in which there is a temperature gradient ΔT: ε = α ΔТ. On the other hand, the same temperature gradient determines the electromotive force εT, which appears in the thermocouple copper-constantan: εТ = αТ ΔТ, where αТ = 42.5 μV/K thermoelectric power coefficient of a copper-constantan thermocouple. 2 Materials and methods p Taking into account the foregoing, the calculation formula for determining the thermoelectric power coefficient of the test samples is presented below: Т Т       (3) (3) To measure the thermoelectric power coefficient of the test samples, a heater and a cooler were used. The scheme for measuring the thermoelectric power coefficient is shown in Fig. 4. Fig. 4. Scheme for measuring the thermoelectric power coefficient. Fig. 4. Scheme for measuring the thermoelectric power coefficient. A microvoltmeter was used to measure the thermoelectric power. Between one of the thermocouple junctions and the sample, a dielectric beryllium plate with a high thermal conductivity was installed, which made it possible to avoid short-circuiting the thermocouple. A microvoltmeter was used to measure the thermoelectric power. Between one of the thermocouple junctions and the sample, a dielectric beryllium plate with a high thermal conductivity was installed, which made it possible to avoid short-circuiting the thermocouple. The list of measuring instruments and certified testing equipment is given in Table 2. y p g p The list of measuring instruments and certified testing equipment is given in Table 2. The list of measuring instruments and certified testing equipment is given Table 2. List of measuring instruments and testing equipment. Table 2. List of measuring instruments and testing equipment. No n/n Name Type Limit of measurement Scale interval Accuracy class 1 Microvolt Nano-Ammeter F 136 5 mV 0.1 mV 1,5 2 Microvolt Nano-Ammeter F 136 500 μV 10 μV 1,5 3 Microvolt Nano-Ammeter F 136 250 μV 10 μV 1,5 4 Microvolt Nano-Ammeter F 136 100 μV 2 μV 1,5 5 Microvolt Nano-Ammeter F 136 50 μV 1 μV 1,5 6 Microvolt Nano-Ammeter F 136 10 μV 0.2 μV 1,5 7 Ammeter M 109 10 A 0.1 A 0,5 8 Ammeter M 109 1 A 0.01 A 0,5 9 Micrometer - 5 cm 0.01 mm - 10 Dial gauge - 1 cm 0.01 mm - 5 5 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 3 Results Using the emission spectrometer ARGON-5SF, a spectral analysis of some samples was preliminary carried out in the OmSRTU research laboratory, the results of which are given in Table 3. Table 3. Spectral analysis of some samples. Table 3. Spectral analysis of some samples. SAMPLE Chemical element and its percentage B C Al Si P S Ca Ti V Cr Mn Fe Co Ni Cu Zn As Zr Nb Mo Sn Sb W Pb Steel 45 0.001 0.271 0.001 0.258 0.009 0.014 0.000 0.005 0.012 0.076 0.573 98.483 0.023 0.100 0.161 0.002 0.005 0.005 0.004 0.000 0.003 0.000 0.009 0.000 Steel 45 (annealed) 0.003 0.140 0.033 0.392 0.024 0.084 0.003 0.000 0.049 0.234 0.778 91.300 0.024 0.537 6.302 0.016 0.016 0.004 0.010 0.039 0.006 0.000 0.028 0.000 Rail R1 0.001 0.761 0.000 0.402 0.008 0.026 0.003 0.002 0.037 0.056 0.954 97.546 0.012 0.049 0.120 0.002 0.004 0.004 0.010 0.007 0.002 0.000 0.011 0.000 Automatic coupler AC1 0.001 0.215 0.043 0.340 0.019 0.018 0.000 0.000 0.013 0.148 1.262 97.555 0.009 0.121 0.222 0.004 0.009 0.004 0.008 0.003 0.009 0.000 0.007 0.000 Using the K-61 structuroscope, the coercive force of all the samples used in the experiment was determined. To do this, a coercimeter was applied along the sample in two opposite directions, and then, the average result was determined according to these indications. The results of measurements are shown in Table 4. Fig. 5 shows the dependence of the thermoelectric power of different samples on the number of loading cycles. Attention should be paid to the fact that the thermoelectric power values of the samples made from the automatic coupler of the freight car (AC1 and AC2), whose chemical and physical properties are listed in Tables 2 and 3, respectively, are significantly lower than those made of rail and steel 45. Fig. 5 shows the dependence of the thermoelectric power of different samples on the number of loading cycles. Attention should be paid to the fact that the thermoelectric power values of the samples made from the automatic coupler of the freight car (AC1 and AC2), whose chemical and physical properties are listed in Tables 2 and 3, respectively, are significantly lower than those made of rail and steel 45. Fig. 5 shows the dependence of the thermoelectric power of different samples on the number of loading cycles. Fig. 7 shows the dependences of the Young's modulus of various samples (automatic coupler - AC-2, steel 45-5 (st. 45), rail-R2 (rail), annealed sample - AN1 (st45)) on the number of loading cycles. It can be seen that the number of load cycles does not influence the values of the Young's modulus due to the small loading amplitude. 3 Results Attention should be paid to the fact that the thermoelectric power values of the samples made from the automatic coupler of the freight car (AC1 and AC2), whose chemical and physical properties are listed in Tables 2 and 3, respectively, are significantly lower than those made of rail and steel 45. Fig. 6 shows the dependences of the specific resistivity of samples made of steel 45 on the number of loading cycles. Samples AN (st 45) and AN4 (st 45)2 were annealed of the first kind without phase recrystallization. It can be seen from Fig. 6 that the specific resistivity of the annealed samples has increased significantly Fig. 6 shows the dependences of the specific resistivity of samples made of steel 45 on the number of loading cycles. Samples AN (st 45) and AN4 (st 45)2 were annealed of the first kind without phase recrystallization. It can be seen from Fig. 6 that the specific resistivity of the annealed samples has increased significantly 6 6 https://doi.org/10.1051/matecconf /201823901027 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 Table 4. Magnetic properties of samples. No Sample Refined name Direction Coercive force, <Нc> direct opposite 1. Automatic coupler AC1 81 79 80,0 AC2 82 82 82,0 AC3 84 83 83,5 AC4 89 87 88,0 AC5 92 93 92,5 2. Rail R1 156 154 155,0 R2 163 159 161,0 R3 162 161 161,5 R4 152 154 153,0 R5 149 151 150,0 3. Steel 45 ST45-5 77 77 77,0 ST 45-6 79 78 78,5 ST 45-2 78 77 77,5 ST 45-3 74 75 74,5 ST 45-5 77 77 77,0 4. Steel 45 (annealed) ST 45-AN1 142 143 142,5 ST 45-AN2 157 157 157,0 ST 45-AN4 141 143 142,0 ST 45-AN3 144 145 144,5 ST 45-AN1 142 143 142,5 Table 4. Magnetic properties of samples. Fig. 7 shows the dependences of the Young's modulus of various samples (automatic coupler - AC-2, steel 45-5 (st. 45), rail-R2 (rail), annealed sample - AN1 (st45)) on the number of loading cycles. It can be seen that the number of load cycles does not influence the values of the Young's modulus due to the small loading amplitude. Fig. 7 shows the dependences of the Young's modulus of various samples (automatic coupler - AC-2, steel 45-5 (st. 45), rail-R2 (rail), annealed sample - AN1 (st45)) on the number of loading cycles. 3 Results It can be seen that the number of load cycles does not influence the values of the Young's modulus due to the small loading amplitude. Fig. 5. Dependence of thermoelectric power of different samples on the number of loading cycles. Fig. 5. Dependence of thermoelectric power of different samples on the number of loading cycles. 7 7 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 , 0 (2018) MATEC Web of Conferences 239 1027 https://doi.org/10.1051/matecconf /201823901027 2018 TransSiberia Fig. 6. Dependence of the specific resistivity of different samples on the number of loading cycles. Fig. 6. Dependence of the specific resistivity of different samples on the number of loading cycles. Fig. 6. Dependence of the specific resistivity of different samples on the number of loading cycles. Fig. 7. Dependence of the Young's modulus of different samples on the number of loading cycles. Fig. 7. Dependence of the Young's modulus of different samples on the number of loading cycles. 4 Results At this stage of studying the structurally sensitive physical characteristics of materials used to fabricate highly loaded elements of rolling stock, the following results were obtained: 1. From the results presented in Fig. 5, it follows that the lowest thermoelectric power values correspond to samples of automatic coupler’s materials (AC1 and AC2), which was previously subjected to embrittlement during operation [15]. 2. The specific resistivity of the annealed samples of steel 45 exceeds the specific resistivity of unannealed samples of the same steel by approximately 80 nOhm • m, which is about 40%. This allows developing a way of input control of the quality of fabricating highly loaded units and parts of the railway transport. 3. The results of measuring the Young's modulus are useful for predicting the residual resource, since they can give an idea of the degree of change in the mechanical characteristics of the objects under study. 4. The value of the coercive force depends on the domain structure of the object under study (see Table 4) and its mechanical properties [14]. Consequently, the degree of increase in 8 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 the external magnetic field is a structural characteristic of the surface under study, which makes it possible to increase the reliability of the determination of the residual resource. the external magnetic field is a structural characteristic of the surface under study, which makes it possible to increase the reliability of the determination of the residual resource. 5. The values of thermoelectric power, specific resistivity, and the Young's modulus do not depend on the number of oscillations, which, in our opinion, indicates a weak external loading of the samples (not more than 1% of the elastic limit). 5. The values of thermoelectric power, specific resistivity, and the Young's modulus do not depend on the number of oscillations, which, in our opinion, indicates a weak external loading of the samples (not more than 1% of the elastic limit). The results of the experimental studies obtained in this paper do not contradict the literature data on the change in the physicochemical and mechanical properties of materials subject to various types of loads. The obtained experimental data should be used in the development of quick methods for diagnosing predefective states in depot conditions, simplifying the preliminary preparation of the studied object. 4 Results It should be noted that the results presented above were obtained under weak mechanical stresses and room temperatures. For the reliable determination of predefective states, it is planned to obtain similar experimental data, taking into account the existing values of the changes in loads and temperatures that arise under real operating conditions of the rolling stock. 16. M.M. Zakirnichnaya, I.R. Kuzeev, V.K. Berdin, N.Yu. Kirillova, Oil and gas business, machines and devices 4-1, 207-214 (2006) machines and devices 4-1, 207-214 (2006) 17. Yu.I. Matyash, Yu.M. Sosnovsky, E.M. Kondrikov, Patent №154462 Russian Federation, G 01 N 3/38, Laboratory installation for accelerated determination of the residual life of metallic ferromagnetic products taking into account the combined load (2015) References 1. R.Yu. Adadurov, Cars and rolling stock 4(44), 24 – 27 (2015) 2. G.S. Shelikhov, Magnetic particle inspection of products (Spectrum, Moscow, 2013) 3. V.F. Krivorudchenko, R.A. Akhmedzhanov, Modern methods of technical diagnostics and non-destructive examination of parts and units of rolling stock of railway transport (Marshrut, Moscow, 2005) 4. V.V. Zuev, L.N. Potselueva, Yu.D. Goncharov, Crystalloenergetics as a basis for evaluating the properties of solid materials (including magnesia cements) (Publishing house of the “Alfapol”, SPb, 2006) 5. E. Clegg, W. G. Blevins, Wheel Impact Load Detector Experience on CN, American Railway Engineering Association Bulletin, 499–523 (1996) 6. J.M. Hart, N. Ahuja, C.P.L. Barkan, D.D. Davis, A Machine Vision System for Monitoring Railcar Health: Preliminary Results, Technology Digest: TD-04-008 (Association of American Railroads, Pueblo, Colorado, 2004) 7. J.R. Edwards, J.M. Hart, S. Todorovic, C.P.L. Barkan, N. Ahuja, Z. Chua, N. Kocher, J. Zeman, Proceedings of the International Heavy Haul Conference Specialist Technical Session - High Tech in Heavy Haul, Kiruna, 745-752 (2007) 8. G. den Buurman, European Railway Review 3, 80 – 86 (2005) 9. V.A. Gapanovich, Rail transport 10, 10-15 (2016) 10. V.V. Bolotin, Forecasting the resource of machines and structures (Mechanical Engineering, Moscow, 1984) 11. V.F. Terentev, S.A. Korableva, Fatigue of metals (Nauka, Moscow, 2015) 12. Yu.G. Matvienko, Models and criteria of the destruction mechanism (Fizmatlit, Moscow, 2006) 13. Yu.I. Matyash, Yu.M. Sosnovsky, A.A. Razhkovskii, E.M. Kondrikov, Omsk Scientific Bulletin. Ser. Mechanical Engineering and Engineering Science 4(154), 9-13 (2017) 14. V. Berzhansky, P. Maruschak, R. Biszak, I. Danilyuk, V. Vishnevsky, A. Prokopov, F. Pankov, N. Lugovskoy, News of TNTU 71-3, 272 – 278 (2015) 15. N.A. Kostenko, Prediction of reliability of transport vehicles (Mashinostroenie, Moscow, 1989) 9 9 , 0 (2018) MATEC Web of Conferences 2018 TransSiberia 239 1027 https://doi.org/10.1051/matecconf /201823901027 17. Yu.I. Matyash, Yu.M. Sosnovsky, E.M. Kondrikov, Patent №154462 Russian Federation, G 01 N 3/38, Laboratory installation for accelerated determination of the residual life of metallic ferromagnetic products taking into account the combined load (2015) 10
W2765662770.txt	https://bmcgastroenterol.biomedcentral.com/track/pdf/10.1186/s12876-017-0667-9	en		The diagnostic value of pepsin detection in saliva for gastro-esophageal reflux disease: a preliminary study from China	BMC gastroenterology	2,017	cc-by	6,336	Du et al. BMC Gastroenterology (2017) 17:107 DOI 10.1186/s12876-017-0667-9 RESEARCH ARTICLE Open Access The diagnostic value of pepsin detection in saliva for gastro-esophageal reflux disease: a preliminary study from China Xing Du1, Feng Wang2, Zhiwei Hu2, Jimin Wu2, Zhonggao Wang1,2, Chao Yan1, Chao Zhang3 and Juan Tang4 Abstract Background: None of current diagnostic methods has been proven to be a reliable tool for gastro-esophageal reflux disease (GERD). Pepsin in saliva has been proposed as a promising diagnostic biomarker for gastro-esophageal reflux. We aimed to determine the diagnostic value of salivary pepsin detection for GERD. Methods: Two hundred and fifty patients with symptoms suggestive of GERD and 35 asymptomatic healthy volunteers provided saliva on morning waking, after lunch and dinner for pepsin determination using the Peptest lateral flow device. All patients underwent 24-h multichannel intraluminal impedance pH (24-h MII-pH) monitoring and upper gastrointestinal endoscopy. Based on 24-h MII-pH and endoscopy study, patients were defined as GERD (abnormal MII-pH results and/or reflux esophagitis) and non-GERD otherwise. Results: Patients with GERD had a higher prevalence of pepsin in saliva and higher pepsin concentration than patients with non-GERD and healthy controls (P < 0.001 for all). The pepsin test had a sensitivity of 73% and a specificity of 88.3% for diagnosing GERD using the optimal cut-off value of 76 ng/mL. Postprandial saliva samples collected when the symptoms occurred had a more powerful ability to identify GERD. Conclusions: Salivary pepsin test had moderate diagnostic value for GERD. It may be a promising tool to replace the use of currently invasive tools with advantages of non-invasive, easy to perform and cost effective. Trial registration: ChiCTR-DDD-16009506 (date of registration: October 20, 2016). Keywords: Gastro-esophageal reflux disease, Pepsin, 24-h multichannel intraluminal impedance pH monitoring, Endoscopy, Diagnosis Background Gastro-esophageal reflux disease (GERD) refers to “a condition that develops when the reflux of stomach contents causes troublesome symptoms and/or complications” in the Montreal Classification [1]. As one of the most common gastrointestinal diseases, its prevalence has increased in the last few decades [2, 3], leading to a considerable healthcare burden and low quality of life. Current methods employed in the diagnosis of GERD include GERD questionnaires, “PPI test”, endoscopy and ambulatory esophageal reflux monitoring. However, the sensitivity and specificity of such Correspondence: zhonggaowang194@sina.com 1 Department of Vascular Surgery, Xuan Wu Hospital, Capital Medical University, Beijing 100053, China 2 Department of Gastroesophageal Reflux Disease, PLA Rocket Force General Hospital, Beijing 100088, China Full list of author information is available at the end of the article methods have been questioned. Recent studies showed that the PPI test and structured questionnaires did not obtain ideal sensitivity and specificity for diagnosis of GERD [4–6]. Endoscopy is not adequate since non-erosive reflux disease (NERD) is more prevalent than erosive reflux disease in the GERD population [7]. Reflux monitoring, including esophageal pH metry and impedance-pH metry, although currently used as the available gold standard, has a lower sensitivity in patients with NERD compared to those with reflux esophagitis and lacks reproducibility [8]. After all, none of the current approaches has been proven to be a reliable tool for GERD, and more specific, non-invasive and cost effective diagnostic methods are warranted. Pepsin, a potential factor contributing to the mucosal tissues when gastro-esophageal reflux (GER) occurs, is a protease originating from pepsinogen synthesized by the © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Du et al. BMC Gastroenterology (2017) 17:107 gastric chief cells. Pepsin has been found in many different tissue samples such as laryngeal mucosa, paranasal sinus mucosa, saliva, middle ear effusion, tracheal secretions and bronchoalveolar lavage fluid [9–13]. The presence of pepsin in esophagus or more proximal sites is indicative of reflux, suggesting that pepsin may be used as a biomarker for the objective assessment of GERD. Some studies have shown that pepsin detection in the sputum and/or saliva can be regarded as a sensitive, non-invasive method for the diagnosis of the proximal reflux of gastric contents or laryngopharyngeal reflux (LPR) [14–17]. Thus, in the present study, we aimed to determine whether the use of pepsin determination in saliva could be useful for diagnosing GERD in the adult cohort from China with Peptest (RD Biomed Ltd., Hull, UK). Methods Subjects Adult participants were consecutively enrolled in this prospective study at the Department of GERD, The General Hospital of the PLA Rocket Force, China. Patients who had at least eight weeks’ history of symptoms suggestive of GERD (for example heartburn, regurgitation, noncardiac chest pain, chronic cough, asthma, throat irritation or clearing, globus sensation) were eligible for enrollment in this prospective study. Before enrollment in the present study, patients received consultancy from the departments of cardiology, pulmonology, or otorhinolaryngology according to their symptoms, and had been fully studied to rule out causes other than GERD. Exclusion criteria were: central system diseases, connective tissue diseases, psychiatric disorders, previous gastric or esophageal surgery, Zollinger-Ellison syndrome, esophageal stricture, achalasia, autoimmune diseases and collagen vascular diseases. Asymptomatic healthy volunteers were recruited from Center of health Examination, the General Hospital of the PLA Rocket Force, China, as normal controls. Subjects were eligible if they were age ≥ 18 years without GERD symptoms. We excluded subjects with a history of previous gastric or esophageal surgery, a known esophageal motor disorder (e.g. achalasia, scleroderma), or psychiatric disorders. A detailed GerdQ questionnaire was completed by the investigations for each patient and asymptomatic healthy subject. Signed informed consent was obtained from all participants before the study and the study protocol was reviewed and approved by the institutional review boards of the General Hospital of the PLA Rocket Force (Beijing, China) and was registered in Chinese Clinical Trial Registry (Registration number: ChiCTR-DDD-16009506). During the study, all patients received 24-h multichannel intraluminal impedance pH (24-h MII-pH) monitoring, upper gastrointestinal endoscopy, esophageal highresolution manometry (HRM) and upper gastrointestinal imaging. Reflux esophagitis was graded based on Los Page 2 of 9 Angeles classification and esophageal motility parameters (including resting lower esophageal sphincter (LES) pressure, resting upper esophageal sphincter (UES) pressure and hiatus hernia (HH)) in HRM were defined according to Chicago classification. 24-h MII-pH monitoring Patients were instructed to discontinue their PPIs medications 7 days prior to reflux monitoring, and H2 receptor antagonists, prokinetic medications, and antacids 3 days prior to the study. After an overnight fast, an experienced staff positioned the MII-pH catheter (Sandhill Scientific, Highlands Ranch, CO, USA) incorporating a pH sensor and six impedance channels located 5 cm above the proximal border of the LES, identified using HRM. The six impedance sensors were positioned 3, 5, 7, 9, 15, and 17 cm above the sphincter. During data acquisition, patients were asked to record mealtimes and activities, and log their symptom events electronically. Data was analyzed with dedicated software (Bioview Analysis; Sandhill Scientific, Highlands Ranch, CO, USA). Each graphical tracting of all events was further scrutinized manually to ensure accurate reflux detection. A pH study was considered abnormal if DeMeester scores were ≥14.7, or acid exposure time (AET) ≥4.2%. A impedance portion was defined as abnormal if percent bolus exposure time (BET) was ≥1.4%, or number of all reflux episodes ≥73 [18–20]. According to the endoscopy and the MII-pH study, patients were defined as GERD if they had reflux esophagitis, or abnormal pH results, or abnormal impedance results, and as non-GERD otherwise. Salivary pepsin Salivary sample collection: Subjects were given 30-mL sterile plastic tubes containing 0.5 ml 0.01 mol/L citric acid, pH 2.5 to collect saliva. Subjects were instructed to cough a few times to clear the saliva from the back of their throat and then spit it into the tubes. Subjects collected saliva on morning waking, 1–2 h after lunch and dinner. Before collecting the early morning sample, subjects were required to refrain from brushing their teeth, drinking or eating. Samples were transferred to the refrigerator at 4 °C and analyzed within 7 days. Pepsin measurement: Saliva specimens were analyzed using the Peptest lateral flow device (LFD) (RD Biomed Ltd., Hull, UK). Plastic tubes were centrifuged for 5 min at 4000 rpm in a bench top centrifuge and 80 μL supernatants were draw up into an automated pipette. The 80 μL sample was then mixed with 240 μL migration butter solution for 10 s. And the 80 μL of the mixture was added to the well of the LFD containing 2 unique human monoclonal antibodies that detected and captured pepsin protein (specific to pepsin-3), with a lower limit of detection of 16 ng/mL and an upper limit of 500 ng/mL. The value of 16 ng/mL Du et al. BMC Gastroenterology (2017) 17:107 was used as a cut-off to consider a saliva sample positive for pepsin. All samples with pepsin concentration below this threshold were regarded to have 0 ng/mL and those above 500 ng/mL had 500 ng/mL in the results. Statistical analysis The SPSS 19.0 statistical software package (IBM, Armonk, NY) and Prism V.5.0, GraphPad were used for data processing. Continuous data were summarized as Mean ± SD if normally distributed and as median (interquartile rang, IQR) otherwise, and categorical variables were summarized as counts and frequencies. The Kruskall-Wallis test was used to conduct multiple group comparisons for nonnormal distributed data and chi-squared test was used for categorical parameter. Correlations between pepsin concentration and reflux variables were assessed using Spearman’s rank correlation as appropriate. Receiver operating characteristic (ROC) curve analysis was performed to determine an optimal cut-off value of salivary pepsin concentration and compare the predictive values of different pepsin cut-off concentrations to diagnose GERD. The optimal cut-off was chosen using Youden index. All tests of significance were 2-sides, with P < 0.05 considered statistically significant. Results Patient characteristic Three hundred and twelve participants were recruited. Eighteen of the patients could not tolerate the endoscopy or reflux monitoring and 9 of the healthy controls chose to drop out of the protocol, and were excluded from the analysis. Finally, one hundred and twenty two symptomatic patients (53 male and 69 female; median age, 53 (44– 60) years), 128 patients (58 male and 70 female; median age, 50 (42–63) years) and 35 asymptomatic subjects (18 male and 17 female; median age, 48 (42–69) years) were included in the GERD, non- GERD and healthy controls. Differences in the ages, body mass index and sex distribution of subjects among the three groups were not significant (P > 0.05 for all) (Table 1). Out of 250 patients, 84% patients reported heartburn and/or regurgitation, 7% patients chest pain, 5% patients throat clearing, 4% patients chronic cough or asthma as primary complains. There were significant differences between non-GERD and Page 3 of 9 GERD patients regarding to the parameters of reflux monitoring and HMR and the presence of HH, except for resting UES pressure (Table 2). Among the GERD patients, there were 31/122(25.4%)subjects with NERD, 91/122 (74.6%) subjects with reflux esophagitis (LA-A = 26, LA-B = 36, LA-C = 17, LA-D = 12), 102/122 (83.6%) with abnormal pH testing and 72/122 (59.0%) with abnormal impedance results, and 35/122 (28.7%) with abnormal pH testing and impedance results. Salivary pepsin results On the basis of the thresholds of 16 ng/mL, there was a significantly stepwise increase in the prevalence of positive pepsin among the 3 cohorts: results were positive in 111/122 (91.0%) of GRED who had at least one saliva sample positive for pepsin, 68/128 (53.1%) of nonGERD, and 15/35 (42.9%) of controls (P < 0.001) (Fig. 1a). The number of subjects having all three samples positive for pepsin was small (16/122 in GERD, 14/ 128 in non-GERD, and 4/35 in controls) (Table 3). The salivary pepsin concentration of each subject was determined as the highest value of all samples. The pepsin concentration was significantly highest in the GERD group (153.3 (70–365.8)) ng/mL, followed by non-GERD (18.5 (0–59.75) ng/mL), and lowest in controls (0 (0–25) ng/mL) (P < 0.001) (Table 3, Fig. 1b). Overall, compared to postprandial samples, the positivity rates of pepsin and concentration of pepsin were significantly lower in the morning waking samples, both in non-GERD and GERD cohorts, while such differences were not found in controls (Table 4). Among patients including subjects with GERD and non-GERD, there were no statistically significant differences between samples after lunch and those after dinner, in terms of the prevalence of positive pepsin samples and concentration of salivary pepsin (Table 4). Interestingly, there were 27 saliva samples collected when symptoms occurred after meals from 27 GERD patients, which had higher positive rates of salivary pepsin and median concentrations of pepsin though statistically significant differences were not obtained compared to the overall postprandial samples (prevalence, 85.2% vs. 74.2%, P = 0.208; concentrations, 130 (36–392) ng/mL vs. 96 (0– 278) ng/mL, P = 0.134). Table 1 Baseline characteristics for subjects in controls and two groups of patients Clinical Variables Controls (n = 35) Non-GERD (n = 128) GERD (n = 122) P Gender (M/F) 18/17 58/70 53/69 0.7 Age (yr), median (IQR) 48 (42–69) 50 (42–63) 53 (44–60) 0.664 Age (yr), rang 18–85 19–81 24–79 – BMI (kg/m ), mean±SD 27.0±3.9 27.7±4.2 28.7±4.0 0.41 GerdQ score, mean±SD 4.1±1.9 6.3±2.6 11.0±2.8 <0.001 2 GERD gastro-esophageal reflux disease, M/F male/female, BMI body mass index, SD standard deviation Du et al. BMC Gastroenterology (2017) 17:107 Page 4 of 9 Table 2 The results of 24-h MII-pH monitoring and esophageal HRM in 2 cohorts Non-GERD (n = 128) GERD (n = 122) P Demeester score, median (IQR) 11.2 (8.8–13) 26.3 (14.0–51.7) <0.001 AET (%), median (IQR) 3.1 (2.1–3.7) 8.9 (4–12.1) <0.001 BET (%), median (IQR) 1.05 (0.8–1.2) 2 (0.4–5.5) 0.006 Number of all reflux episodes, median (IQR) 63 (51.3–69) 75 (55.8–117.3) <0.001 12.4 (7.3–18) 8.4 (4.4–12) <0.001 57.2 (34.2–84.9) 58.4 (39.6–80.7) 0.605 24.2 73.0 <0.001 Parameters 24-h MII-pH monitoring HRM Resting LES pressure (mmHg), median ( IQR) Resting UES pressure (mmHg), median (IQR) # HH (%) 24-h MII-pH 24-h multichannel intraluminal impedance pH, IQR interquartile rang, GERD gastro-esophageal reflux disease, AET acid exposure time, BET bolus exposure time, HRM high resolution manometry, LES lower esophageal sphincter, UES upper esophageal sphincter, HH hiatal hernia # HH was detected by endoscopy combined with HRM The features of different values of pepsin concentration in saliva to differentiate patients with GERD from patients with non-GERD Using the ROC curve, we identified the optimal cut-off value of salivary pepsin concentration to differentiate GERD patients from non-GERD patients (Fig. 2). The area under the ROC curve was 0.868±0.023 (95% CI, 0.822 to 0.914, P < 0.001). When the best pepsin test cut-off value was determined to be 76 ng/mL, the value of Youden index was biggest (61.3%). And the sensitivity of the Peptest test was 73%, and the specificity was 88.3% at the measured optimal cut-off value (Fig. 3). In Table 5, we displayed a range of Fig. 1 a The prevalence of positive pepsin in controls and two groups of patients. b The concentrations of pepsin in controls and two groups of patients. c The concentrations of pepsin in GERD patients with NERD, LA-A, LA-B and LA-C + D based on Los Angeles classification. d The concentrations of pepsin in GERD patients with or without HH. GERD: gastro-esophageal reflux disease, NERD: non-erosive reflux disease, HH: hiatus hernia Du et al. BMC Gastroenterology (2017) 17:107 Page 5 of 9 Table 3 The prevalence and concentrations of pepsin in saliva for controls and two groups of patients Parameters Controls (n = 35) Non-GERD (n = 128) GERD (n = 122) P Prevalence of positive samples (%) 29.5 32.3 62.0 <0.001 Proportion of subjects having at least one positive samples (%) 42.9 53.1 91.0 <0.001 Proportion of subjects having all three positive samples (%) 11.4 10.9 13.1 0.864 Concentrations of salivary pepsin (ng/mL), median (IQR) 0 (0–25) 18.5 (0–59.75) 153.3 (70–365.8) <0.001 GERD gastro-esophageal reflux disease, IQR interquartile rang salivary pepsin concentrations and compared their predictive values to diagnose GERD using the endoscopy and MIIpH metry as the gold standard. Correlation analyses between concentration of salivary pepsin, 24-h MII-pH monitoring and HRM Spearman’s rank correlation analyses were used to evaluate potential correlations between concentration of salivary pepsin, reflux parameters and HRM. A complete list of the associations tested was presented in Table 6. These cases included the moderately positive correlation between the pepsin level and DeMeester score (rs = 0.665, P < 0.001), the low positive correlation between the pepsin level and AET (rs = 0.423, P < 0.001), and number of all reflux episodes (rs = 0.424, P < 0.001), the very low positive correlation between the pepsin level and BET (rs = 0.178, P = 0.005), the low negative correlation between the pepsin level and LES (rs = -0.181, P = 0.004). There was no significant correlation between the pepsin level and UES (P = 0.376). Salivary pepsin in GERD Among patients with GERD, there was also a stepwise increase in the concentration of salivary pepsin: highest in those with LA-C + D, followed by those with LA-B, lowest in those with LA-A and NERD (P < 0.001) (Fig. 1c). Similarly, subjects with HH had a high level of pepsin in saliva than those without HH (P = 0.005) (Fig. 1d). Discussion GERD is a common but often misdiagnosed disease in clinical practice. Studies [21–28] showed that GERD was the underlying factor of many pulmonary and otolaryngology diseases and conditions, such as asthma, chronic cough, pneumonia, laryngitis, pharyngitis, hoarseness, and even malignant tumor of head and neck. Because of the nonspecific symptoms of reflux disease, the definitive diagnosis of GERD is still challenging. Currently, the most reliable test for GERD diagnosis is ambulatory 24h MII-pH monitoring. But it has inherent limitations. First, a considerable proportion of GERD patients could not be detected via the MII-pH metry [29], and dietary modifications and activity restrictions during reflux monitoring may lead to false negative results. Second, there are some asymptomatic cases of reflux who will escape reflux-related monitoring. Third, it is an invasive, expensive technique and a proportion of patients could not tolerate this test. Therefore, the development of a noninvasive, convenient, effective and sensitive method is warranted in a clinical setting. Pepsin has been proposed as a promising biomarker for GER [24, 30–32]. Several studies [33–37] have indicated that pepsin is a major cause of GER, particularly in weakly acidic and non-acidic reflux. Pepsin may be present in the saliva/sputum of patients during episodes of GER and can keep stable below pH 8.0 [38]. Its presence in the oropharynx suggests GER. The Peptest test is a convenient, office-based, noninvasive and quick technique for pepsin determination with the use Table 4 The prevalence and concentrations of pepsin in saliva at different sampling time points for three cohorts Parameters Morning waking After lunch After dinner P 17.1 37.1 34.3 0.14 0 (0–0) 0 (0–21) 0 (0–25) 0.095 Control (n = 35) Prevalence (%) # Concentrations (ng/mL) Non-GERD (n = 128) Prevalence (%) 22.7 36.7 37.5 0.017 Concentrations (ng/mL) # 0 (0–0) 0 (0–31.5) 0 (0–39.5) 0.005 37.7 73.8 74.6 <0.001 0 (0–75.3) 103.5 (0–274.8) 92 (0–288.3) <0.001 GERD (n = 122) Prevalence (%) Concentrations (ng/mL) # GERD gastro-esophageal reflux disease # presented as median (IQR) Du et al. BMC Gastroenterology (2017) 17:107 Fig. 2 Receiver operating characteristic curve analysis for determining an optimal cut-off value of salivary pepsin concentration to identify patients with GERD. GERD: gastro-esophageal reflux disease of two unique human monoclonal antibodies to capture pepsin-3b independent of pH, which is superior to the digestion assay [39]. Several studies have shown that the measurement of pepsin in saliva/sputum may be used as a promising tool for diagnosing LPR using the Peptest test [40, 41]. This article is the largest-scale, prospective study in which we assessed the diagnostic value of the Peptest test for objective GERD confirmed by 24-h MII pH monitoring combined with endoscopy. Our study showed that (1) Fig. 3 Comparison of salivary pepsin results with 24-h MII-pH monitoring combined with endoscopy. GERD: gastro-esophageal reflux disease, 24-h MII-pH: 24-h multichannel intraluminal impedance pH Page 6 of 9 patients with GERD had a higher prevalence of pepsin in saliva and higher pepsin concentration than patients with non-GERD and healthy controls, (2) Postmeal saliva samples collected at the time of symptomatic episodes had a higher positive rate for pepsin and level of pepsin than overall postprandial samples in GERD patients, (3) About 40% of healthy asymptomatic subjects had salivary pepsin with a low concentration of less than 130 ng/mL, (4) Using the cut-off value of 76 ng/mL, the sensitivity and specificity of the Peptest test were determined to be 73.0% and 88.3% respectively. Kim et al. [14] used the western blot analysis to detect the pepsin in the sputum/saliva in patients with clinically suspected atypical GERD symptoms. The results found that the sensitivity and negative predictive value of pepsin determination were excellent in most of atypical symptom groups (100%), whereas its specificity and positive predictive value were relatively low (76%). Similar researches are few, so the significance of pepsin detection for adult patients with atypical symptoms should be further studied. To date, three studies have assessed the value of salivary pepsin for GERD with heartburn in adult cohorts using the Peptest test [42–44]. In these three studies, MII-pH metry [43, 44] and pH metry combined with endoscopy [42] were utilized as diagnostic criteria for GERD or reflux-related symptoms. AET with or without reflux number was the only parameter that was used to determine whether reflux monitoring was abnormal, which would result in false negative results. By contrast, we used pH monitoring data (Demeester scores and AET), impedance study (BET and number of all reflux) and endoscopy as the basis for test characteristics, which had a higher sensitivity for detecting GERD. In the study by Saritas Yuksel [42], salivary pepsin was positive in 12% of controls and 50% of objective GERD (abnormal AET and/or esophagitis). Pepsin assay had a low sensitivity of 50% and a high specificity of 92% using the cut-off of 50 ng/mL with only one saliva sample collected at a random time point. In the published study by Bortoli [43], the Peptest test was positive in 94% of NERD, similar to the results obtained by us, and the pepsin analysis was found to have a sensitivity of 100% and a specificity of 80%, superior to our results. So the authors proposed that salivary pepsin determination was a convenient, economic, accurate and highly specific test to detect GERD without esophagitis. More recently, Hayat et al. [44] took three salivary samples for each participant on waking, 1 h after lunch and dinner during reflux monitoring period. They found that pepsin could be found saliva in healthy subjects and patients, and the postprandial saliva samples were more likely to be positive for pepsin and have a higher level of pepsin compared to the morning saliva samples. Both the Du et al. BMC Gastroenterology (2017) 17:107 Page 7 of 9 Table 5 The predictive values of a range of salivary pepsin concentrations to identify patients with GERD Concentrations (ng/mL) Sen (%) Spe (%) Youden index (%) PPV (%) NPV (%) PLR NLR ≥16 85.1 47.1 32.2 60.5 76.8 1.61 0.32 ≥76 73.0 88.3 61.3 85.6 77.4 6.24 0.30 ≥125 54.8 90.0 44.8 83.9 67.6 5.48 0.50 ≥200 44.3 97.2 41.5 93.8 64.7 15.82 0.57 GERD gastro-esophageal reflux disease, Sen sensitivity, Spe specificity, PPV positive predictive value, NPV negative predictive value, PLR positive likelihood ratio, NLR negative likelihood ratio prevalence and concentrations of salivary pepsin were significantly higher in patients with GERD and hypersensitive esophagus (HE) compared to those with functional heartburn (FH) and controls, with higher concentrations predicting a greater probability of reflux. These results were consistent with those obtained by us. However, there were some differences needed to be issued between the two studies. Compared to the study by Hayat et al., the prevalence and median concentrations of pepsin in saliva of GERD patients seemed to be higher in our study (prevalence, 78% vs. 91%; concentrations, 126 (49.7–246.3) ng/mL vs. 153.3 (70– 365.7) ng/mL). Our optimal cut-off value of pepsin was lower than that obtained by Hayat et al. (207 ng/mL vs. 76 ng/mL), and the area under the ROC and the Youden index were bigger in the study by us. Namely, the results about the diagnostic value of the Peptest test in our study were superior to those obtained by Hayat. The differences might be explained by some reasons. First, we used MII-pH combined with endoscopy as gold standard to define GERD, which can low the false negative results. Such more “true” GERD patients could be detected with more significant differences on the pepsin concentrations between GERD patients and non-GERD patients. Second, postprandial saliva samples tend to have a higher positive rate of pepsin and level of pepsin compared to the morning waking samples by increased gastric pepsin concentration and volume of reflux after meals [24, 44]. In addition, Kim et al. [14] found that pepsin was significantly more likely to be detected at the time of symptoms than on the waking. In the present Table 6 Correlation analyses between concentrations of salivary pepsin, 24-h MII pH monitoring and HRM rs P vs. DeMeester score 0.665 <0.001 vs. AET 0.423 <0.001 vs. BET 0.178 0.005 vs. Number of all reflux episodes 0.424 <0.001 vs. LES -0.181 0.004 vs. UES −0.056 0.376 Parameters Concentrations of pepsin HRM high resolution manometry, AET acid exposure time, BET bolus exposure time, LES lower esophageal sphincter, UES upper esophageal sphincter, 24-h MII-pH 24-h multichannel impedance pH study, 27 samples taken at the symptoms after meals from GERD patients had a higher prevalence of pepsin and level of pepsin compared to the overall postprandial samples. Based on the evidences above, we think that postprandial salivary samples when the symptoms occur may have a more powerful ability of differentiating GERD patients from non-GERD patients and we recommend postprandial saliva sampling during the symptomatic phase in the clinical application. Third, the study protocol in our study might be different from that of Hayat. Pepsin was found in saliva of 40% of healthy control subjects with a low concentration of pepsin as a result of physiological reflux. A higher concentration of pepsin and positive rate is more likely to capture GERD. In our study, all three negative saliva samples suggested a 76% probability of diagnosing non-GERD, whereas a positive samples ≥200 ng/mL suggested GERD with a probability of 94%. Additionally, our study demonstrated that there were correlations between the level of pepsin and reflux parameters and esophageal motility (LES), suggesting that the pepsin test can be used as an indicator of degree of reflux. The Peptest analysis achieved the accepted sensitivity and specificity for diagnosing GERD. Though far from perfect, its intrigue is that it provides a non-invasive, easy to perform, and inexpensive technique different from the currently available tools. There were some limitations in our study. First, a small number of healthy asymptomatic subjects were enrolled into this prospective study. Further large-scale populationbased study is required to establish an appropriate normal value for healthy subjects. Second, our study did not measure reflux-symptom association analysis and did not classify non-GERD into HE and FH via this parameter because of the lack of definite consensus about the diagnostic criteria for HE and FH. Third, there was no follow-up data to observe pepsin change before and after antireflux therapy and we could not assess its predictive value for clinical outcome. Conclusions In summary, as a rapid, convenient, cost effective and non-invasive method, the detection of salivary pepsin had moderate diagnostic value for GERD and may be a promising tool to replace the use of currently invasive tools. Du et al. BMC Gastroenterology (2017) 17:107 Page 8 of 9 Abbreviations 24-h MII-pH: 24-h multichannel intraluminal impedance pH; AET: Acid exposure time; BET: Bolus exposure time; FH: Functional heartburn; GER: Gastro-esophageal reflux; GERD: Gastro-esophageal reflux disease; HE: Hypersensitive esophagus; HH: Hiatus hernia; HRM: High-resolution manometry; IQR: interquartile rang; LES: Lower esophageal sphincter; LFD: Lateral flow device; LPR: Laryngopharyngeal reflux; NERD: Non-erosive reflux disease; NLR: Negative likelihood ratio; NPV: Negative predictive value; PLR: positive likelihood ratio; PPV: positive predictive value; ROC: Receiver operating characteristic; SD: Standard deviation; Sen: Sensitivity; Spe: Specificity; UES: Upper esophageal sphincter 5. Acknowledgements Not applicable. 8. Funding There is no funding for the study. 9. Availability of data and materials The data generated and analyzed during this study are available from the corresponding author on reasonable request. 10. Authors’ contributions All authors read and approved the final version of the manuscript, including the authorship. Study concept and design: XD, FW, ZWH, JMW, and ZGW; acquisition of data: XD, FW, CY, CZ, and JT; analysis and interpretation of data: ZWH, JMW, ZGW, CY, CZ, and JT; manuscript draft: XD, JMW, ZGW, CY, and CZ; critical revision of the manuscript for important intellectual content: XD, FW, ZWH, and JT. Ethics approval and consent to participate Approval for this study was obtained from the institutional review boards of the General Hospital of the PLA Rocket Force (Beijing, China) (No. KY2016021), and written informed consent was acquired from all participants. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 6. 7. 11. 12. 13. 14. 15. 16. 17. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W4306916078	https://zenodo.org/records/7231656/files/7.%20Pradeep%20Bhanudas%20Girhe.pdf	English	null	WOMEN DISCRIMINATION IN PRIVATE ORGANIZATION	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	2,291	Understanding of Results It is already known that the human society consists of male and female, women and men, girls and boys. There are clear biological differences between them, i.e. differences carried from birth, and these differences have originated in the early stages of the human genesis. Therefore we can also say that “sex is determined by biological, natural factors or attributes, i.e. born”. Taking into account these biological differences between the sexes, different theories a economic “differences”. So, theories arise which “proved” the men as more intelligent, stronger, and therefore “naturally” more dominant, based on gender inequality which of course is in favor of men hence human society must be managed by them. Differences begin from toys that are bought for children, as through them we understand that boys are being prepared by parents for their role outside the house, It is already known that the human society consists of male and female, women and men, girls and boys. There are clear biological differences between them, i.e. differences carried from birth, and these s have originated in the early stages of the human genesis. Therefore we can also say that “sex is determined by biological, natural factors or attributes, i.e. born”. Taking into account these biological differences between the sexes, different theories arise regarding their different social, psychological and economic “differences”. So, theories arise which “proved” the men as more intelligent, stronger, and therefore “naturally” more dominant, based on gender inequality which of course is in favor of men hence human society must be managed by them. Differences begin from toys that are bought for children, as through them we understand that boys are being prepared by parents for their role outside the house, It is already known that the human society consists of male and female, women and men, girls and boys. There are clear biological differences between them, i.e. differences carried from birth, and these s have originated in the early stages of the human genesis. Therefore we can also say that “sex is determined by biological, natural factors or attributes, i.e. born”. Taking into account these biological rise regarding their different social, psychological and economic “differences”. So, theories arise which “proved” the men as more intelligent, stronger, and therefore “naturally” more dominant, based on gender inequality which of course is in favor of men, hence human society must be managed by them. WOMEN DISCRIMINATION IN PRIVATE ORGANIZATION WOMEN DISCRIMINATION IN PRIVATE ORGANIZATION WOMEN DISCRIMINATION IN PRIVATE ORGANIZATION Pradeep Bhanudas Girhe Abstract This research will help us to focus on a role and place of women in the overall privet/corporate sector after we got freedom in India. inthisissue the employment process and other current problems, which women are facing atevery single p address immediately. Women, who organize half of humanity, But because of male dominating culture they have to face discrimination, in social as well as economic and political aspects, and since then appeared barriers to their career development. This problem is present even today, in almost across the country. However this problem in privet/Corporate sector appears to becomes more critical. AlsoIn recent period, especially during in 21st era, women‘s participation in everyday life has beg from the desirable place. Our findings, which helped the completion of this research, lead us to Aarhat Publication & Aarhat Journals Keywords: discrimination; equality; gender; gender equality; male Introduction This research will help us to focus on glass ceiling (women Discrimination).It gives us a detailed picture of a role and place of women in the overall privet/corporate sector after we got freedom in India. inthisissue the employment process and other current problems, which women are facing atevery single p address immediately. Women, who organize half of humanity, But because of male dominating culture they have to face discrimination, in social as well as economic and political aspects, and since then appeared barriers lopment. This problem is present even today, in almost across the country. However this problem in privet/Corporate sector appears to becomes more critical. AlsoIn recent period, especially during in era, women‘s participation in everyday life has begun to improve in all sectors of life, however it is still far from the desirable place. Our findings, which helped the completion of this research, lead us to Aarhat Publication & Aarhat Journals licensed Based on a work at http://www.aarhat.com/eiirj/ discrimination; equality; gender; gender equality; male-controlled; male dominating culture (women Discrimination).It gives us a detailed picture of a role and place of women in the overall privet/corporate sector after we got freedom in India. inthisissue the employment process and other current problems, which women are facing atevery single place, needs to be address immediately. Women, who organize half of humanity, But because of male dominating culture they have to face discrimination, in social as well as economic and political aspects, and since then appeared barriers lopment. This problem is present even today, in almost across the country. Introduction The gender inequality in the allocation of senior positions has also a theoretical According to the social dominance theory, there are institutional level contributors that help in gender inequality. These contributors influence to further strengthen and maintain the existing inequality of groups. However, the theory explain society that they are no longer seen by the society. In sectors and institutions that are dominated by men, the latter compile vacancy announcements in order to attract the attention of th eventually helps to preserve gender inequality, keeping women outside areas which are led by men (the dominant group). The main purpose of this research is to identify methods to be used in order to improve the position of women in the private sector. The gender inequality in the allocation of senior positions has also a theoretical According to the social dominance theory, there are institutional level contributors that help in gender inequality. These contributors influence to further strengthen and maintain the existing inequality of groups. However, the theory explains that these contributors are so deeply rooted in the structures of society that they are no longer seen by the society. In sectors and institutions that are dominated by men, the latter compile vacancy announcements in order to attract the attention of th eventually helps to preserve gender inequality, keeping women outside areas which are led by men (the dominant group). The main purpose of this research is to identify methods to be used in order to improve ivate sector. The gender inequality in the allocation of senior positions has also a theoretical explanation. According to the social dominance theory, there are institutional level contributors that help in gender inequality. These contributors influence to further strengthen and maintain the existing inequality of s that these contributors are so deeply rooted in the structures of society that they are no longer seen by the society. In sectors and institutions that are dominated by men, the latter compile vacancy announcements in order to attract the attention of the men only - that eventually helps to preserve gender inequality, keeping women outside areas which are led by men (the dominant group). The main purpose of this research is to identify methods to be used in order to improve ISSN–2277- 8721 ISSN–2277- 8721 EIIRJ Abstract However this problem in privet/Corporate sector appears to becomes more critical. AlsoIn recent period, especially during in un to improve in all sectors of life, however it is still far from the desirable place. Our findings, which helped the completion of this research, lead us to http://www.aarhat.com/eiirj/ controlled; male dominating culture Keywords: discrimination; equality; gender; gender equality; male Introduction discrimination; equality; gender; gender equality; male-controlled; male dominating culture controlled; male dominating culture Understanding of Results Differences begin from toys that are bought for children, as through them we understand that boys are being prepared by parents for their role outside the house, Peer Reviewed Refereed Refereed Journal Peer Reviewed Refereed Refereed Journal SJIF Impact Factor 6.806 SJIF Impact Factor 6.806 29 ISSN–2277- 8721 whereas the girls for a life that takes place inside the house. Throughout their lives, men and women are pressured to behave as required by the gender differences with a social nature. Therefore men are encouraged to be more active, more decision-makers, more leaders, whereas girls are asked to be more submissive, laconic and serve to others. For women, it is usually thought that they should play a reproductive role, raise and care for children, the elderly, the disabled, to cook and do the housework, whereas the production activities are reserved for men. Regarding the second hypothesis, which has to do with the women‘s level of education in comparison to male education, this research showed that women comprise more than 50% of unemployed with low-education (no education or just elementary education). Most of the vacancies for loweducation persons are filled by men, this because these jobs usually require physical skills, for example in construction, and this may be one of the causes for male domination compared to women in the jobs that require low-education. In addition, this situation also results from the fact that women in general are less educated than men. Conclusion Private employers still have male-controlled mentality, considering that women and men are biologically "destined" for certain professions. 1. Private employers still have male-controlled mentality, considering that women and men are biologically "destined" for certain professions. 2. The unsatisfactory education for women greatly affects the employment of women in the private sector, where women mostly are satisfied with a High School education, as the thought that female is born for biological reproduction and not for economic and scientific achievements is embedded. 3. Laws adopted should quality laws in regards to the role and position of women in privet companies. 4. The salary of women employees in the private sector is not even close to meet their demands, nevertheless they seem to be "satisfied", because they have managed to have a job and this enables them somehow to enjoy the social life, compared to many other women who have little or no such a life at all. Conclusion From the results obtained, we see that women, despite the adoption of high quality laws such as the Law on Labor, Law on protection from discrimination(POSH), etc., which clearly define the role and position of women, i.e. they "disable" gender discrimination of women in daily life, in the field we see that the opposite happens, hence women continue to be discriminated and deprived of many rights, be that their fundamental rights, such as the right to work because these good and quality laws are not implemented, in brief they remain only written on paper. From the results obtained it may be concluded that: 1. Private employers still have male-controlled mentality, considering that women and men are biologically "destined" for certain professions. 1. Private employers still have male-controlled mentality, considering that women and men are biologically "destined" for certain professions. 2. The unsatisfactory education for women greatly affects the employment of women in the private sector, where women mostly are satisfied with a High School education, as the thought that female is born for biological reproduction and not for economic and scientific achievements is embedded. 3. Laws adopted should quality laws in regards to the role and position of women in privet companies. 4. The salary of women employees in the private sector is not even close to meet their demands, nevertheless they seem to be "satisfied", because they have managed to have a job and this enables them somehow to enjoy the social life, compared to many other women who have little or no such a life at all. 1. Private employers still have male-controlled mentality, considering that women and men are biologically "destined" for certain professions. 2. The unsatisfactory education for women greatly affects the employment of women in the private sector, where women mostly are satisfied with a High School education, as the thought that female is born for biological reproduction and not for economic and scientific achievements is embedded. 3. Laws adopted should quality laws in regards to the role and position of women in privet companies. 4. The salary of women employees in the private sector is not even close to meet their demands, nevertheless they seem to be "satisfied", because they have managed to have a job and this enables them somehow to enjoy the social life, compared to many other women who have little or no such a life at all. 1. List of references : 1) POSH ; (Prevention of Sexual Harassment) Act, 2013 5) 2017 SCC Online Del 11327 Peer Reviewed Refereed Journal SJIF Impact Factor 6.806 Peer Reviewed Refereed Journal 30 ISSN–2277- 8721 EIIRJ Electronic International Interdisciplinary Research Journal (EIIRJ) Volume–X, Issues–II March - April 2021 ISSN–2277- 8721 ISSN–2277- 8721 ISSN–2277- 8721 EIIRJ Electronic International Interdisciplinary Research Journal (EIIRJ) Volume–X, Issues–II March - April 2021 6) Sec 3(2) of the Act 7)_ Sec 4 of the Act 8) Sec 5, 6 and 7 of the Act 9) 2018 SCC Online Del 9340 10) Sec 10 of the Act 11) Sec 11 of the Act 12) Sec 12 of the Act 13) Sec 13 of the Act 14) Sec 14 of the Act 15) Sec 18 of the Act 16) Shreya Khaitan, IndiaSpend.com Mar 07, 2021 · 01:30 pm , p 6) Sec 3(2) of the Act 7)_ Sec 4 of the Act 8) Sec 5, 6 and 7 of the Act 9) 2018 SCC Online Del 9340 10) Sec 10 of the Act 11) Sec 11 of the Act 12) Sec 12 of the Act 13) Sec 13 of the Act 14) Sec 14 of the Act 15) Sec 18 of the Act 16) Shreya Khaitan, IndiaSpend.com Mar 07, 2021 · 01:30 pm Peer Reviewed Refereed Journal SJIF Impact Factor 6.806 Peer Reviewed Refereed Journal 31
https://openalex.org/W2913908916	https://hal.archives-ouvertes.fr/hal-03098794/document	English	null	Nuclear nanomedicine using Si nanoparticles as safe and effective carriers of 188Re radionuclide for cancer therapy	Scientific reports	2,019	cc-by	7,413	To cite this version: V. Petriev, V. Tischenko, A. Mikhailovskaya, A. Popov, G. Tselikov, et al.. Nuclear nanomedicine using Si nanoparticles as safe and effective carriers of 188Re radionuclide for cancer therapy. Scientific Reports, 2019, 9 (1), 10.1038/s41598-018-38474-7. hal-03098794 Nuclear nanomedicine using Si nanoparticles as safe and effective carriers of 188Re radionuclide for cancer therapy V. M. Petriev1,2, V. K. Tischenko2, A. A. Mikhailovskaya2, A. A. Popov3, G. Tselikov3, I. Zelepukin4, S. M. Deyev1,4,7, A. D. Kaprin2, S. Ivanov2, V. Yu. Timoshenko1,5, P. N. Prasad1,6, I. N. Zavestovskaya1 & A. V. Kabashin1,3 Received: 16 October 2018 Accepted: 19 November 2018 Published: xx xx xxxx Received: 16 October 2018 Accepted: 19 November 2018 Published: xx xx xxxx Nuclear nanomedicine, with its targeting ability and heavily loading capacity, along with its enhanced retention to avoid rapid clearance as faced with molecular radiopharmaceuticals, provides unique opportunities to treat tumors and metastasis. Despite these promises, this field has seen limited activities, primarily because of a lack of suitable nanocarriers, which are safe, excretable and have favorable pharmacokinetics to efficiently deliver and retain radionuclides in a tumor. Here, we introduce biodegradable laser-synthesized Si nanoparticles having round shape, controllable low-dispersion size, and being free of any toxic impurities, as highly suitable carriers of therapeutic 188Re radionuclide. The conjugation of the polyethylene glycol-coated Si nanoparticles with radioactive 188Re takes merely 1 hour, compared to its half-life of 17 hours. When intravenously administered in a Wistar rat model, the conjugates demonstrate free circulation in the blood stream to reach all organs and target tumors, which is radically in contrast with that of the 188Re salt that mostly accumulates in the thyroid gland. We also show that the nanoparticles ensure excellent retention of 188Re in tumor, not possible with the salt, which enables one to maximize the therapeutic effect, as well as exhibit a complete time-delayed conjugate bioelimination. Finally, our tests on rat survival demonstrate excellent therapeutic effect (72% survival compared to 0% of the control group). Combined with a series of imaging and therapeutic functionalities based on unique intrinsic properties of Si nanoparticles, the proposed biodegradable complex promises a major advancement in nuclear nanomedicine. Сancer therapy using radiopharmaceutical products has become increasingly important over the last decades, promising an attractive and powerful alternative to conventional chemotherapy1. This nuclear medicine modal- ity implies an injection of short decay time radionuclides in vivo (systemically or intratumorally), while their ionizing radiation (α, β, γ) is used to damage the DNAs of actively proliferating cancer cells, thus causing their selective death while keeping normal cells weakly affected1. The radionuclide therapy becomes especially efficient when one can achieve a high tumor/non-tumor radionuclide contrast, which enables to minimize side effects related to the irradiation of healthy issues. In a conventional approach, one employs vectoring molecules (specific antibodies, etc.) to target radionuclides to the tumor, but these molecules are typically small (less than 60–65 kDa) and can carry only a few chelates linked to radionuclide atoms2,3. HAL Id: hal-03098794 https://hal.science/hal-03098794v1 Submitted on 6 Jan 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. www.nature.com/scientificreports www.nature.com/scientificreports Received: 16 October 2018 Accepted: 19 November 2018 Published: xx xx xxxx Results and Discussion Fabrication, characterization and functionalization of SiNPs. Bare (ligand-free) SiNPs were fab- ricated by femtosecond laser ablation in deionized water11–13, as shown schematically in Fig. 1a and described in details in the Methods section. Being composed of crystalline Si covered by a 1–2 nm thick oxide shell13, laser-synthesized SiNPs have an ideal spherical shape and are relatively monodispersed, with their mean size being about 25 nm (Fig. 1b). The SiNPs were coated with PEG according to our newly developed protocol (see methods section), in order to minimize the immune response of the biological system. Due to high hydrophilicity, PEG is known to form a water cloud around the NPs, which protects them from the interaction with antibodies and opsonic proteins, and dramatically increases the circulation of nanomaterials in the blood streem16. Finally, we conjugated Si NPs-PEG complex with 188Re ions using coordination with the carboxyl group available on the PEG surface, as described in the Methods section. Biodistribution of SiNPs-PEG-188Re conjugates under systemic administration. In our tests, biodistribution of the nanoparticle carrier-based SiNPs-PEG-188Re conjugate was compared with that of freely circulating radioactive rhenium using its salt sodium perrhenate form, Na188ReO4. Five sub-groups of 4 Wistar female rats from the “signal” group, implanted with liver cholangioma RS-1, were intravenously administered with a single dose of 56.8–62.5 µg/kg of animal weight of SiNPs-PEG-188Re conjugates. Similar number of ani- mals from the “control” group were injected with water-dissolved Na188ReO4, containing radioactive rhenium atoms at the same concentration.t As shown in Fig. 2, the maximal level of radioactivity in blood was recorded after 5 minutes of injection of both SiNPs-PEG-188Re and Na188ReO4 solutions which then gradually decreased. For free 188Re (in 188ReO4 -), the level of radioactivity in blood was much lower (<0.5%, after 5 min; <0.2% after 1 hour, <0.1% after 24 hours, and finally not detectable after 48 hours). At the same time, the injection of free Na188ReO4 was accompanied by an immediate increase of the 188Re concentration mainly in the thyroid gland, reaching its maximum values of 17% 3 hours after the radionuclide injection. The accumulation of 188Re in other organs was much lower (Fig. 2), although we recorded a certain concentration of 188Re in the stomach just after the injection (1.2% after one hour), and its smaller concentrations in lungs and kidneys (less than 0.25% and 0.3%, respectively, after five minutes). www.nature.com/scientificreports/ www.nature.com/scientificreports/ side effects, taking into account that the efficiency of molecular targeting typically does not exceed 10–12%. In addition, the size of most targeting molecules appears to be within the renal glomerular filtration range (<7 nm)4, which leads to too fast accumulation of radionuclide complexes in the kidney, causing consequent interstitial nephritis or renal failure problems5,6. p p Recently, there has been a great deal of interest in developing nuclear nanomedicine which utilizes nano- particles (NPs) as carriers of radionuclides7,8. When functionalized by biopolymers such as polyethylene glycol (PEG), NPs promise safe and controllable transport of radionuclides in the blood stream, as well as offer a passive vectoring mechanism for targeting tumors based on their selective size accumulation (enhanced permeability and retention (EPR) effect)2. In addition, NPs can be more heavily loaded with radionuclides to ensure an enhanced therapeutic outcome in the tumor region7,8. However, some stringent requirements to make nuclear medicine safe and effective, have been challenging. The challenges to be met are: (1) NPs-based carrier should be large enough (>20–30 nm) to avoid immediate renal filtration and ensure efficient delivery of radionuclides to the intended site; (II) the NP –radiopharmaceutical conjugate should be safe and excretable from the organism to minimize toxicity and residual accumulation risks4,9; (III) the NP –conjugate should have targeting ability to effectively localize in high concentrations in the tumor; (IV) the coupling to the radioactive nuclei should be fast compared to their half life in order to maximize radiation therapy. Despite the presence of several classes of highly biocom- patible nanomaterials, these challenges are very difficult to meet, as the required large size of NPs beyond the renal filtration range drastically complicates their further bioelimination4,10. i g y p In this article, we propose a pathway to meet these challenges by introducing silicon (Si) NPs (SiNPs), syn- thesized by pulsed laser ablation in liquids11–13, as a nearly ideal carrier of radionuclides for nuclear nanomed- icine. The uniqueness of such SiNPs is based on their biodegradability, which makes possible elimination of these structures from the organism within several days, even if their initial size is large (30–80 nm)12,13 under absence of any toxic effects, which was earlier confirmed in a mice model12. www.nature.com/scientificreports/ In addition, in contrast to Si nanos- tructures prepared by conventional electrochemical14 or chemical15 routes, laser-synthesized SiNPs have ideal round shape, controllable size with a small size dispersion, and are free of any toxic impurities11, which promises a better transport in vivo and no side effects. Here, we demonstrate the possibility for coating of laser-synthesized SiNPs by PEG and a fast conjugation of the SiNPs-PEG complex with the Rhenium-188 (188Re) radionuclide, which is one of most promising generator-type therapeutic beta-emitters with the energy of positron emission of 1.96 MeV (16.7%) and 2.18 MeV (80%) and half-decay time of 17 hours1. We show that such SiNPs-PEG-188Re conjugates can efficiently deliver the radionuclide through the blood stream and retain it in the tumor region. We also demonstrate strong therapeutic effect under intratumoral administration of the conjugate. Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 Nuclear nanomedicine using Si nanoparticles as safe and effective carriers of 188Re radionuclide for cancer therapy V. M. Petriev1,2, V. K. Tischenko2, A. A. Mikhailovskaya2, A. A. Popov3, G. Tselikov3, I. Zelepukin4, S. M. Deyev1,4,7, A. D. Kaprin2, S. Ivanov2, V. Yu. Timoshenko1,5, P. N. Prasad1,6, I. N. Zavestovskaya1 & A. V. Kabashin1,3 Consequently, one has to deliver very high con- centrations of radionuclide-carrying molecules to achieve any sufficient therapeutic effect, but this leads to severe 1MEPhI, Institute of Engineering Physics for Biomedicine (PhysBio), 115409, Moscow, Russia. 2National Medical Research Radiological Center of the Ministry of Health of the Russian Federation, Obninsk, Russia. 3Aix Marseille Univ, CNRS, LP3, Campus de Luminy – Case 917, 13288, Marseille, France. 4Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St, Moscow, 117997, Russia. 5Lomonosov Moscow State University, Physics Department, Leninskie Gory 1, 119991, Moscow, Russia. 6Department of Chemistry and Institute for Lasers, Photonics, and Biophotonics, University at Buffalo, The State University of New York, Buffalo, New York, 14260, United States. 7National Research Tomsk Polytechnic University, Tomsk, Russia. Correspondence and requests for materials should be addressed to P.N.P. (email: pnprasad@buffalo. edu) or A.V.K. (email: kabashin@lp3.univ-mrs.fr) 1 Results and Discussion Notice that the recorded biodistribution and pharmcokinetics with much preferable accumulation of the product in thyroid gland and stomach is typical for free 188Re and other radionuclides1. However, the biodistribution and pharmacokinetics were quite different for nanoparticle carrier-based SiNPs-PEG-188Re conjugates. First, the maximal level of 188Re in the blood was much higher (1.95% and 1.8% after 5 min and 1 hour, respectively) and easily detectable, even 48 hours after the injection (0.5%). In contrast to the free 188Re case, there was no preferential accumulation of radionuclide in any particular organ or tissue. Here, we also recorded certain radionuclide signal in the thyroid gland and stomach (5.5% and 0.65% after 5 min, with a further rapid decrease), but we attributed this signal to washing out of some 188Re atoms from the SiNPs-PEG- 188Re complexes due to possible non-optimized protocol of their conjugation. Surprisingly, the accumulation of radionuclide in liver and spleen was very weak (less than 2.5% and 0.3% after 5 minutes and then rapidly Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 2 www.nature.com/scientificreports/ Figure 1. Synthesis and functionalization of Si nanoparticles for nuclear medicine tasks. (a) Schematic of laser synthesis of SiNPs. Crystalline Si microcolloids (~0.5 μm in size), preliminarily prepared by mechanical milling of a Si wafer, are dispersed in deionized water and illuminated by focused radiation from fs laser. The laser-ablative process leads to the formation of spherical, small size-dispersed SiNPs exempt of any toxic impurity; (b) Typical transmission electron microscopy image (inset) and corresponding size distribution of SiNPs prepared by fs laser ablation; (c) Schematic presentation of functionalization protocol for the coating of SiNPs by polyethylene glycol (PEG) and subsequent decoration by radioactive 188Re atoms. All images were designed and drawn by authors of this manuscript. Figure 1. Synthesis and functionalization of Si nanoparticles for nuclear medicine tasks. (a) Schematic of laser synthesis of SiNPs. Crystalline Si microcolloids (~0.5 μm in size), preliminarily prepared by mechanical milling of a Si wafer, are dispersed in deionized water and illuminated by focused radiation from fs laser. The laser-ablative process leads to the formation of spherical, small size-dispersed SiNPs exempt of any toxic impurity; (b) Typical transmission electron microscopy image (inset) and corresponding size distribution of SiNPs prepared by fs laser ablation; (c) Schematic presentation of functionalization protocol for the coating of SiNPs by polyethylene glycol (PEG) and subsequent decoration by radioactive 188Re atoms. Results and Discussion All images were designed and drawn by authors of this manuscript. decreased down to 0.5–0.7% and 0.05% after 24 hours, respectively). The absence of any significant accumulation of SiNPs-PEG-188Re conjugates in organs of reticuloendothelial system (liver, spleen) can only be explained by their invisibility to the immune system, which was obviously due to the PEG-based coating of SiNPs. As follows from Fig. 2, such a coating led to prolonged circulation of SiNPs-PEG-188Re conjugates in the blood stream and their efficient delivery to most organs. It is also important that the concentration of 188Re gradually increased in the kidneys, reaching its maximal value 24 hours after the injection (almost 3%), which is consistent with gradual dissolution of nanoformulations and their time-delayed elimination via renal clearance12,13. For comparison, in the case of free rhenium (injection of Na188ReO4 solutions) its concentration in the kidney was maximal just after the injection (5 min), which can lead to undesirable kidneys damage. Biodistribution of SiNPs-PEG-188Re conjugates under intratumoral administration. Three sub-groups of 4 Wistar rats with implanted cholangioma RS-1 from the “signal” group were intratumorally administered with a single dose of 56.8–62.5 µg/kg of nanoparticle carrier-based SiNPs-PEG-188Re complexes, while the same number of animals from the “control” group were intratumorally administered Na188ReO4 solu- tions having a similar concentration of 188Re atoms. Different sub-groups of animals from the “signal” and the “control” groups were sacrificed 5 minutes, 3 hours and 24 hours after the injection and examined for 188Re dis- tribution in different organs. As shown in Fig. 3, in the case of free 188Re atoms, we recorded a drastic (4-fold) decrease of 188Re concentration in the tumor during the first 3 hours (from 25% to 6%), while after 24 hours, it was not detectable in this area, suggesting a fast washing out of the radionuclide. At the same time, we recorded a fast increase of 188Re concentration in blood (3.75% after 5 min),with a further slow decrease down to 2.75% after 3 hours and 0.05% after 24 hours. After 3 hours, 188Re mostly migrated into the thyroid gland (14%) that looks Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 3 www.nature.com/scientificreports/ consistent with typical biodistribution for this radionuclide. Significant concentrations of 188Re w Figure 2. Biodistribution of 188Re under its systemic administration in Wistar rats with the na SiNPs-PEG-188Re conjugate. Results and Discussion Different colors show relative amounts of radioactivity for differ and tissues (blood, thyroid gland, lungs, stomach, liver, spleen, kidneys, heart, muscle, tumor) with implanted liver cholangioma RS-1 after 5 min, 1 hour, 3 hours, 24 hours and 48 hours of in administration of SiNPs-PEG-188Re complexes. The blue color shows the relative amount of r in organs and tissues for control group, in which 188Re was systemically administered in the fre dissolved sodium perrhenate Na188ReO4 molecules). Figure 2. Biodistribution of 188Re under its systemic administration in Wistar rats with the nanocarrier-based SiNPs-PEG-188Re conjugate. Different colors show relative amounts of radioactivity for different organs and tissues (blood, thyroid gland, lungs, stomach, liver, spleen, kidneys, heart, muscle, tumor) of Wistar rats with implanted liver cholangioma RS-1 after 5 min, 1 hour, 3 hours, 24 hours and 48 hours of intravenous administration of SiNPs-PEG-188Re complexes. The blue color shows the relative amount of radioactivity in organs and tissues for control group, in which 188Re was systemically administered in the free state (with dissolved sodium perrhenate Na188ReO4 molecules). Figure 2. Biodistribution of 188Re under its systemic administration in Wistar rats with the nanocarrier-based SiNPs-PEG-188Re conjugate. Different colors show relative amounts of radioactivity for different organs and tissues (blood, thyroid gland, lungs, stomach, liver, spleen, kidneys, heart, muscle, tumor) of Wistar rats with implanted liver cholangioma RS-1 after 5 min, 1 hour, 3 hours, 24 hours and 48 hours of intravenous administration of SiNPs-PEG-188Re complexes. The blue color shows the relative amount of radioactivity in organs and tissues for control group, in which 188Re was systemically administered in the free state (with dissolved sodium perrhenate Na188ReO4 molecules). consistent with typical biodistribution for this radionuclide. Significant concentrations of 188Re were also recorded in lungs, kidneys and liver (2.75%, 2.5% and 1% after 3 hours, respectively), while its concentration in the stomach was much lower compared with intravenous injection (0.4% after 3 hours). In general, our data on intratumoral Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 4 www.nature.com/scientificreports/ i j i f f 188R h d i di hi f h di lid f h Figure 3. Biodistribution of 188Re under its intratumoral administration with the nanocarrier-base PEG-188Re conjugate. Results and Discussion Different colors show relative amount of radioactivity in the organs and tissue rats (blood, thyroid gland, lungs, stomach, liver, spleen, kidneys, heart, muscle, tumor) with implan cholangioma RS-1 after 5 min, 3 hours and 24 hours of intratumoral administration of the SiNPs-P complexes.The blue color shows relative amount of radioactivity in organs and tissues for control gr subjected to intratumoral injection of Na188ReO4. Figure 3. Biodistribution of 188Re under its intratumoral administration with the nanocarrier-based SiNPs- PEG-188Re conjugate. Different colors show relative amount of radioactivity in the organs and tissues of Wistar rats (blood, thyroid gland, lungs, stomach, liver, spleen, kidneys, heart, muscle, tumor) with implanted liver cholangioma RS-1 after 5 min, 3 hours and 24 hours of intratumoral administration of the SiNPs-PEG-188Re complexes.The blue color shows relative amount of radioactivity in organs and tissues for control group subjected to intratumoral injection of Na188ReO4. injection of free 188Re showed immediate washing out of the radionuclide from the tumor area and its further accumulation preferably in the thyroid gland. As shown in Fig. 3, nanoparticle carrier-based SiNPs-PEG-188Re conjugate demonstrated a radically different biodistribution and pharmacokinetics. Here, we did not observe any injection of free 188Re showed immediate washing out of the radionuclide from the tumor area and its further accumulation preferably in the thyroid gland. As shown in Fig. 3, nanoparticle carrier-based SiNPs-PEG-188Re conjugate demonstrated a radically different biodistribution and pharmacokinetics. Here, we did not observe any Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 5 www.nature.com/scientificreports/ Figure 4. Assessment of therapeutic effect. Survival curves for Wistar rats with implanted cholangioma RS-1 after intratumoral injection of the SiNPs-PEG-188Re conjugates providing different doses of radioactivity (37 and 74 MBq) and for control group injected with physiological solutions. Each group was composed of 10 animals. Figure 4. Assessment of therapeutic effect. Survival curves for Wistar rats with implanted cholangioma RS-1 after intratumoral injection of the SiNPs-PEG-188Re conjugates providing different doses of radioactivity (37 and 74 MBq) and for control group injected with physiological solutions. Each group was composed of 10 animals. decrease of the 188Re concentration in the tumor during the first 3 hours (its value was always higher than 30%) and the concentration of the radionuclide in this area was very high (>15%) even after 24 hours. Thus, due to the employment of SiNPs-based carrier, we had very good retention of 188Re over its half-decay time, enabling maximal therapeutic effect. Results and Discussion On the other hand, the migration of 188Re to other organs was very weak, although we recorded certain accumulation of the radionuclude in the thyroid gland (less than 2.8%), blood (less than 0.3%), lungs (less than 0.2%), liver (less than 0.6%), stomach (less than 0.15%) and spleen (less than 0.1%). We believe that a relatively strong signal in the thyroid gland could arise from washing out of some 188Re atoms from SiNPs-PEG-188Re conjugates, similarly to what happened after intravenous injection, while the increase of 188Re concentration in other organs can be due to the interjection of certain number of SiNPs-PEG-188Re conjugates from the tumor to the blood stream. Of particular attention, we can mention a gradual increase of 188Re concen- tration in the kidneys, with a maximal value of 3% reached 24 hours after the injection, which contrasts the data for free rhenium atoms. To summarize, our tests established a very good retention of 188Re in the tumor, which shows promise for successful use of SiNPs as carriers of radionuclides. Therapeutic efficiency using SiNPs-188Re conjugates. The therapeutic efficiency of SiNPs-188Re conjugates was assessed by using Wistar rats with cholangioma RS-1 implanted in the right femoral muscle. We used 30 rats divided into three sub-groups of 10 animals: the 1st and 2nd “signal” groups were intratumorally administered with a single dose of 37 and 74 MBq of NPs carrier-based SiNPs-PEG-188Re conjugates, respec- tively, while the 3rd “control” group were intratumorally injected by 0.1 mL of physiological solutions. Figure 4 shows results of survival tests for these 3 groups. One can see that after 20 days, only 40% of rats from the control group survived, while the survival rate for the 1st and 2nd “signal” groups was 100%. After 30 days, all animals from the control group were dead, while the survival rate for the 1st and 2nd groups was 50% and 72%, respectively. Thus, our experiments clearly demonstrate a remarkable therapeutic effect under intratumoral injection of the SiNPs-PEG-188Re conjugates. It should be noted that the accomplished injection protocol was not optimized to maximize the therapeutic effect. We believe that the efficiency of the treatment can still be improved, e.g., by using 2 and more injections and further optimization of dose radioactivity. Biodegradation, bioelimination and safety of conjugates. As shown in Fig. Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 Methods S th i Synthesis and characterization of SiNPs. SiNPs were prepared by ultra-short (fs) laser fragmenta- tion in water ambience, as described in our recent publications11,13. Briefly, a powder of 0.5 µm Si microparticles, preliminarily prepared by mechanical milling of a Si wafer, was introduced into a glass cuvette at 0.35 g/L and dispersed in deionized water by a sonication bath step for 30 minutes. The dispersed Si microparticles were then fragmented under laser irradiation for one hour using a Yb:KGW (fs) laser (Avesta Inc., Russia, 1030 nm, 270 fs, 1–30 kHz). The laser beam was focused at 1 cm below the water level, while the solution was continually homog- enized by a magnetic stirrer. In addition, the initial concentration was varied in the range 0.15 g/L to 0.5 g/L in order to control the mean size of the NPs according to the protocol proposed in ref.11. To determine the size char- acteristics of nanoparticles, a high-resolution transmission electron microscopy (HR-TEM) system (JEOL JEM 3010) was employed in the imaging and diffraction modes. A droplet of solution containing laser-synthesized nanoparticles was deposited onto the surface of a carbon-coated TEM copper grid, dried and finally examined by the TEM system. Chemical modification and functionalization of SiNPs. Materials. Silane-PEG-COOH (aver- age Mw 5000) were purchased from Biochempeg Scientific Inc. Ethanol and 30% ammonium hydroxide were obtained from Sigma-Aldrich. MilliQ-grade water was used in the preparation of buffers and aqueous solutions. PEG-based coating of SiNPs. The functionalization of laser-synthesized SiNPs with polyethylene glycol was performed as follows. The SiNPs were dispersed in 10 mL of 96% ethanol to a final concentration of 2 g/L. Then, 200 mg of the silane-PEG-COOH solution in 20 mL of ethanol was added to the NPs under continuous stirring at room temperature. Since only dense coating of the NPs by PEG is able to provide stealth properties, we used a large molar excess of the silane-PEG chains in this reaction. The resulting mixture was ultra-sonicated for 1 min, and 1 mL of 3% ammonium hydroxide was quickly drop added into the mixture under vigorous stirring to catalyze the hydrolysis and condensation of the silane groups on the surface of the SiNPs. Then we tested if the pH of the mixture had reached 9–10, and heated this solution for 2.5 h at 70 °C. Results and Discussion 2, intravenous admin- istration with the SiNPs-PEG-188Re conjugates leads to at least a 10-fold higher concentration of 188Re in the kidneys compared to the case of free 188Re atoms (injection of Na188ReO4 solutions). This unambiguously indi- cates that the radionuclide comes to the kidneys in the conjugated state. The pharmacokinetics of 188Re is also completely different in the case of the SiNPs-PEG-188Re conjugates, as the radionuclide comes not immediately, but after some delay (24 hours). t y It should be noted that by themselves, the SiNPs prepared by laser ablation present a highly safe product for biomedical use, as follows from the results of our recent tests in a mouse model12,17. Here, we considered the worst “stress” scenario, when the NPs are bare (non-PEGylated) and should be immediately sequestrated by the reticuloendothelial system. Indeed, after systemic administration, almost 100% of SiNPs immediately accumulated in the liver and the spleen, but in contrast to silica (SiO2) and many other nanomaterials whose accumulation in the liver causes a series of damaging effects (hyperplasia of Kupffer cells, hepatic inflammation, oxidative stress etc.18), we observed only minor inflammation effects which completely disappeared 48 h after the injection, as evidenced by a histopathological investigation of tissues12. At the same time, we recorded stability of blood parameters (aminotrasferases, alkaline phosphatase, bilirubin, cholesterol, etc.)17 and absence of any liver or kidney toxicity, as was confirmed by ALAT, ASAT and the serum creatinine levels, and negligible changes Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 6 www.nature.com/scientificreports/ of oxidative stress parameters including catalase, SOD, GPx activities, Vit A and E7212. Furthermore, SiNPs started to decompose into orthosilicic acid Si(OH)4 soon after the administration and then migrated to kidneys where the decomposition process continued to reduce the NPs size down to a renal glomerular filtration range (<7 nm), rendering possible their excretion with the urine. The complete bioelimination process took 5–7 days, as was controlled by monitoring the Si content in the urine12. In the presented study, SiNPs were additionally PEGylated, which prolonged the circulation time in the organism and radically changed biodistribution, giving access to most organs. Nevertheless, the SiNPs-PEG-188Re conjugates similarly migrated to kidneys where they were supposed to decompose and finally excrete via renal filtration. Results and Discussion It is important that such filtration starts only after some delay (typically, after 24 hours), which should minimize damaging effects in the kidney as the radioac- tivity of 188Re is already much lower after the half-decay time.f y yt y To summarize, we established the merits of the SiNPs as safe and effective carriers of 188Re radionuclide for nuclear therapy. Our study has revealed a quite different biodistribution and pharmacokinetics of the SiNPs-PEG-188Re conjugates compared to the free 188Re atoms (water-dissolved sodium perrhenate Na188ReO4) in a Wistar rat model. Our tests on intravenous administration showed that the NPs-based carrier conjugate can freely circulate in the blood stream and target tumors, while the free 188Re atoms mostly accumulate in the thyroid gland. In addition, intratumoral administration tests evidenced very good retention of the radionuclide in the tumor for more than 24 hours, while the free 188Re rapidly washed out form the tumor under similar con- ditions. Under both administrations, we recorded at least a 24-hour delayed delivery of 188Re to kidney for the SiNPs-PEG-188Re conjugates, which is consistent with gradual decomposition of these complexes; it promises much reduced side effects in the kidneys. Finally, our tests evidenced a considerable increase of the rat survival rate for the groups of animals intratumorally administered with the SiNPs-PEG-188Re conjugates; the radioactive doses of the SiNPs-PEG-188Re conjugates compared to the control group were: (i) 100% for both groups com- pared to 40% after 20 days; (ii) 50% and 72% compared to 0% after 30 days. pt y pt y We believe that the SiNPs-based transport vehicle complex can be considered as a general biodegradable platform for targeted delivery of radionuclides for nuclear therapy. We demonstrated its successful conjugation with 188Re, which is one of the very efficient beta-emitters that can be synthesized in portable 188W/188Re gen- erators, making possible its low-cost fabrication and worldwide distribution19. Nevertheless, other promising diagnostic (64Cu, 68Ga) or therapy (e.g., 90Y) radionuclides can equally be conjugated with biodegradable SiNPs carriers to maximize the efficacy of imaging or therapy. It is also important that crystalline nano-Si is a IV group semiconductorwhose intrinsic properties make possible a series of unique imaging and therapy functionalities, including room temperature photoluminescence for bioimaging20–22, light-induced generation of singlet oxygen for photodynamic therapy of cancer23, and infrared24, radio frequency17, and ultrasound-induced25 cancer hyper- thermia. Results and Discussion In fact, the choice of SiNPs as radionuclide carrier means that all these imaging and therapy modalities can be utilized in parallel with the main nuclear medicine modality to produce image guided therapy, and thus maximize the final therapeutic outcome. One of the most promising tandem therapeutic approaches, we see, is radio frequency-induced hyperthermia using SiNPs as sensitizers of local heating17 which can be used even for the treatment of deep tissues due to good transparency of the body to the RF radiation. As another additional functionality, one can imagine the use of fluorescence properties of SiNPs to track the localization of therapeutic agents in the tumor, although this modality is limited by superficial tissues due to low transmission of light even in the spectral range of relative tissue transparency (750–900 nm). Methods S th i To prevent further hydrolysis and self-aggregation of unreacted silanes, the mixture was cooled down to the room temperature and the NPs were washed by centrifugation (15 min, 5000 g), firstly with pure ethanol and further with water 3 times. After Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 7 www.nature.com/scientificreports/ washing, the product was redispersed in 20 mL of PBS (pH 7.4). The obtained NPs suspension did not contain any aggregates and had long-term colloidal stability at physiological conditions, as was confirmed by Dynamic Light Scattering (Zetasizer Nano ZS, Malvern Instruments, UK). Also, we observed a strong negative zeta-potential after coupling of the PEG-COOH chains with the silicon surface. All the measurements were conducted in MilliQ water. Preparation of the SiNPs-PEG-188Re conjugates. Preparation of radioactive 188Re solutions. 188Re was obtained in the form of a Na188ReO4 solution by elution with saline from a column of a 188Re/188W generator (A. I. Leipunsky Institute of Physics and Power Engineering, Joint Stock Company, Obninsk, Russia). The radio- active purity of the 188Re eluate exceeded 99%. Volume activity of eluate 188Re was 185 MBq·ml−1 (5,0 mCi·ml−1). Conjugation of 188Re with SiNPs carrier and assessment of its efficiency. The conjugation was performed according to the protocol developed in ref.26. Briefly, 5 ml of the SiNPs-PEG complexes having a concentration of 1 mg/ml was added to 1.5 ml of distilled water and 7 mg of ascorbic acid. The ingredients were then mixed for 5 min via ultrasound, to which was added 9.5 mg of SnCl2 2H2O (5.0 mg for Sn2+) in 0.1 ml 0.1 M HCl. After 5-minutes of mixing by ultrasound for 5 min, the 74 MBq 188Re eluate in 0.2 ml of physiological solution was added to it and then mixed once again for 1 hour. Then, a centrifugation step was applied to wash out the unconjugated 188Re elute. Then, the superficial layer was removed, while the precipitate was re-suspended in 4 ml of physiologi- cal solution. The centrifugation procedure was repeated once again to remove the superficial layer. The precip- itate was once again re-suspended in 2 ml physiological solution and examined for radiochemical purity using chromatography methods. The efficiency of conjugation of 188Re with the SiNPs-PEG complex was determined by paper chromatography using a Whatman 1 paper (Germany). 3.0 µl samples of the reaction mixture were applied with a micropipette onto chromatographic paper stripes (10110 mm). Methods S th i The stripes were placed vertically in a beaker, and elution was performed with acetone buffer. The SiNPs-PEG-188Re conjugate stayed at the bot- tom start region of the stripes (Rf = 0.1–0.15), while the free 188ReO4 − ions ascended with the eluent front, until Rf = 0.8–0.9. The amounts of the SiNPs-PEG-188Re and free 188Re were determined by radiometry of chromato- graphic paper stripes using an automatic gamma counter, “Wizard 2480” (Perkin Elmer/Wallac, Finland). Our tests showed that the radiometric outcome of SiNPs-PEG-188Re was 59.2 MBq in 2 ml (29.6 MBq/ml), which corresponds to 80% of the initial radioactivity of the 188Re eluate (Supplementary Fig. 1Sa). We also found that the 188Re-based nanoconjugates were stable for more than 48 hours, while the impact of radionuclide impurities after the washing procedure was less than 5% (Supplementary Fig. 1Sb). Methodology of animal tests. Implantation of RS-1 tumor. All experiments were carried out using female Wistar rats with a body weight of 120–140 g (branch of “Stolbovaya” of the Scientific Center of Biomedical Technologies of Federal Medical-Biological Agency (FMBA)). As a tumor model, we used cholangioma RS-1 (Tumor strain depository of N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation). To obtain an initial sample of the solid tumor, a donor rat with the tumor was sacrificed and the tumor tissue was extracted. Then, the tumor tissue was fragmented, diluted in physiological solution in the proportion of 1:3, and implanted into the right femoral muscle. Each injection was about 100 µg of the tissue in 0.1 ml per animal, which was optimal for cholangioma RS-1, as it ensured 100% implantation of the tumor and its good growth, as well as provided maximal lifetime for the animals. Every three days, the tumor volume of every animal was examined and its volume was assessed. 8–10 days after the implantation, when the tumor volume was about 0.7–0.8 cm3, all animals were subdivided into “signal” and control groups, according to the planned experiments. Intravenous administration of SiNPs-PEG-188Re and free 188Re. 40 Wistar rats were divided into 2 groups (20 rats in each). Rats from the first group were intravenously injected in the jugular vein (under isofluorane anes- thesia) with a single dose of 0.74–1.11 MBq of SiNPs-PEG-188Re in 0.1 ml of physiological solution (12.5 µg of SiNPs-PEG-188Re), which provided 56.8–62.5 µg per kg of animal weight. Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 Methods S th i Rats from the second group were injected in the jugular vein with a single dose of 0.74–1.11 MBq of free Na188ReO4) in 0.1 ml of physiological solution, which provided 3.7–5.55 MBq per kg of animal weight. Intratumoral administration of SiNPs-PEG-188Re and free 188Re. 24 Wistar rats were divided into 2 groups (12 rats in each). Rats from the first group were intratumorally (in the tumor center) injected with a single dose of 0.74–1.11 MBq of SiNPs-PEG-188Re in 0.1 ml of physiological solution (12.5 µg of SiNPs-PEG-188Re), which pro- vided 56.8–62.5 µg per kg of animal weight. Rats from the second group were intratumorally injected in a similar way with a single dose of 0.74–1.11 MBq of free Na188ReO4) in 0.1 ml of physiological solution, which provided 3.7–5.55 MBq per kg of animal weight. Methodology of 188Re biodistribution experiments. Different sub-groups of animals (4 of each) were sacrificed 5 minutes, 1 hour, 3 hours, 24 hours and 48 hours after the injection of both solutions. Samples of key organs and tissues were collected, weighted in electronic balance (“Sartorius”, Germany) and placed in plastic boxes to assess the intensity of ionizing emission from 188Re by a gamma counter (2480 Wizard, Perkin Elmer-Wallac, Finland). 0.1 mL of the Si*NPs-PEG-188Re solution was collected at the moment of NPs administration in mice, and placed in a separate cuvette to serve as a calibration standard. Based on the radiometric data for every observation point, we calculated the relative radioactivity per 1 g of organs or tissues as well as the total radioactivity of all organs or tissues. All experimental procedures and animal care were carried out in accordance with the legislation of Rus Federation, directive 2010/63/EU of European Parliament and EU Council from 22 September 2010, as we Scientific Reports \| (2019) 9:2017 \| https://doi.org/10.1038/s41598-018-38474-7 8 www.nature.com/scientificreports/ with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publ. No. 80–23, revised 1996). Experiments and animal care were performed at the National Medical Research Radiological Center (NMRRC) of the Ministry of Health of the Russian Federation, Obninsk, Russia. All experimental proto- cols were approved by the Scientific Council and Committee of Ethics on Animal experiments of NMRRC. with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publ. No. 80–23, revised 1996). References 103, 193110 (2013). 26. Petriev, V. M. Influence of reactant concentrations and solution acidity on the complexation of 188Re with 1-hydroxyethane-1,1- 25. Sviridov, A. P. et al. Porous Silicon Nanoparticles as Sensitizers for Ultrasonic Hyperthermia. Appl. Phys. Lett. 103, 193110 (2013). 26. Petriev, V. M. Influence of reactant concentrations and solution acidity on the complexation of 188Re with 1-hydroxyethane-1,1- diphosphonic acid Radiochemistry 50 203 207 (2008) 25. Sviridov, A. P. et al. Porous Silicon Nanoparticles as Sensitizers for Ultrasonic Hyperthermia. Appl. Phys. Lett. 103, 193110 (2013). 26 P i V M I fl f i d l i idi h l i f 188R h h d h Acknowledgementsh The authors are grateful to V. Shipunova for her help during the implementation of PEGylization protocol. The authors acknowledge support from the MEPhI Academic Excellence Project (Contract No. 02.a03.21.0005). A.V.K. acknowledges the contribution from the ITMO Cancer AVIESAN (National Alliance for Life Sciences & Health) within the framework of the Cancer Plan (GRAVITY Project). V.Y.T. acknowledges the support from the Russian Science Foundation (project No. 16-13-10145). S.M.D. acknowledges the support from the Russian Foundation for Basic Research (project No. 17-00-00121). Methods S th i Experiments and animal care were performed at the National Medical Research Radiological Center (NMRRC) of the Ministry of Health of the Russian Federation, Obninsk, Russia. All experimental proto- cols were approved by the Scientific Council and Committee of Ethics on Animal experiments of NMRRC. Author Contributions A.V.K. conceived and designed the research. A.A.P., G.T., V.Y.T. and A.V.K. designed laser fabrication setup, fabricated and characterized Si NPs. I.Z. and S.M.D. chemically modified and functionalized Si NPs. V.M.P., V.K.T. and A.A.M. performed all animal tests. V.M.P., V.K.T., A.A.M., A.A.P., G.T., I.Z., S.M.D., A.D.K., S.I., V.Y.T., P.N.P., I.N.Z., A.V.K. analyzed and discussed obtained data. A.V.K. and P.N.P. prepared the manuscript using data from co-authors. A.V.K., I.N.Z. and P.N.P. guided the project. All authors have given approval to the final version of the manuscript. References References 1. Volkert, W. A. & Hoffman, T. J. Therapeutic Radiopharmaceuticals. Chem. Rev. 99, 2269–2292 (1999). fh p p 2. Matsumura, Y. & Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropi accumulation of proteins and the antitumor agent smancs. Cancer Res. 46, 6387–6392 (1986). p g 3. Deyev, S. M., Waibel, R., Lebedenko, E. N., Schubiger, A. P. & Plückthun, A. Design of multivalent complexes using the barnase barstar module. Nature Biotech. 21, 1486–1492 (2003). Yu, M. & Zheng, J. Clearance pathways and tumor targeting of imag 4. Yu, M. & Zheng, J. 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Tamarov, K. P. et al. Radio Frequency Radiation-Induced Hyperthermia Using Si Nanoparticle-Based Sensitizers for Mild Cance Therapy. Sci. Rep. 4, 7034 (2014). h py p 18. Xie, G. et al. Biodistribution and toxicity of intravenously administered silica nanoparticles in mice. Arch. Toxicol. 84, 183–190 (2010). 19. Pillai, M. R., Dash, A. & Knapp, F. F. Rhenium-188: availability from the (188)W/(188)Re generator and status of current applications. Current Radiopharm. 5, 228–43 (2012). pp p 20. Erogbogbo, F. et al. Biocompatible Luminescent Silicon Quantum Dots for Imaging of Cancer Cells. ACS Nano 2, 873–878 (20 21 Gu L et al In Vivo Time Gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles 20. Erogbogbo, F. et al. Biocompatible Luminescent Silicon Quantum Dots for Imaging of Cancer Cells. ACS Nano 2, 873–878 (2008). 21. Gu, L. et al. In Vivo Time-Gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles. Nat. 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https://openalex.org/W2068121450	https://projecteuclid.org/journals/journal-of-applied-mathematics/volume-2013/issue-SI27/Asymptotic-and-Dynamical-Analyses-of-Heat-Transfer-through-a-Rectangular/10.1155/2013/987327.pdf	English	null	Asymptotic and Dynamical Analyses of Heat Transfer through a Rectangular Longitudinal Fin	Journal of applied mathematics	2,013	cc-by	5,311	Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 987327, 8 pages http://dx.doi.org/10.1155/2013/987327 Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 987327, 8 pages http://dx.doi.org/10.1155/2013/987327 Hindawi Publishing Corporation Journal of Applied Mathematics Volume 2013, Article ID 987327, 8 pages http://dx.doi.org/10.1155/2013/987327 C. Harley Centre for Differential Equations, Continuum Mechanics and Applications, School of Computational and Applied Mathematics, University of the Witwatersrand, Johannesburg, Private Bag 3, Wits 2050, South Africa Correspondence should be addressed to C. Harley; charis.harley@wits.ac.za Correspondence should be addressed to C. Harley; charis.harley@wits.ac.za Received 12 November 2012; Accepted 19 November 2012 Academic Editor: Fazal M. Mahomed Copyright © 2013 C. Harley. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The steady heat transfer through a rectangular longitudinal fin is studied. The thermal conductivity and heat transfer coefficient are assumed to be temperature dependent making the resulting ordinary differential equation (ODE) highly nonlinear. An asymptotic solution is used as a means of understanding the relationship between key parameters. A dynamical analysis is also employed for the same purpose. 1. Introduction has yet to be fully understood. It is the purpose of asymptotic solutions to reveal the dominant physical mechanisms of the model. In Moitsheki and Harley [7] and Harley and Moitsheki [8], the impact of the thermogeometric parameter was discussed with regards to its proportionality to the length of the fin, 𝐿. It was noticed that for small values of M that there seemed to be unstable heat transfer in the fin—this was thought to be related to the fact that M ∝ 𝐿. Investigating an asymptotic solution to the steady heat transfer in a rectangular longitudinal fin allows us to validate this relationship and more firmly establish the importance of the length of the fin. A dynamical system analysis was conducted in Harley and Moitsheki [8], however, not to the extent conducted here. In this work, the analysis in [8] is improved upon and the behaviour, particularly a the tip of the fin, more meticulously investigated and documented. This dynamical systems analysis also functions as a means of investigating the effect of the thermo-geometric parameter. In this paper we consider the temperature profile in a longitudinal rectangular fin attached to a stationary base surface. Fins are defined as extended surfaces used to enhance the heat dissipation from a hot surface [1, 2]. They are used in multiple different contexts such as in air-conditioning, air-cooled craft engines, refrigeration, and the cooling of computer processors and oil carrying pipe lines. There are many documented mathematical models which describe the heat transfer in fins of different geometries and profiles with a variety of boundary conditions [1]. Many methods have been implemented as a means of obtaining solutions to such equations, analytical and numerical alike. In much research both the thermal conductivity and heat transfer coefficient have been considered as constants (see e.g., [3–5]). For engineering applications and physical phenomena thermal conductivity of a fin is assumed to be linearly dependent on temperature (see e.g., [6]). In this paper we have chosen the thermal conductivity as a linear function of the temperature and the heat transfer coefficient as a nonlinear function of the temperature. 2. Model As far as the author knows, there is no or very little work which has been done on obtaining asymptotic solutions to a problem of the form presented here. An investigation of such solutions is of value given the prevalence of many parameters whose impact and relationship with each other We consider a rectangular longitudinal one-dimensional fin with a cross-sectional area 𝐴𝑐. The perimeter of the fin is denoted by 𝑃and the length of fin by 𝐿. The fin is attached to a fixed base surface of temperature 𝑇𝑏and extends into a fluid of temperature 𝑇𝑎. The energy balance for a longitudinal 2 Journal of Applied Mathematics fin is given by an ordinary differential equation (ODE) [1] as follows: exponent. The thermal conductivity coefficient can be written in dimensionless variables as 𝑘(𝜃) = 1 + 𝛽𝜃with 𝛽= 𝜆(𝑇𝑏− 𝑇𝑎), where 𝑇𝑏is the dimensional fin base temperature and 𝑇𝑎 is the dimensional ambient temperature. From these choices of 𝑘(𝜃) and ℎ(𝜃), we obtain the one-dimensional nonlinear heat conduction equation: 𝐴𝑐 𝑑 𝑑𝑋(𝐾(𝑇) 𝑑𝑇 𝑑𝑋) = 𝑃𝐻(𝑇) (𝑇−𝑇𝑎) , 0 ≤𝑋≤𝐿, (1) where 𝐾and 𝐻are the nonuniform thermal conductivity and heat transfer coefficient both of which depend on the temperature (see, e.g., [3, 4, 9, 10]). The fin length is measured from the tip to the base as shown in Figure 1 (see also, [1, 3, 4]). An insulated fin at one end with the base temperature at the other leads to the following boundary conditions [1]: 𝑑 𝑑𝑥[(1 + 𝛽𝜃) 𝑑𝜃 𝑑𝑥] = M2𝜃𝑛+1, 0 ≤𝑥≤1. (7) (7) The dimensionless boundary conditions become The dimensionless boundary conditions become 𝑑𝜃 𝑑𝑥 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑥=0 = 0 at the fin tip, 𝜃(1) = 1, at the base. (8) (8) 𝑇(𝐿) = 𝑇𝑏, 𝑑𝑇 𝑑𝑋 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑋=0 = 0. (2) (2) These conditions ensure an insulated fin tip and a con- stant base temperature. The heat transfer coefficient may be given as the power law [3] 𝐻(𝑇) = ℎ𝑏( 𝑇−𝑇𝑎 𝑇𝑏−𝑇𝑎 ) 𝑛 (3) 3. Asymptotic Analysis for Steady Heat Transfer (3) 3.1. M ≪𝜖. In this section we will assume that M ≪𝜖 (i.e., the thermogeometric parameter is very much smaller than some small parameter 𝜖) as a means of investigating the nature of the solutions of the steady state case of (7) as follows: as is done for most industrial applications. Furthermore, the thermal conductivity of the fin may be assumed to vary linearly with temperature for many engineering applications [3, 6] and as such may be written as the following: 𝑑 𝑑𝑥[(1 + 𝛽𝜃) 𝑑𝜃 𝑑𝑥] = M2𝜃𝑛+1, 0 ≤𝑥≤1, 𝜏≥0, (9) 𝐾(𝑇) = 𝑘𝑎(1 + 𝜆(𝑇−𝑇𝑎)) . (4) (4) (9) In the above equations, 𝐻is the heat transfer, ℎ𝑏indi- cates the heat transfer at the base of fin, 𝐾is the thermal conductivity, 𝑘𝑎is the thermal conductivity of the fin at the ambient temperature, and the exponent 𝑛is defined to be dependent upon the heat transfer mode. Here the exponent represents laminar film boiling or condensation when 𝑛= −1/4, laminar natural convection when 𝑛= 1/4, turbulent natural convection 𝑛= 1/3, nucleate boiling when 𝑛= 2, radiation when 𝑛= 3, and 𝑛vanishes for constant heat transfer coefficient. The constant 𝑛may vary between −6.6 and 5, however, in most practical applications it lies between 3 and −3 [11]. where 𝑓(𝑥) = 1 with boundary conditions 𝜕𝜃 𝜕𝑥 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑥=0 = 0 at the fin tip, 𝜃(1) = 1, at the base. (10) (10) We assume a solution of the form 𝜃(𝑥) = 𝜃0(𝑥) + M2𝜃1(𝑥) + M4𝜃2(𝑥), where M → 0. After substitution the term −M2𝜃(𝑥)𝑛+1 is expanded using a series expansion so that −M2𝜃(𝑥)𝑛+1 = −M2 −(𝑛+ 1) 𝜃1 (𝑥) M4 −(1 2 (𝑛2 + 𝑛) 𝜃1(𝑥)2 + (𝑛+ 1) 𝜃2 (𝑥)) M6 + 𝑂(M7) . 3. Asymptotic Analysis for Steady Heat Transfer (11) Introducing the following dimensionless variables: 𝑥= 𝑋 𝐿, 𝜃= 𝑇−𝑇𝑎 𝑇𝑏−𝑇𝑎 , ℎ= 𝐻 ℎ𝑏 , 𝑘= 𝐾 𝑘𝑎 , M2 = 𝑃ℎ𝑏𝐿2 𝑘𝑎𝐴𝑐 , (5) (11) (11) (5) In this manner, we are able to separate (11) according to powers of M and obtain the following system of equations: 𝑂(M0) : 𝛽(𝑑𝜃0 𝑑𝑥) 2 + 𝑑2𝜃0 𝑑𝑥2 (1 + 𝛽𝜃0) , (12) 𝑂(M2) : −1 + 2𝛽𝑑𝜃0 𝑑𝑥 𝑑𝜃1 𝑑𝑥+ 𝛽𝜃1 𝑑2𝜃0 𝑑𝑥2 + 𝑑2𝜃1 𝑑𝑥2 (1 + 𝛽𝜃0) , (13) 𝑂(M4) : −(𝑛+ 1) 𝜃1 + 𝛽(𝑑𝜃1 ) 2 + 2𝛽𝑑𝜃0 𝑑𝜃2 𝑂(M0) : 𝛽(𝑑𝜃0 𝑑𝑥) 2 + 𝑑2𝜃0 𝑑𝑥2 (1 + 𝛽𝜃0) , (12) 𝑂(M0) : 𝛽(𝑑𝜃0 𝑑𝑥) 2 + 𝑑2𝜃0 𝑑𝑥2 (1 + 𝛽𝜃0) , (12) 𝑂(M2) : −1 + 2𝛽𝑑𝜃0 𝑑𝑥 𝑑𝜃1 𝑑𝑥+ 𝛽𝜃1 𝑑2𝜃0 𝑑𝑥2 + 𝑑2𝜃1 𝑑𝑥2 (1 + 𝛽𝜃0) , (13) 𝑂(M4) : −(𝑛+ 1) 𝜃1 + 𝛽(𝑑𝜃1 𝑑𝑥) 2 + 2𝛽𝑑𝜃0 𝑑𝑥 𝑑𝜃2 𝑑𝑥 + 𝛽𝜃2 𝑑2𝜃0 𝑑𝑥2 + 𝛽𝜃1 𝑑2𝜃1 𝑑𝑥2 + 𝑑2𝜃2 𝑑𝑥2 (1 + 𝛽𝜃0) . (14) allows (1) to be reduced to the following nondimensional partial differential equation: (12) 𝑑 𝑑𝑥[𝑘(𝜃) 𝑑𝜃 𝑑𝑥] = M2𝜃ℎ(𝜃) , 0 ≤𝑥≤1. (6) 𝑂(M2) : −1 + 2𝛽𝑑𝜃0 𝑑𝑥 𝑑𝜃1 𝑑𝑥+ 𝛽𝜃1 𝑑2𝜃0 𝑑𝑥2 + 𝑑2𝜃1 𝑑𝑥2 (1 + 𝛽𝜃0) , (13) 𝑂(M2) : −1 + 2𝛽𝑑𝜃0 𝑑𝑥 𝑑𝜃1 𝑑𝑥+ 𝛽𝜃1 𝑑2𝜃0 𝑑𝑥2 + 𝑑2𝜃1 𝑑𝑥2 (1 + 𝛽𝜃0) , (6) In (6) 𝜃is defined as the dimensionless temperature and 𝑥as the dimensionless spatial variable, where 𝑘(𝜃) is the dimensionless thermal conductivity, ℎ(𝜃) is the heat transfer coefficient, and M is termed the thermo-geometric parameter. In dimensionless variables the heat transfer coef- ficient is chosen as ℎ(𝜃) = 𝜃𝑛with 𝑛termed as the 𝑂(M4) : −(𝑛+ 1) 𝜃1 + 𝛽(𝑑𝜃1 𝑑𝑥) 2 + 2𝛽𝑑𝜃0 𝑑𝑥 𝑑𝜃2 𝑑𝑥 + 𝛽𝜃2 𝑑2𝜃0 𝑑𝑥2 + 𝛽𝜃1 𝑑2𝜃1 𝑑𝑥2 + 𝑑2𝜃2 𝑑𝑥2 (1 + 𝛽𝜃0) . (14) (14) 3 Journal of Applied Mathematics 3 Fin base Fin tip 𝑊 𝐿 𝑋= 𝐿 𝑋= 0 𝛿 Figure 1: Schematic representation of a longitudinal fin with a rectangular profile. Fin tip Figure 1: Schematic representation of a longitudinal fin with a rectangular profile. Given these expansions the boundary conditions (10) become As such, this asymptotic solution would not be valid for the instance when 𝛽= −1. 3. Asymptotic Analysis for Steady Heat Transfer 𝜕𝜃0 𝜕𝑥 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑥=0 = 𝜕𝜃1 𝜕𝑥 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑥=0 = 𝜕𝜃2 𝜕𝑥 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨𝑥=0 = 0 at the fin tip, (15) 𝜃0 (1) = 1, 𝜃1 (1) = 0, 𝜃2 (1) = 0 at the base. (16) 3.2. Boundary Layer Analysis: M ≫𝜖. In this section we will consider the case where M is large and as such define it as M = 1/𝜖for 𝜖small. Thus we can write (9) as the following: (16) Solving these equations we firstly obtain 𝜃0(𝑥) = 1 given that the boundary condition given in (16) needs to be satisfied. In Moitsheki and Harley [7], the solutions for small M flatten and the temperature at 𝑥= 0 tends to 1. As such the solution 𝜃0(𝑥) = 1 is a reasonable approximation. We solve (13) and (14) via MATHEMATICA and obtain the following solution: 𝜖2 𝑑 𝑑𝑥(𝑓(𝑥) (1 + 𝛽𝜃) 𝑑𝜃 𝑑𝑥) = 𝜃𝑛+1. (18) (18) If 𝜖is small as assumed then the solution can be approximated as 𝜃≈0. Since this solution, in and of itself, is not useful in understanding our problem we transform variables from 𝑥∈[0, 1] into 𝑋∈[0, ∞] through the transformation 𝑋= (1 −𝑥)/𝛿. Here we define 𝛿as the distance from the point where the temperature ceases to be constant, say 𝑥⋆, and starts to increase as we move along the length of the fin, to 𝑥𝑁= 1. We assume the solution to be constant except for the range [𝑥⋆, 𝑥𝑁], where 𝛿= 𝑥𝑁−𝑥⋆. This transformation of the independent variable gives If 𝜖is small as assumed then the solution can be approximated as 𝜃≈0. Since this solution, in and of itself, is not useful in understanding our problem we transform variables from 𝑥∈[0, 1] into 𝑋∈[0, ∞] through the transformation 𝑋= (1 −𝑥)/𝛿. Here we define 𝛿as the distance from the point where the temperature ceases to be constant, say 𝑥⋆, and starts to increase as we move along the length of the fin, to 𝑥𝑁= 1. We assume the solution to be constant except for the range [𝑥⋆, 𝑥𝑁], where 𝛿= 𝑥𝑁−𝑥⋆. This transformation of the independent variable gives 𝜃(𝑥) 𝜃(𝑥) = 1 + ( 𝑥2 −1 2 (𝛽+ 1)) M2 + ( (𝑥2−1) ((𝑥2−5) (1+𝑛(1+𝛽)) −2𝛽(𝑥2−1)) 24(1+𝛽)3 ) M4. 3. Asymptotic Analysis for Steady Heat Transfer (17) 𝜖2 𝛿2 𝑑 𝑑𝑋(𝑓(1 −𝛿𝑋) (1 + 𝛽Θ) 𝑑Θ 𝑑𝑋) = Θ𝑛+1, (19) (19) (17) This solution verifies the notion that the length of the fin has a proportional relationship to the thermogeometric parameter. In assuming that M ≪𝜖, we found an asymptotic solution which when plotted has a high temperature at the fin tip—see Figure 2. Simplistically our results seem to indicate that when the thermo-geometric parameter has a small value, then the fin is short which only increases the temperature profile of the entire fin. where Θ = Θ(𝑋) and the boundary conditions become Θ(0) = 1 and Lim𝑋→∞Θ(𝑋) = 0. In order to obtain a nontrivial solution, we require that at least two leading-order terms in this equation have the same order of magnitude. Balancing the terms we find that 𝜖2 𝛿2 = 1, (20) (20) i It needs to be noted that the solution does not allow for the case 𝛽= −1, even though the assumption that one has a decreasing gradient seems a plausible one. Were one to con- duct the same calculations done to obtain the above solution (17) with an initial choice of 𝛽= −1, one would obtain a contradiction when separating according to powers of the thermo-geometric parameter such that for M2 : −1 = 0. which means that 𝜖 = 𝛿 = 1/M. In this manner we have obtained a relationship between the thermo-geometric parameter and 𝛿. 3.3. Remark. The method of asymptotics is effectively used here to inspect the behaviour of the solution for small M. In Journal of Applied Mathematics 0 0.2 0.4 0.6 0.8 1 0.9975 0.998 0.9985 0.999 0.9995 1 Θ 𝑋 Figure 2: Plot of the asymptotic solution (17) with M = 0.1 and 𝛽= 𝑛= 1. Journal of Applied Mathematics Figure 2: Plot of the asymptotic solution (17) with M = 0.1 and 𝛽= 𝑛= 1. with 𝑢(1) = 1 and V(0) = 0. We now use these equations and obtain the following equilibrium points: previous work (see [7, 8]) the thermo-geometric parameter was suspected to be proportional to the length of the fin, 𝐿. It was noticed that for small values of M there seemed to be unstable heat transfer in the fin—this was thought to be related to the fact that M ∝𝐿. 3. Asymptotic Analysis for Steady Heat Transfer Through our asymptotic analysis, we are now able to establish that for M ≪𝜖the temperature at the fin tip (i.e., 𝑥= 0) is quite high at 0.999975. This makes sense given that the value attached to 𝜃0(𝑥) is one. This behaviour of the solution also supports the idea that M ∝𝐿given that the overall temperature profile would increase if the fin were short. 𝑧1 = (0, 0) , 𝑧2 = (−1 𝛽, −M(−1)(𝑛+1)/2 𝛽(𝑛+2)/2 ) , 𝑧3 = (−1 𝛽, M(−1)(𝑛+1)/2 𝛽(𝑛+2)/2 ) , (22) (22) and nullclines which are curves drawn in the phase portrait along which one of the state variables does not change in time i For the case where M ≫ 𝜖we found an inverse relationship between the thermo-geometric parameter and the distance 𝛿defined above. This is again another indication of the proportional relationship between this parameter and the length of the fin, 𝐿, given that for large M, 𝛿is small which implies that 𝐿must be large. The reason for this is twofold. Only when 𝐿is large (1) would the temperature at the tip be ≪𝜖and (2) would the temperature gradient →0 for a long enough period of time to allow 𝛿= 𝑥𝑁−𝑥⋆to be small. V = 𝑢3/2, V = −𝑢3/2. (23) (23) The Jacobian matrix 𝐽for the system (21) is given by 𝐽(𝑢, V) = [ 𝛽V 1 + 𝑢𝛽 M2 (𝑛+ 1) 𝑢𝑛 −2𝛽V ] . (24) (24) To be able to do a phase plane analysis of the relevant equation we need to linearise the system. This is done via the calculation of the Jacobian (24), where the elements in 𝐽need to be linear with respect to V. We now consider the Jacobian (24) for the equilibrium points given in (22). Firstly, we consider the point 𝑧1 = (0, 0) which produces the following Jacobian equation: In order to investigate the dynamics of a system in which M ∝ 𝐿, we consider a dynamical system analysis. The purpose of this is to verify the results obtained thus far and also investigate the findings of [7, 8] with regards to possibly thermally unstable behaviour. 𝐽(𝑢1, V1) = [0 1 0 0] . (25) 4. Dynamical System Analysis (25) As a means of further investigating the behaviour of the system we conduct a dynamical system analysis. We do this by rewriting (7) as a system of first-order ordinary differential equations through the transformations 𝜃(𝑥) = 𝑢(𝑥) and V(𝑥) = 𝑑𝜃/𝑑𝑥such that 𝑑2𝜃/𝑑𝑥2 = V(𝑑V/𝑑𝜃). This gives the following system: This in turn produces the eigenvalues 𝜆(1) 1 = 0, 𝜆(1) 2 = 0. (26) (26) The phase diagrams produced for this equilibrium point—to be seen in Figure 3—indicate that the point is a centre which is neutrally stable for 𝑢< 0 joined to a saddle which is unstable for 𝑢> 0. Furthermore, given that the Jacobian matrix has zero eigenvalues this critical point is a nonhyperbolic and degenerate singular point. More precisely, the linear part of the vector field can now be said to be doubly degenerate and the reduced system on the center manifold is 𝑑V 𝑑𝑥= M2𝑢𝑛+1 −𝛽V2, 𝑑𝑢 𝑑𝑥= V (1 + 𝛽𝑢) , (21) (21) Journal of Applied Mathematics 5 0 0.2 0.4 0 0.2 0.4 𝑣 𝑢 𝑢 𝑉 −0.2 −0.4 −0.2 −0.4 (a) 0 0.2 0.4 0 0.2 0.4 𝑣 𝑢 𝑢 𝑉 −0.2 −0.4 −0.2 −0.4 (b) Figure 3: Plot of the phase trajectories for system (25) with M = 1 (a) and M = 10 (b) with 𝛽= 𝑛= 1. 0 0.2 0.4 0 0.2 0.4 𝑣 𝑢 𝑢 𝑉 −0.2 −0.4 −0.2 −0.4 (b) 0 0.2 0.4 0 0.2 0.4 𝑣 𝑢 𝑢 𝑉 −0.2 −0.4 −0.2 −0.4 (a) 𝑣 𝑣 (b) (a) Figure 3: Plot of the phase trajectories for system (25) with M = 1 (a) and M = 10 (b) with 𝛽= 𝑛= 1. defined by 𝜙= 𝜂cos 𝜓, 𝜌= 𝜂sin 𝜓—the following vector field is obtained [12]: two-dimensional [12]. The classification and unfolding of this (nilpotent) type was done simultaneously and independently by Takens [13] and Bogdanov [14]. The normal form of the case (25) can be obtained as per Takens [13] and done in Guckenheimer and Holmes [12] and written as ̇𝜂= 𝜂2 (−𝑏cos3 𝜓+ 2 cos2 𝜓sin 𝜓 −sin3 𝜓+ 𝑏cos 𝜓sin2 𝜓⋅⋅⋅) , ̇𝑢= V + 𝑎𝑢2, ̇V = 𝑏𝑢2. (27) ̇𝜓= 𝜂(𝑏cos3 𝜓sin 𝜓−3 cos 𝜓sin2 𝜓+ 𝑏cos2 𝜓sin 𝜓⋅⋅⋅) . 4. Dynamical System Analysis 6 Journal of Applied Mathematics 6 0 0.5 1 1.5 0 0.5 −0.5 −1 −1.5 −0.5 −1 −1.5 𝑣 𝑢 𝑢 𝑉 (a) 0 5 10 0 0.5 −10 −5 −0.5 −1 −1.5 𝑣 𝑢 𝑢 𝑉 (b) Figure 4: Plot of the phase trajectories for system (25) with M = 0.1 (a) and M = 10 (b) with 𝛽= 𝑛= 1. 0 5 10 0 0.5 −10 −5 −0.5 −1 −1.5 𝑣 𝑢 𝑢 𝑉 (b) 0 0.5 1 1.5 0 0.5 −0.5 −1 −1.5 −0.5 −1 −1.5 𝑣 𝑢 𝑢 𝑉 (a) 𝑣 (a) (b) Figure 4: Plot of the phase trajectories for system (25) with M = 0.1 (a) and M = 10 (b) with 𝛽= 𝑛= 1. We now turn to our other two equilibrium points. Evaluating 𝐽at 𝑧2 produces the following: In the first instance we turn to the Hartman-Grobman theorem which states that if 𝐽= 𝐷𝑓(𝑥) has no zero or purely imaginary eigenvalues, then there is a homeomorphism ℎ defined on some neighbourhood 𝑈of 𝑥in R𝑛locally taking orbits of the nonlinear flow (21) to those of the linear flow (31)–(33) [12, 15]. The homeomorphism preserves the sense of orbits and can also be chosen to preserve parametrization by time. 𝐽(𝑢2, V2) = [−M(−1)(𝑛+1)/2𝛽(−𝑛/2) 0 M2 (𝑛+ 1) (−𝛽)−𝑛 2M(−1)(𝑛+1)/2𝛽(−𝑛/2)] , (31) with eigenvalues y For case (a) where 𝐽has no eigenvalues with zero real part, 𝑥is called a hyperbolic or nondegenerate fixed point and the asymptotic behaviour of solutions near it (and hence its stability type) is determined by the linearisation. For this to be the case we must have that (𝑛+ 1)/2 is even. In Figure 4 we obtain eigenvalues which produce unstable saddle points; these results are similar to those obtained in Harley and Moitsheki [8] where the system was found to have two unstable saddle points for 𝑛= 1. As M increases this behaviour becomes more visually apparent, however when the thermo-geometric parameter takes on a small value it becomes more difficult to analyse the stability of the system visually. 𝜆(2) 1 = −M(−1)(𝑛+1)/2𝛽(−𝑛/2), 𝜆(2) 2 = 2M(−1)(𝑛+1)/2𝛽(−𝑛/2). 4. Dynamical System Analysis (29) (27) ) (29) Division of the vector field by 𝜂will leave the phase portrait of (29) unaffected (except possibly at 𝜂= 0) and since the common factor 𝜂occurs in both components we may consider the “divided out” vector field given by the following: Division of the vector field by 𝜂will leave the phase portrait of (29) unaffected (except possibly at 𝜂= 0) and since the common factor 𝜂occurs in both components we may consider the “divided out” vector field given by the following: Takens [13] went on to show that (27) determines the local topological type of any vector field ̇𝑢= V + 𝑎𝑢2 + O (󵄨󵄨󵄨󵄨𝑥, 𝑦󵄨󵄨󵄨󵄨 3) , ̇V = 𝑏𝑢2 + O (󵄨󵄨󵄨󵄨𝑥, 𝑦󵄨󵄨󵄨󵄨 3) (28) ̇𝜂= 𝜂(−𝑏cos3 𝜓+ 2 cos2 𝜓sin 𝜓 −sin3 𝜓+ 𝑏cos 𝜓sin2 𝜓⋅⋅⋅) , ̇𝜂= 𝜂(−𝑏cos3 𝜓+ 2 cos2 𝜓sin 𝜓 (28) −sin3 𝜓+ 𝑏cos 𝜓sin2 𝜓⋅⋅⋅) , provided that 𝑏̸= 0. The predominant tool is a technique called blowing-up. Singular changes to the coordinates are introduced which expand the degenerate fixed points into circles containing a finite number of fixed points. If these are hyperbolic after the first blow-up, then the local flow near the circle, and, hence, near the original fixed point is stable with respect to higher-order terms. In our case three blow-ups are required for the transformed vector field to become stable. The first transformation is to polar coordinates given by 𝑢= 𝑟cos 𝜃, V = 𝑟sin 𝜃, and still leaves us with a degenerate vector field. After two further blow-ups however—(𝑟, 𝜃) →(𝜌, 𝜙) defined by 𝜃= 𝜌cos 𝜙, 𝑟= 𝜌sin 𝜙, and (𝜌, 𝜙) →(𝜂, 𝜓) ̇𝜓= 𝑏cos3 𝜓sin 𝜓−3 cos 𝜓sin2 𝜓+ 𝑏cos2 𝜓sin 𝜓⋅⋅⋅, (30) ̇𝜓= 𝑏cos3 𝜓sin 𝜓−3 cos 𝜓sin2 𝜓+ 𝑏cos2 𝜓sin 𝜓⋅⋅⋅, (30) , (30) which has six hyperbolic fixed points at 𝜂= 0, 𝜓= 0, 𝜋/2, 𝜋, 3𝜋/2, and 𝜓= arctan(2𝑏/3). Hence the flow of (30) is stable to small (higher-order) perturbations and consequently the flow of (29) near 𝜂= 0 is similarly stable. If we now “blow- down” three times (𝜂, 𝜓) →(𝜌, 𝜙) →(𝑟, 𝜃) →(𝑥, 𝑦), we are able to conclude that the flow of (27) near the degenerate fixed point (𝑥, 𝑦) = (0, 0) is indeed stable with respect to the addition of (small) higher-order terms provided that 𝑏̸= 0. 4. Dynamical System Analysis (32) (32) When we consider 𝐽at 𝑧3 we find that When we consider 𝐽at 𝑧3 we find that 𝐽(𝑢3, V3) = [M(−1)(𝑛+1)/2𝛽(−𝑛/2) 0 M2 (𝑛+ 1) (−𝛽)−𝑛 −2M(−1)(𝑛+1)/2𝛽(−𝑛/2)] , (33) ] (33) where the eigenvalues are where the eigenvalues are If however, as in the case of (b), any one of the eigenvalues has zero real part, then stability cannot be determined by linearisation. Thus, given that the linearised system does not describe the nonlinear system, we consider phase diagrams for specific values of our parameters as a means of under- standing the dynamics of the system. If we choose 𝑛= 2 we end up with the case in question and obtain the following equilibrium points: 𝑧1 = (0, 0), 𝑧2 = (−1, 𝑖) and 𝑧3 = (−1, −𝑖), where 𝑖= √−1. In Figure 5 we see the phase diagrams for the system (25) for M = 0.1, 1, and 10. The real parts of the points 𝑧2 and 𝑧3 lie on top of each other to the left of the origin. 𝜆(3) 1 = −2M(−1)(𝑛+1)/2𝛽(−𝑛/2), 𝜆(3) 2 = M(−1)(𝑛+1)/2𝛽(−𝑛/2). (34) (34) For these two equilibrium points we find that we have two cases to consider: (a) eigenvalues with no zero or purely imaginary eigen- values, (a) eigenvalues with no zero or purely imaginary eigen- values, (b) eigenvalues which are purely imaginary. Journal of Applied Mathematics 7 7 pp 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑉 𝑢 (a) 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑉 𝑢 (b) 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑉 𝑢 (c) Figure 5: Plot of the phase trajectories for system (25) with M = 0.1 (a), M = 1 (b), and M = 10 (c) with 𝛽= 1 and 𝑛= 2. 5. Concluding Remarks In this paper, we were able to obtain an asymptotic solution which clarifies the behaviour of the system when M ≪𝜖. The solution curve also verified our suspicions that the thermo- geometric parameter is proportional to the length of the fin. Furthermore, when we consider large M—that is, when M ≫ 𝜖—we found that M ∝𝐿via a boundary layer analysis. This justifies the work conducted in [7, 8] with regards to this relationship.f [6] A. Aziz and T. Y. Na, “Periodic heat transfer in fins with variable thermal parameters,” International Journal of Heat and Mass Transfer, vol. 24, no. 8, pp. 1397–1404, 1981. [7] R. J. Moitsheki and C. Harley, “Transient heat transfer in longitudinal fin of various profiles with temperature-dependent thermal conductivity and heat transfer coefficient,” Pramana: Journal of Physics, vol. 77, no. 3, pp. 519–532, 2011. p As a means of further investigating the effects of M on the dynamics of the system we conducted a dynamic system analysis. In doing so we found that when the temperature gradient is zero—this is true specifically at the tip of the fin where 𝜃󸀠\|𝑥=0 = 0—the system is in fact stable, with respect to the addition of (small) higher-order terms, even though the behaviour exhibited may indicate otherwise. Furthermore, the behaviour of the other equilibrium points is that of unstable saddle points when 𝑛is odd, which according to the Hartman-Grobman theorem can be ascertained via a consideration of the linearised system in this instance. The system maintains unstable (or neutrally stable when M = 0.1) behaviour when 𝑛is even—the cases of M = 4 and 6 were also considered—as can be witnessed when considering the phase diagrams. Lastly, we find that while it is quite a complex endeavour to investigate the stability at the tip of the fin—that is, when 𝜃󸀠= 0—it is even more complicated to consider the dynamics of the system when M becomes small, that is, when the fin is short. [8] C. Harley and R. J. Moitsheki, “Numerical investigation of the temperature profile in a rectangular longitudinal fin,” Nonlinear Analysis: Real World Applications, vol. 13, no. 5, pp. 2343–2351, 2012. [9] R. J. Moitsheki, T. Hayat, and M. Y. Malik, “Some exact solutions of the fin problem with a power law temperature-dependent thermal conductivity,” Nonlinear Analysis: Real World Applica- tions, vol. 11, no. 5, pp. 3287–3294, 2010. [10] R. J. Acknowledgments [14] V. I. Bogdanov, “Lectures on bifurcations and versal families,” Russian Mathematical Surveys, vol. 27, no. 5, pp. 54–123, 1972. C. Harley, thanks H. Ockendon, C. Hall, and C. Please for useful discussion and acknowledges support from the National Research Foundation, South Africa, under Grant no. 79184. Furthermore, this paper was made possible (in part) by a grant from the Carnegie Corporation of New York. The statements made and views expressed are, however, solely the responsibility of the author. [15] P. Hartman, Ordinary Differential Equations, John Wiley & Sons, New York, NY, USA, 1964. [16] B. Hasselblatt, “Phase portraits of linear systems,” http://ci- teseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.184.8666. Journal of Applied Mathematics 8 as 𝑡→∞the solutions may not remain close or actually tend to the equilibrium points we cannot claim that the critical points are asymptotically stable either. [4] F. Khani, M. Ahmadzadeh Raji, and H. Hamedi-Nezhad, “A series solution of the fin problem with a temperature- dependent conductivity,” Communications in Nonlinear Science and Numumerical Simulation, vol. 14, no. 7, pp. 3007–3017, 2009. [5] A. A. Joneidi, D. D. Ganji, and M. Babaelahi, “Differential trans- formation method to determine fin efficiency of convective straight fins with temperature dependent thermal conductivity,” International Communications in Heat and Mass Transfer, vol. 36, pp. 757–762, 2009. 5. Concluding Remarks Moitsheki, “Steady one-dimensional heat flow in a lon- gitudinal triangular and parabolic fin,” Communications in Nonlinear Science and Numerical Simulation, vol. 16, no. 10, pp. 3971–3980, 2011. [11] H. C. ¨Unal, “The effect of the boundary at a fin tip on the performance of the fin with and without internal heat generation,” International Journal of Heat and Mass Transfer, vol. 31, no. 7, pp. 1483–1496, 1998. [12] J. Guckenheimer and P. Holmes,Nonlinear Oscillations, Dynam- ical Systems, and Bifurcations of Vector Fields, vol. 42 of Applied Mathematical Sciences, Springer, New York, NY, USA, 1983.i [13] F. Takens, “Singularities of vector fields,” Publications Math´ematiques de l’IH´ES, no. 43, pp. 47–100, 1974. 4. Dynamical System Analysis 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑢 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑉 𝑢 𝑉 (a) (b) 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑉 𝑢 (c) (a) (b) 0 0.5 1 1.5 0 5 10 −0.5 −1 −1.5 −10 −5 𝑉 𝑢 (c) F g 5 Pl t f th h t j t i f t (25) ith M 0 1 ( ) M 1 (b) d M 10 ( ) ith 𝛽 1 d (a) (b) (c) Plot of the phase trajectories for system (25) with M = 0.1 (a), M = 1 (b), and M = 10 (c) with 𝛽= 1 and 𝑛= 2. When M = 1 we notice that 𝑧2 and 𝑧3 seem to create a centre (neither attracting nor repelling) for 𝑢< −1 attached to a saddle point for 𝑢> −1. However, when M increases to 10 the points 𝑧2 and 𝑧3 are now positioned on top of each other which causes the system to behave in a manner indicative of a plane of equilibria. This means that nothing moves and every solution curve is a point with the form 𝑥= 𝑥0 and 𝜃= 𝜃0. For M = 0.1 the dynamics of 𝑧2 and 𝑧3 are akin to that of a saddle point. The representation of the equilibrium point 𝑧1 = (0, 0) seems to be influenced by the points 𝑧2 and 𝑧3 and behaves in a manner corresponding with what is seen when considering some linear systems [16]. In fact what we see visually corresponds to the case when 𝑧1 only has one linearly independent eigenvector in which case the line determined by the eigenvector consists of fixed points, and all other solution curves are parallel to this line, moving in opposite directions on either side of the line [16]. This behaviour is local and is consistent with the analysis conducted above in which the degenerate point 𝑧1 was found to be stable with respect to the addition of (small) higher-order terms. As such, for these values of M the system does not behave in a stable manner around 𝑧2 and 𝑧3—at best we find neutrally stable behaviour. Even if a solution were to start sufficiently near these equilibrium points, it would not necessarily remain bounded. Furthermore, given that References [1] A. D. Kraus, A. Aziz, and J. Welty, Extended Surface Heat Transfer, John Wiley and Sons, New York, NY, USA, 2001. [2] B. Sunden and P. J. Heggs, Eds., Recent Advances in Analysis of Heat Transfer for Fin Type Surfaces, WIT Press, Boston, Mass, USA, 2000. [3] F. Khani, M. Ahmadzadeh Raji, and H. Hamedi Nejad, “Analytic solutions and efficiency of the nonlinear fin problem with temperature-dependent thermal conductivity and heat transfer coefficient,” Communications in Nonlinear Science and Numeri- cal Simulation, vol. 14, pp. 3327–3338, 2009.
https://openalex.org/W4386092765	https://www.nature.com/articles/s41598-023-40979-9.pdf	English	null	Seasonally-reversed trends in the subtropical Northwestern Pacific linked to asymmetric AMO influences	Scientific reports	2,023	cc-by	9,087	OPEN Yong‑Fu Lin 1, Chuen‑Teyr Terng 2, Chau‑Ron Wu 3,4 & Jin‑Yi Yu 1* This study identifies seasonally-reversed trends in Kuroshio strength and sea surface temperatures (SSTs) within the western North Pacific (WNP) since the 1990s, specifically in the 22° N–28° N region. These trends are characterized by increases during summer and decreases during winter. The seasonally-reversed trends are a result of the asymmetric responses of the WNP to a shift towards the positive phase of the Atlantic multidecadal oscillation (AMO) around the same period. The positive AMO induces an anomalous descent over the North Pacific during summer, leading to the direct strengthening of the gyre. However, during winter, it triggers an anomalous descent over the tropical Pacific, which excites a poleward wavetrain impacting the WNP and causing gyre weakening. The associated responses of the East Asian monsoon and China Coastal Current contribute to the observed seasonally-reversed SST trends. It is noteworthy that the seasonally-reversed trends in gyre strength and SSTs are predominantly observed north of 20° N in the WNP. This limitation arises because the anomalous cyclone within the winter poleward wavetrain is located north of this latitude boundary. Specifically, the clearest trends in gyre strength are observed in the northern segment of the Kuroshio, while the manifestation of SST trends in the Taiwan Strait could potentially be attributed to the influence and enhancement of the East Asian monsoon and the China Coastal Current. Due to the limited length of observational data, statistical significance of some of the signals discussed is rather limited. A CESM1 pacemaker experiments is further conducted to confirm the asymmetric responses of the North Pacific to the AMO between the summer and winter seasons. An increasing number of studies indicate that Pacific climate has experienced significant changes since the 1990s1–4. In the tropical Pacific, the location of El Niño-Southern Oscillation (ENSO) events moved from the eastern Pacific to the central Pacific4–9. Beginning around the same time, there was a southward migration of the mean locations of the North Equatorial Current (NEC) and the North Equatorial Counter Current (NECC)2. Sea level rise in the western Pacific have also accelerated during this period1. Furthermore, the interannual variability in monsoon rainfall has significantly decreased since 19933. gi y In the western North Pacific (WNP), the North Pacific subtropical gyre (NPSG) is a key feature that shapes regional climate. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Earth System Science, University of California, Irvine, CA, USA. 2Central Weather Bureau, Taipei, Taiwan. 3Department of Earth Sciences, National Taiwan Normal University, Taipei, Taiwan. 4Research Center for Environmental Changes, Academia Sinica, Taipei, Taiwan. email: jyyu@uci.edu OPEN Variations in the strength of the NPSG modulate regional circulations in the East China Sea and South China Sea, the strengths of the Mindanao, Taiwan Strait, and Luzon Strait currents10–12, typhoon development13, and even fish migration14–20. For example, a weakening (strengthening) of the Kuroshio, which is the western boundary current of the NPSG, can induce more (less) intrusion events into East China Sea and South China Sea, affecting fish migration, as well as mass, heat, salinity, and nutrient balances11,12,21–25. As another example, a weakening (strengthening) of the NEC component of the NPSG can weaken (strengthen) the Mindanao Current26. In this study, we focus on contrasting the summer and winter trends in the WNP since the 1990s with a particular focus on the region around the Taiwan Strait (Fig. 1a). The strait is a narrow passage connecting the broad and shallow East China Sea to the north of the Strait to the much deeper South China Sea to the south. It is located right off the East Asian continent, and the currents and sea surface temperatures (SSTs) there can 1Department of Earth System Science, University of California, Irvine, CA, USA. 2Central Weather Bureau, Taipei, Taiwan. 3Department of Earth Sciences, National Taiwan Normal University, Taipei, Taiwan. 4Research Center for Environmental Changes, Academia Sinica, Taipei, Taiwan. email: jyyu@uci.edu \| https://doi.org/10.1038/s41598-023-40979-9 Scientific Reports \| (2023) 13:13735 w.nature.com/scientificreports/ Figure 1. (a) Climatology of sea surface temperature (colors, in °C) and upper-ocean (0–200 m) current speed (vectors) from the JCOPE2 reanalysis data during the period 1993–2018; (b,c) boreal summer and winter linear trends in SSTs (in °C/year) of the JCOPE2 reanalysis data and (d,e) the NOAA OISST observational data. (f,g) boreal summer and winter linear trends in currents of the JCOPE2 reanalysis data during the period; and (h,i) summer and winter geostrophic surface velocity differences (color, in cm/s) between two sub-periods (1997– 2018 minus 1993–1996) of the AVIOS observational data. In (a), the bold blue arrow marks the mean location of the North Equatorial Current (NEC)-Kuroshio component of the North Pacific Subtropical Gyre. Also marked are the locations of the Taiwan Strait (TS), East China Sea (ECS), South China Sea (SCS), Mindanao Current (MC), and the south and north segments of the Kuroshio (KC-S and KC-N, respectively). The blue line next to KC-N marks the area (24.5–27° N and 122–124° E) used to calculate the area-average KC-N current speed. OPEN Vectors are the summer and winter current climatology calculated from the JCOPE2 reanalysis for the period 1993 to 2018. In (f–i), the vectors represent the summer and winter current climatology. Only currents exceeding 10 cm/s in (f,g) and 20 cm/s in (h,i) are shown. These climatologies were calculated from the JCOPE2 and AVISO data for the period 1993 to 2018. In (b–i), stippled areas indicate significance at the 90% confidence level determined using a Student’s t-test. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. (a) Climatology of sea surface temperature (colors, in °C) and upper-ocean (0–200 m) current speed (vectors) from the JCOPE2 reanalysis data during the period 1993–2018; (b,c) boreal summer and winter linear trends in SSTs (in °C/year) of the JCOPE2 reanalysis data and (d,e) the NOAA OISST observational data. (f,g) boreal summer and winter linear trends in currents of the JCOPE2 reanalysis data during the period; and (h,i) summer and winter geostrophic surface velocity differences (color, in cm/s) between two sub-periods (1997– 2018 minus 1993–1996) of the AVIOS observational data. In (a), the bold blue arrow marks the mean location of the North Equatorial Current (NEC)-Kuroshio component of the North Pacific Subtropical Gyre. Also marked are the locations of the Taiwan Strait (TS), East China Sea (ECS), South China Sea (SCS), Mindanao Current (MC), and the south and north segments of the Kuroshio (KC-S and KC-N, respectively). The blue line next to KC-N marks the area (24.5–27° N and 122–124° E) used to calculate the area-average KC-N current speed. Vectors are the summer and winter current climatology calculated from the JCOPE2 reanalysis for the period 1993 to 2018. In (f–i), the vectors represent the summer and winter current climatology. Only currents exceeding 10 cm/s in (f,g) and 20 cm/s in (h,i) are shown. These climatologies were calculated from the JCOPE2 and AVISO data for the period 1993 to 2018. In (b–i), stippled areas indicate significance at the 90% confidence level determined using a Student’s t-test. be influenced by trends in the East Asian monsoon27. Over the region, southwestly monsoon winds dominate during the summer and northeastly monsoon winds dominate during the winter.il be influenced by trends in the East Asian monsoon27. Results d i Trends in the western North Pacific SSTs and currents during 1993–2018. We first examined the summer (June–July–August; JJA) and winter (December–January–February; DJF) linear trends in SST and upper-ocean (0–200 m) current speed in the WNP during 1993–2018. While warming trends are found during both summer and winter in most of the WNP, a summer warming trend but a winter cooling trend appear off the East Asian Coast to the north of 20° N (Fig. 1b,c). This phenomenon of seasonally-reversed trends around the Taiwan Strait is verified by the high-resolution OISST data (Fig. 1d,e). A similar seasonal reversal is also found in the trends in upper-ocean current speeds along the Kuroshio component of the NPSG (Fig. 1f,g), which exhibit a strengthening trend during summer but a weakening trend during winter, with the most noticeable effect occur- ring in the 22° N–28° N region. g g To further examine the current trend, we divided the Kuroshio into two sectors (see Fig. 1a): the southern sector (KC-S; hereafter) that is upstream of the Taiwan Strait and the northern sector (KC-N; hereafter) that is downstream of the Taiwan Strait. As depicted in Fig. 1f,g, the seasonally reversed trends most apparent in the KC-N sector, still observable south of KC-N along the Kuroshio from 22° to 28° N, diminishing south of Taiwan, and fading over the KC-S sector. Smaller weakening trends are found in KC-S and NEC during both summer and winter. Most pronounced seasonally-reversed trends from 22° to 28° N in the WNP, including the Taiwan Strait and KC-N sector of the Kuroshio. We further quantify in Fig. S1 the seasonal trends in the Taiwan Strait SST and two major currents of the North Pacific subtropical gyre near the Taiwan Strait (i.e., the NEC and Kuroshio) (see Fig. 1a). We examine the current trends in KC-S using the current averaged around 18° N and 122–124° E and the current trends in KC-N using the current averaged around 24.5–27° N and 122–124° E. We find weak linear trends in the NEC for both the summer (0.0008 cm/s/year, P > 0.1) and winter (− 0.02 cm/s/year, P > 0.1) seasons (Fig. S1a,b). The seasonally-reversed trends that we observed around the Taiwan Strait did not occur in the NEC. The current trends of KC-S (Fig. Results d i S1c,d) are similar to the NEC trends, which are very weak during summer (0.005 cm/s/year, P > 0.1) and slightly negative during winter (trend = − 0.06 cm/s/year, P > 0.1). However, a seasonally-reversed trend appears in the KC-N segment of the Kuroshio (Fig. S1e,f). An increasing current trend is found in the sum- mer (0.07 cm/s/year, P > 0.1), but there is a decreasing trend in the winter (− 0.05 cm/s/year, P > 0.1). Although the trends are not statistically significant, they provide indications that KC-N segment of the Kuroshio have seasonally reversal trends. This phenomenon also occurs in the SST of the Taiwan Strait. The variations in the SST anomalies, after being averaged across the TS transect line, indicate a linear warming trend of 0.29 °C/decade (P < 0.01) during the summer (Fig. S1g) and a cooling trend of − 0.17 °C/decade (P > 0.1) during the winter from 1993 to 2018 (Fig. S1h). The cooling trend is not statistically significant because it is affected by strong interan- nual variability, such as ENSO. Nonetheless, it shows that the Taiwan Strait has become cooler, exhibiting the opposite trend compared to summer. pp p Our analyses indicate that the seasonally-reversed trend appears in the Taiwan Strait SSTs and Kuroshio strength. A large-scale driving mechanism is likely to exist to cause such a seasonally-reversed trend. Our analysis of the different segments of the Kuroshio also suggests that the reversed trend does not exist in the equatorial part of the NWP but becomes apparent somewhere around 22° N–28° N. The driving mechanism has to explain not only the seasonally-revering feature of the trend but also this latitudinal dependence of the forcing. Trans‑basin influences of the Atlantic Ocean on the Pacific climate. To uncover the large-scale driving mechanism behind the reversed trends described in “Trends in the western North Pacific SSTs and cur- rents during 1993–2018” section, we first calculated the correlation coefficients between monthly upper-ocean (0–200 m) Kuroshio Current speed averaged along the KC-N line (see Fig. 1a) and wind stress curl (WSC) over the Western North Pacific during 1993–2018. The correlation (Fig. OPEN Over the region, southwestly monsoon winds dominate during the summer and northeastly monsoon winds dominate during the winter.il In addition to the Pacific Decadal Oscillation (PDO), the WNP can be influenced by the Atlantic Multi- decadal Oscillation (AMO) over decadal or longer time scales via trans-basin processes. Previous studies have suggested that the AMO can influence the North Pacific climate either directly through the mid-latitudes28–30 or indirectly through the tropics30–34. In this study, we show with observational analyses and pacemaker coupled model experiments that trends (since the 1990s) in SSTs and the NPSG strength in the WNP have an interesting seasonally-reversed character due to different trans-basin influences from the Atlantic Ocean between summer and winter. https://doi.org/10.1038/s41598-023-40979-9 Scientific Reports \| (2023) 13:13735 \| www.nature.com/scientificreports/ Results d i S2) indicates that WSC anomalies (WSCAs) over 125° E–165° W and 18–35° N of the WNP are most correlated to surface current variations in the KC-N sector We then calculated the 21-year running correlations between the summer and winter WSCA averaged over this region and the PDO and AMO indices during 1948–2018 to explore the roles of these two decadal vari- ability modes in the current trends. We found that the summer and winter WSCA correlations with the PDO index never pass the 90% significance interval during the analysis period (Fig. 2a), while the correlations with the AMO index both exceeds this interval after the 1990s (Fig. 2b). Most interestingly, the summer WSCA and winter WSCA show reversed correlations with the AMO index during the decades after the 1990s. The AMO has a significant negative correlation with the summer WSCA after the 1990s but a significant positive correlation with the winter WSCA. The 1990s is the time when the AMO switched from a negative phase to a positive phase (Fig. S3). Based on the correlations, the positive AMO phase since the 1990s is expected to result in negative summer WSCAs over the WNP, which strengthen the gyre circulation within the region where the WSCAs were averaged. Similarly, during the same positive AMO phase, positive winter WSCAs are anticipated to weaken the gyre circulation in the region. The seasonally reversed trends in the WNP region since the 1990s can be explained as a result of the seasonally-asymmetric responses of the WNP wind stress curl to the positive phase of the AMO. To further verify that the AMO can induce the seasonally reversed trends in the Kuroshio, we used the geo- strophic surface current velocity derived from AVISO observational data to examine the velocity differences between a negative-AMO period (i.e., 1993–1996) and a positive-AMO period (i.e., 1997–2018) during both summer and winter (Fig. 1h,i). The figures also show the summer and winter current climatologies, averaged from 1993 to 2018. The differences observed confirm that surface currents in the Kuroshio, have strengthened during summer (Fig. 1h) and weakened during winter (Fig. 1i) after the AMO changed from a negative to posi- tive phase around the mid-1990s. Results d i S3) during summer (red line) and winter (black line). Grey-dashed lines mark the 90% confidence level determined using a Student’s t-test. The red and blue shadings at bottom of (a,b) respectively indicate the positive and negative phases of the PDO and AMO determined using the 7-year running mean values of the indices. Figure 2. The 21-year running correlation between the yearly (a) Pacific decadal oscillation (PDO) and (b) Atlantic multi-decadal oscillation (AMO) index with the wind stress curl anomalies averaged over the region of the Western North Pacific (125° E–165° W an 18–35° N, shown by the green box in Fig. S3) during summer (red line) and winter (black line). Grey-dashed lines mark the 90% confidence level determined using a Student’s t-test. The red and blue shadings at bottom of (a,b) respectively indicate the positive and negative phases of the PDO and AMO determined using the 7-year running mean values of the indices. We next performed atmospheric analyses with the longer analysis period (i.e., 1948–2018) to understand the reasons why the AMO can induce different ocean current and SST responses in the WNP between winter and summer. To achieve this, we regressed the 7-year low-pass filtered sea level pressure (SLP) anomalies and WSCAs over the Atlantic–Pacific region onto the filtered AMO index during 1948–2018. We found that the atmosphere over the North Pacific responds differently to the AMO during summer and winter (Fig. 3). During summer, the AMO-regressed SLP anomalies reveal a strengthened subtropical high that covers almost the entire Pacific basin (Fig. 3a). Interestingly, negative SLP anomalies appear over the North Atlantic. This SLP regression pat- tern suggests that the positive AMO may induce anomalous ascent (and therefore negative SLP anomalies) over the North Atlantic and anomalous descent over the North Pacific (therefore positive SLP anomalies) though a mid-latitude trans-basin mechanism such as the one suggested by some previous studies29,30. This is confirmed in Fig. S4a where we regress the filtered omega velocity anomalies in a vertical cross-section averaged along the subtropical (15–30° N) Pacific–Atlantic sector onto the filtered AMO index. The regression is generally char- acterized by ascending motions over the North Atlantic and descending motions over the North Pacific. The anomalous descent intensifies the Pacific subtropical High during summer (Fig. 3a) and extends it further into the Western Pacific, giving rise to overall negative WSCA (color shadings in Fig. 3c) in the WNP. Results d i We then calculated the 21-year running correlations between the summer and winter WSCA averaged over this region and the PDO and AMO indices during 1948–2018 to explore the roles of these two decadal vari- ability modes in the current trends. We found that the summer and winter WSCA correlations with the PDO index never pass the 90% significance interval during the analysis period (Fig. 2a), while the correlations with the AMO index both exceeds this interval after the 1990s (Fig. 2b). Most interestingly, the summer WSCA and winter WSCA show reversed correlations with the AMO index during the decades after the 1990s. The AMO has a significant negative correlation with the summer WSCA after the 1990s but a significant positive correlation with the winter WSCA. The 1990s is the time when the AMO switched from a negative phase to a positive phase (Fig. S3). Based on the correlations, the positive AMO phase since the 1990s is expected to result in negative summer WSCAs over the WNP, which strengthen the gyre circulation within the region where the WSCAs were averaged. Similarly, during the same positive AMO phase, positive winter WSCAs are anticipated to weaken the gyre circulation in the region. The seasonally reversed trends in the WNP region since the 1990s can be explained as a result of the seasonally-asymmetric responses of the WNP wind stress curl to the positive phase of the AMO. To further verify that the AMO can induce the seasonally reversed trends in the Kuroshio, we used the geo- strophic surface current velocity derived from AVISO observational data to examine the velocity differences between a negative-AMO period (i.e., 1993–1996) and a positive-AMO period (i.e., 1997–2018) during both summer and winter (Fig. 1h,i). The figures also show the summer and winter current climatologies, averaged from 1993 to 2018. The differences observed confirm that surface currents in the Kuroshio, have strengthened during summer (Fig. 1h) and weakened during winter (Fig. 1i) after the AMO changed from a negative to posi- tive phase around the mid-1990s. https://doi.org/10.1038/s41598-023-40979-9 Scientific Reports \| (2023) 13:13735 \| www.nature.com/scientificreports/ Figure 2. The 21-year running correlation between the yearly (a) Pacific decadal oscillation (PDO) and (b) Atlantic multi-decadal oscillation (AMO) index with the wind stress curl anomalies averaged over the region of the Western North Pacific (125° E–165° W an 18–35° N, shown by the green box in Fig. Results d i The overall negative WSCAs should intensify the NPSG and Kuroshio. Additionally, we present in Fig. 3e the regression of the filtered summer Sverdrup streamfunction over the North Pacific onto the filtered AMO index. A positive Sverdrup streamfunction in the subtropical region (between 15° and 30° N) indicates an increase in the strength of the Kuroshio (Fig. 1d,f). ( g ) During winter, the AMO-regressed SLP anomalies (Fig. 3b) are dominated by a poleward-propagating wave- train pattern that emanates from the western tropical Pacific. This pattern suggests that the positive phase of the AMO may induce anomalous descent in the tropical Pacific during the winter season, in contrast to the descent in the North Pacific that occurred during the summer season. This is confirmed in Fig. S5b where we regress the filtered omega velocity anomalies over a vertical cross-section averaged along the tropical (0°–10° N) Pacific-Atlantic sector onto the filtered AMO index. The regression shows that anomalous descent occurred in the tropical central Pacific between 150° and 180° E, although the statistical significance area is limited to a small region. Since the winter atmosphere is barotropically more unstable (due to the strong vorticity gradient associated with the jet in winter and the strong upper-level divergent flow over the warm pool; not shown), the AMO-induced descent can excite a Rossby wavetrain propagating poleward into the North Pacific.h y p p g g pi The AMO-regressed barotropic streamfunction and Z200 anomalies further reveal this Rossby wave pattern in the North Pacific during winter (Fig. S6b), which does not appear in summer (Fig. S6a). The wavetrain produces SLP anomaly centers over the NWP region (around 160° E and 25° N), the central North Pacific (around 150° Scientific Reports \| (2023) 13:13735 \| https://doi.org/10.1038/s41598-023-40979-9 www.nature.com/scientificreports/ Figure 3. Regression of 7-year running-mean anomalies in SLP (contour, unit is hPa) onto the 7-year running- mean AMO index during summer (a) and winter (b) for the period 1948–2018. (c,d) Same as (a,b), but for wind stress curl (shading, unit is 10–7 N/m3). (e,f) Same as (a,b), but for Sverdrup streamfunction (unit is 106 Sv). Red (blue) colors in (e,f) indicate clockwise (counter-clockwise) circulations. The red-dashed lines in (a–d) indicate the location of the equator, while the green boxes in all figures indicate the region bounded by 125° E–165° W and 18–35° N. Results d i We nudged SSTAs in the North Atlantic (0–70° N) to simulate the positive AMO phase in one experiment (AMO-positive experiment) and the negative AMO phase in another experiment (AMO-negative experiment). Figure 4a,b displays the ensemble mean SST differences between the two experi- ments. In the North Atlantic, the SSTA differences resemble the observed AMO pattern, characterized by tropi- cal and subpolar warming bands separated by a weaker-warming band in between. Figure 4c–f also shows the SLP and Z200 differences between the two experiments. During summer, the subtropical high across the entire Pacific basin strengthened, and negative SLP anomalies appeared over the North Atlantic (Fig. 4c). In winter, the SLP anomalies (Fig. 4d) were dominated by a poleward-propagating wavetrain pattern emanating from the western tropical Pacific (west of 150° E). The Z200 difference further revealed this Rossby wave pattern in the North Pacific during winter (Fig. 4f), but it was weaker during summer (Fig. 4e). i g g g g Recent studies have reported on the respective roles of the tropical and extra-tropical parts of the AMO’s SST anomaly pattern in affecting Pacific climate30,34,36–38. These studies suggested that the tropical Atlantic part of the AMO SST anomalies can first induce an anomalous descent over the tropical central Pacific, which the excite a poleward-propagating Rossby wave pattern into the North Pacific, similar to the PNA pattern39 (see Figs. 3b, 4d,f, Fig. S6b). However, the extra-tropical Atlantic part of the AMO SST anomalies can directly produce an atmospheric bridge from the North Atlantic to the North Pacific, with an anomalous ascent over the North Atlantic and an anomalous descent over the North Pacific (see Figs. 3a, 4c). i g To further examine these seasonally varying forcing mechanisms, we examined the AMO-regressed omega vertical velocity anomalies along vertical cross-sections averaged over the subtropical (15–30° N) and tropical (0–10° N) Pacific-Atlantic Oceans during summer and winter from 1948 to 2018 (Figs. S4, S5). Our results reveal that the positive phase of the AMO induces similar anomalous descent in the tropical Pacific during both summer (Fig. S5a) and winter (Fig. S5b). However, the positive AMO forcing only lead to anomalous descent in the subtropical Pacific during summer (Fig. S4a), while no such descent is observed during winter (Fig. S4b). Results d i The main factor contributing to this discrepancy is the barotropic instability of the winter atmosphere, which enables the AMO-induced anomalous descent in the tropical Pacific to excite a poleward-propagating Rossby wave. This wave weakens the expected anomalous descent in the subtropical Pacific (around 150° E–150° W), which should have occurred due to the positive AMO forcing (see Fig. S4b). In contrast, during summer, the atmosphere exhibits greater barotropic stability, preventing the excitation of a similar propagating wave by the AMO-induced anomalous descent in the tropical Pacific. Consequently, the positive AMO-induced anomalous descent mainly occurs over the tropical Pacific during winter and over the subtropical Pacific during summer.hfi ii The seasonally reversed trend in SSTs off the East Asian coast can also be explained as an asymmetric Pacific response to the AMO modulated by the East Asian Monsoon. During summer, the anomalous surface high across the North Pacific in association with the positive AMO should intensify the summer southwesterly monsoon winds. We confirm in Fig. S8a that the East Asian summer monsoon winds exhibit a strengthening trend since the 1990s which can bring more moist air from the South China Sea region into the Taiwan Strait, reducing surface evaporation there. This explains how the summer warming trend is produced off the East Asian Coast (Fig. 1b). The suggested roles of surface heat fluxes are confirmed in Fig. S9, where we regress onto the AMO index the 7-year low-pass filtered anomalies of summer net heat flux (NHF) and its four components (i.e., latent heat flux, sensible heat flux, and shortwave and longwave radiation flux). The regressions show that the latent heat flux (Fig. S9g) and sensible heat flux (Fig. S9i) are the main contributor to the positive NHF forcing (Fig. S9a) of the summer SST warming trend off the East Asian coast. Apparently, the moister and warmer air imported by the anomalously strong summer monsoon winds dominates the effect of the stronger wind speeds which would be to cause a decrease in the surface evaporation and surface warming. During winter, the AMO-induced wavetrain produces anomalously low surface pressures to the east of Tai- wan, resulting in anomalous northerlies on its western flank, which strengthens the winter monsoon. We also confirmed that the East Asian winter monsoon has a strengthening trend since 1990s (Fig. S8b). The regression analysis with the winter surface heat flux components (Fig. Results d i Based on this result, it is evident that the AMO may indeed induce the seasonally reversed trends in the subtropical Northwestern Pacific. Affected by the length limitation of the observation data, the statistical significance area of the result is also limited. We further employed a coupled climate model to validate the plausibility of our theory.l W and 35° N), Gulf of Alaska (around 135° W and 50° N), and Northeastern Canada (around 55° W and 65° N). Such a wavetrain response in the North Pacific to the AMO has been documented in previous studies34,35. The anomalous low over the WNP, together with the slower mid-latitude westerlies caused by the anomalous high over the North Pacific, generate the positive WSCAs within the WNP region (Fig. 3d) associated with the seasonally reversed trends discussed earlier. The positive WSCAs should weaken the subtropical gyre circulation over the region, including the Kuroshio. Figure 3f confirms that, associated with the positive WSCAs over the 20° N–35° N region of the WNP, a weakened Sverdrup streamfunction appears. The negative Sverdrup streamfunc- tion anomalies in the subtropical region indicate a weakened Kuroshio. When comparing the summer season (Fig. 3c), the most significant difference in the WSCA is observed in the 20° N–28° N region. Consequently, the greatest difference in the Sverdrup streamfunction also occurs in the same region. Therefore, it can be concluded that the seasonally reversed trends of the Kuroshio are specifically observed in the 20° N–28° N region due to these variability in the atmosphere. We also construct composites of the SLP, WSC, and Sverdrup streamfunction anomalies during the positive-AMO period (i.e., 1997–2018) for both summer and winter (see Fig. S7) to dem- onstrate that the AMO can trigger the seasonally reversed trends in the subtropical gyre. These figures exhibit a similar pattern to Fig. 3, showing a trans-basin impact pattern of the positive AMO over the North Pacific. Based on this result, it is evident that the AMO may indeed induce the seasonally reversed trends in the subtropical Northwestern Pacific. Affected by the length limitation of the observation data, the statistical significance area of the result is also limited. We further employed a coupled climate model to validate the plausibility of our theory.l p y p p y y We conducted pacemaker experiments using CESM1 to further investigate the trans-basin influences of the AMO on the Pacific climate. Results d i The stippled areas in all figures indicate significance at the 90% confidence level determined using a Student’s t-test. Figure 3. Regression of 7-year running-mean anomalies in SLP (contour, unit is hPa) onto the 7-year running- mean AMO index during summer (a) and winter (b) for the period 1948–2018. (c,d) Same as (a,b), but for wind stress curl (shading, unit is 10–7 N/m3). (e,f) Same as (a,b), but for Sverdrup streamfunction (unit is 106 Sv). Red (blue) colors in (e,f) indicate clockwise (counter-clockwise) circulations. The red-dashed lines in (a–d) indicate the location of the equator, while the green boxes in all figures indicate the region bounded by 125° E–165° W and 18–35° N. The stippled areas in all figures indicate significance at the 90% confidence level determined using a Student’s t-test. https://doi.org/10.1038/s41598-023-40979-9 Scientific Reports \| (2023) 13:13735 \| www.nature.com/scientificreports/ W and 35° N), Gulf of Alaska (around 135° W and 50° N), and Northeastern Canada (around 55° W and 65° N). Such a wavetrain response in the North Pacific to the AMO has been documented in previous studies34,35. The anomalous low over the WNP, together with the slower mid-latitude westerlies caused by the anomalous high over the North Pacific, generate the positive WSCAs within the WNP region (Fig. 3d) associated with the seasonally reversed trends discussed earlier. The positive WSCAs should weaken the subtropical gyre circulation over the region, including the Kuroshio. Figure 3f confirms that, associated with the positive WSCAs over the 20° N–35° N region of the WNP, a weakened Sverdrup streamfunction appears. The negative Sverdrup streamfunc- tion anomalies in the subtropical region indicate a weakened Kuroshio. When comparing the summer season (Fig. 3c), the most significant difference in the WSCA is observed in the 20° N–28° N region. Consequently, the greatest difference in the Sverdrup streamfunction also occurs in the same region. Therefore, it can be concluded that the seasonally reversed trends of the Kuroshio are specifically observed in the 20° N–28° N region due to these variability in the atmosphere. We also construct composites of the SLP, WSC, and Sverdrup streamfunction anomalies during the positive-AMO period (i.e., 1997–2018) for both summer and winter (see Fig. S7) to dem- onstrate that the AMO can trigger the seasonally reversed trends in the subtropical gyre. These figures exhibit a similar pattern to Fig. 3, showing a trans-basin impact pattern of the positive AMO over the North Pacific. Data and methods Data sets. To study ocean current and SST variations in the WNP region, we use high-resolution monthly reanalysis data provided by the Japan Coastal Ocean Predictability Experiment Reanalysis (JCOPE2). This ocean data set has a domain that covers the WNP from 10.5° to 62° N and from 108° to 180° E with a resolution of 1/12° in the horizontal and 46 sigma levels in the vertical45. We verify the results related to ocean surface cur- rents with the observations provided by the Archiving, Validation and Interpretation of Satellite Oceanographic Data (AVISO) and SSTs with the optimum interpolation SST (OISST) from the NOAA National Centers for Environmental Information. These two observational datasets have a global horizontal resolution of 0.25°× 0.25°. For the analyses related to the trans-basin interactions between Pacific and Atlantic Oceans, atmospheric information over these two basins are provided by National Centers for Environmental Prediction/National Center for Atmospheric Research (NCEP/NCAR) reanalysis 1. The NCEP/NCAR reanalysis 1 has a 1.875° × 1.875° horizontal resolution. In this study, the analysis periods are: 1993–2018 for the regional analyses using the JCOPE2, AVISO, OISST and 1948–2018 for the trans-basin analyses using NCEP/NCAR reanalysis I. Anomalies are defined as the deviations from the monthly climatology calculated using the respective analysis periods (i.e., 1993–2018 or 1948–2018). In addition, the linear trends during 1948–2018 were removed from NCEP/NCAR reanalysis I data (see Figs. 2, 3, Figs. S2, S4–S9). Climate indices. Four climate indices were used in the study: the AMO, PDO, East Asia summer monsoon index, and East Asia winter monsoon index. The AMO index46 is obtained from NOAA’s Physical Sciences Divi- sion and is calculated as the de-trended SST anomalies averaged over the North Atlantic from the equator to 70° N. The PDO index is obtained from the Joint Institute for the Study of the Atmosphere and Ocean (JISAO) and is the principal component of the first leading Empirical Orthogonal Function mode of the North Pacific SST anomalies47. The East Asia summer and winter monsoon indices48–51 are used to quantify the strength of the monsoon during the two seasons. The East Asia summer monsoon index is defined as surface meridional wind averaged over the region between 10–40° N and 110–140° E, whereas the East Asia winter monsoon index is defined as surface meridional wind averaged over the region between 20–40° N and 100–140° E. The CEMS1 pacemaker experiments. www.nature.com/scientificreports/ in the humidity. Although Fig. S9 is almost not statistically significant around the Taiwan Strait, these findings suggest a potential influence of the AMO around the Taiwan Strait.l l In addition to the surface heat flux, the East Asian monsoon also plays a role in modulating the strength of the China Coastal Current (CCC), which could contributes to the cooling trend in winter40. The CCC is a narrow band of southward currents that appears only during winter off the China coast (Fig. S10). Driven by the East Asian winter monsoon, the CCC can transport cool water from the Yellow Sea and East China Sea into the South China Sea through the Taiwan Strait41,42. We found that the winter CCC speed averaged over the TS transect line (black line in Fig. S10) shows a strengthening trend (of − 0.01 cm/s/year) during the 1993–2018 (Fig. S8c). The CCC current speed and East Asia winter monsoon index are significantly correlated (R = 0.52 with P < 0.01) in the TS transect line. A stronger East Asian winter monsoon causes a stronger CCC, which resulted in more cold water flowing southward into the Taiwan Strait, contributing to the observed winter cooling trend there. The CCC can serves as a conduit for transmitting the monsoon-induced changes more prominently to SST, thereby influencing the alteration of SST. As the CCC can affects SST in the Taiwan Strait, the AMO’s influence on Western Pacific SST trends becomes particularly pronounced in this region. Discussion and summaryi y Using statistical analyses, we find seasonally reversed trends in surface currents and SSTs in the WNP from 22° to 28° N since the 1990s. However, given the constrained length of the observational data, we extend our analysis by employing CESM1 pacemaker experiments. These experiments serve to validate that the observed trends stem from the North Pacific’s asymmetric responses to the AMO during both the summer and winter seasons. Figure 5 summarizes and illustrates the series of processes involved in the asymmetric trans-basin interactions. As the AMO changed to a positive phase beginning in the middle 1990s, the warmer SSTs in the North Atlantic induced an anomalous descent over the North Pacific during the summer season. This subtropical trans-basin interaction mechanism directly strengthens the Pacific subtropical High, which then produces a strengthening of the gyre in the WNP through changes in wind stress curls and a warming trend in ocean surface temperatures off the East Asian coast (Fig. 5a) through enhancement of East Asian summer monsoon. During winter (Fig. 5b), the positive phase of the AMO induces an anomalous descent over the Tropical Pacific that subsequently excites a poleward-propagating Rossby wavetrain into the North Pacific. The wave train produced by this tropical trans-basin interaction mechanism consists of an anomalous surface cyclone north of 20° N in the WNP. This anomalous cyclone produces a gyre weakening trend through wind stress curl changes and a cooling trend off the Asian coast through enhancement of the East Asian winter monsoon and the associated CCC. The seasonally reversed trends observed in the WNP are thus a result of asymmetric Atlantic-Pacific trans-basin interactions between summer and winter. Previous studies have pointed out that global warming can enhance inter-basin interaction between the pan- tropical (including Pacific) and Northern Atlantic regions, especially after the 1990s29,43,44. Therefore, it is likely that climate change in the Atlantic will become increasingly important for the Pacific climate. Results d i S9 right panels) further indicates that the strength- ened winter monsoon increases surface evaporation, primarily through the latent heat flux component, result- ing in winter cooling of SSTs observed off the East Asian coast. Unlike the strengthened summer monsoon, the strengthened winter monsoon affects the surface evaporation through changes in wind speed rather than changes Scientific Reports \| (2023) 13:13735 \| https://doi.org/10.1038/s41598-023-40979-9 www.nature.com/scientificreports/ om/scientificreports/ Figure 4. Ensemble mean SST (unit is °C) difference between positive and negative AMO experiments during summer (a) and winter (b). (c–f) Same as (a,b), but for SLP (unit is hPa) and Z200 (unit is m). The red-dashed lines in all figures indicate the location of the equator, while the green boxes in (c,d) indicate the region bounded by 125° E–165° W and 18–35° N. The stippled areas in all figures indicate significance at the 99% confidence level determined using a Student’s t-test. Figure 4. Ensemble mean SST (unit is °C) difference between positive and negative AMO experiments during summer (a) and winter (b). (c–f) Same as (a,b), but for SLP (unit is hPa) and Z200 (unit is m). The red-dashed lines in all figures indicate the location of the equator, while the green boxes in (c,d) indicate the region bounded by 125° E–165° W and 18–35° N. The stippled areas in all figures indicate significance at the 99% confidence level determined using a Student’s t-test. https://doi.org/10.1038/s41598-023-40979-9 Scientific Reports \| (2023) 13:13735 \| www.nature.com/scientificreports/ Data and methods All the experiments in this study were conducted using the CEMS1.2.2 (for more details, see https://www.cesm.ucar.edu/models/cesm1.2/). The experiments utilized the B1850C5CN (fully coupled) compset of the CESM1.2.2, with the f19_g16 model configuration. This config- uration has a horizontal resolution of approximately 2° and a vertical resolution of 30 vertical levels in the https://doi.org/10.1038/s41598-023-40979-9 Scientific Reports \| (2023) 13:13735 \| www.nature.com/scientificreports/ tificreports/ Figure 5. Diagram illustrating the processes enable the asymmetric Atlantic–Pacific trans-basin interactions during boreal summer (a) and winter (b). During summer (a), the positive AMO induces anomalous ascent over the North Atlantic and anomalous descent over the subtropical North Pacific. This subtropical trans-basin interaction mechanism directly intensifies the Pacific Subtropical High, which then produces a strengthening trend in the gyre circulation in the WNP through changes in wind stress curls and a warming trend in SST off the East Asian coast via the strengthening of the East Asian summer monsoon. During winter (b), the positive AMO first induces anomalous descent over the tropical North Pacific. This tropical trans-basin interaction mechanism subsequently excites a wavetrain that propagates into the North Pacific. The wave train consists of an anomalous cyclone north of 20° N in the WNP, which causes a gyre weakening trend through wind stress curl anomalies and a SST cooling trend off the Asia coast via a strengthening of the East Asian winter monsoon and associated China Coastal Current. Figure 5. Diagram illustrating the processes enable the asymmetric Atlantic–Pacific trans-basin interactions during boreal summer (a) and winter (b). During summer (a), the positive AMO induces anomalous ascent over the North Atlantic and anomalous descent over the subtropical North Pacific. This subtropical trans-basin interaction mechanism directly intensifies the Pacific Subtropical High, which then produces a strengthening trend in the gyre circulation in the WNP through changes in wind stress curls and a warming trend in SST off the East Asian coast via the strengthening of the East Asian summer monsoon. During winter (b), the positive AMO first induces anomalous descent over the tropical North Pacific. This tropical trans-basin interaction mechanism subsequently excites a wavetrain that propagates into the North Pacific. The wave train consists of an anomalous cyclone north of 20° N in the WNP, which causes a gyre weakening trend through wind stress curl anomalies and a SST cooling trend off the Asia coast via a strengthening of the East Asian winter monsoon and associated China Coastal Current. Data and methods Community Atmospheric Model version 5.3 (CAM5.3), and a horizontal resolution of approximately 1° and 60° vertical levels for the Parallel Ocean Program version 2 (POP2).h The experiments consisted of an AMO-positive and an AMO-negative experiment. In the pacemaker experi- ments, the North Atlantic (0–70° N) SST was restored to the model climatology plus the observed AMO-positive or AMO-negative SST pattern. The AMO’ SSTA pattern was obtained by regressing North Atlantic SSTAs onto the AMO index using 1900–2018 observations. The restoring timescale was set as 10 days, and 5 ensemble members were generated for each AMO-positive and AMO-negative experiment in this study.h g g y The climatological mean states and the external forcings (e.g., greenhouse gas, solar forcing, and aerosol) were fixed at preindustrial levels. Each member of the experiments was integrated for 110 years, with the model output from the last 80 years analyzed. Significance test. The statistical significance of correlation is examined using the two-tailed P-values from a Student’s t-test, and the degrees of freedom used in the test is determined by considering the auto-correlation of the tested variables. The calculation of the effective degrees of freedom (df) is determined according to the method proposed by Bretherton et al.52 and can be expressed as: df = N × ((1 −r1r2)/(1 + r1r2)) −2. Scientific Reports \| (2023) 13:13735 \| https://doi.org/10.1038/s41598-023-40979-9 www.nature.com/scientificreports/ The length of the year analyzed is denoted by N, while r1 and r2 represent the lag one autocorrelation coef- ficients of the two series under consideration in the regression or correlation analysis. Referencesi Oceans 118, 4305–4316 (2013).ii p y 2. Wu, C.-R. Interannual modulation of the Pacific decadal oscillation (PDO) on the low-latitude western North Pacific. Prog Oceanogr. 110, 49–58 (2013). g 3. Wu, C.-R., Chang, Y.-L., Oey, L.-Y., Chang, C.-W.J. & Hsin, Y.-C. Air-sea interaction between tropical cyclone Nari and Kuroshio Geophys. Res. Lett. 35, 3942 (2008). p y 14. Tsukamoto, K. Spawning of eels near a seamount. Nature 439, 929–929 (2006). p g 15. Han, Y. et al. Biogeographic distribution of the eel Anguilla luzonensis: Dependence upon larval duration and oceanic currents. Mar. Ecol. Prog. Ser. 551, 227–238 (2016). g 6. 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The AVISO, OISST, and NCEP r1 data were downloaded from https://www.aviso.altimetry.fr/en/data.html, and https://data.nodc.noaa.gov, respectively. The AMO and PDO indices were obtained from http://www.esrl. noaa.gov/psd/data/timeseries/AMO/, and http://research.jisao.washington.edu/pdo/, respectively. The CESM1 pacemaker simulations data used in this study can be obtained by contacting Yong-Fu Lin at yongfulin0711@ gmail.com. Received: 18 March 2023; Accepted: 19 August 2023 Referencesi References 1. Merrifield, M. A. A shift in Western Tropical pacific sea level trends during the 1990s. J. Clim. 24, 4126–4138 (2011).i 1. Merrifield, M. A. A shift in Western Tropical pacific sea level trends during the 1990s. J. Clim. 24, 4126 4138 (2011). 2. Qiu, B. & Chen, S. Multidecadal sea level and gyre circulation variability in the Northwestern tropical pacific ocean. J. Phys. Oceanogr. 42, 193–206 (2012). 3. Lee, E.-J., Ha, K.-J. & Jhun, J.-G. 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This research was supported by a contract from Central Weather Bureau of Taiwan under Grant IISI-202924. Y.-F. Lin and J.-Y. Yu were also supported by NSF Climate and Large‐Scale Dynamics Program under Grant 2109539. They sincerely acknowledge Dr. Tien-Yiao Hsu, Ms. Ting-Hui Lee and Mr. He‑Ming Xiao for fruitful discussion and designed the model experiments. Author contributions J.Y.Y., and Y.F.L. developed the initial concept for the study, and drafted the paper. Y.F.L. was responsible for executing the simulations, analyzing the data and preparing all figures. All authors contributed to the data inter- pretation, discussion of the dynamics, and improvement of this paper. Referencesi et al. Phytoplankton and bacterial responses to monsoon-driven water masses mixing in the Kuroshio Off the East Coas of Taiwan. Front. Mar. Sci. 8, 807 (2021). 25. Lui, H.-K. et al. Intrusion of Kuroshio helps to diminish coastal hypoxia in the coast of Northern South China Sea. Front. Mar. 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Geophys. Res. Lett. 28, 2077–2080 (2001).i p y 7. Mantua, N. J., Hare, S. R., Zhang, Y., Wallace, J. M. & Francis, R. C. A Pacific interdecadal climate oscillation with impacts on Salmon production. Bull. Am. Meteorol. Soc. 78, 1069–1079 (1997). © The Author(s) 2023 Competing interests h p g The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-40979-9. Correspondence and requests for materials should be addressed to J.-Y.Y. Correspondence and requests for materials should be addressed to J.-Y.Y. 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 nstitutional affiliations. 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https://openalex.org/W2531065778	https://www.nature.com/articles/srep34498.pdf	English	null	Effect of methyl salicylate (MeSA), an elicitor on growth, physiology and pathology of resistant and susceptible rice varieties	Scientific reports	2,016	cc-by	10,129	Kandaswamy Kalaivani1, Marimuthu Maruthi Kalaiselvi1 & Sengottayan Senthil-Nathan2 Methyl salicylate (MeSA) is a volatile organic compound synthesized from salicylic acid (SA) a plant hormone that helps to fight against plant disease. Seed treatment with MeSA, is an encouraging method to the seed industry to produce more growth and yield. The aim of our study is to find out the growth, development and disease tolerance of rice seed treated with different concentrations of MeSA. Also the seed treatments were studied to determine whether they directly influenced seedling emergence and growth in rice (Oryza sativa L) cultivars ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46’ under greenhouse condition. MeSA seed treatments at 25, 50, 75 and 100 mg/L significantly increased seedling emergence. Effects were stronger in IR 50, and IR 64 and the effects were dose dependent, although the relationship between dose and effect was not always linear. MeSA seed treated rice plant against bacterial blight were analyzed. Bacterial blight was more effectively controlled by the seed treated with 100 mg/L than others. These results suggest that seed treatment with MeSA alters plant physiology in ways that may be useful for crop production as well as protection. Plants are one of the basic staple food, for all forms of life. We depend on plants for food and medicine, plant derivatives, regulation of the hydrologic cycle. Plants are essential to the balance of nature1. For more than half of the world’s population “Rice is life”. Rice is an economic crop for the farmers and major food staple for the world. Rice provides the main source of food energy for at least one third of the world’s population2–4. Rice is an essential part of the daily meal in Southern and Eastern parts of India and Asia. However most the rice plant are affected by the Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) and it is one of the most important bacterial diseases of rice. Heavy yield losses due to X. oryzae was reported during last one decade3,5,6. y y y p g Seed germination is one of the early and vital stages in the plant life cycle. Improved germination of seeds is one process which can improve the economic success of crop by ultimately enhancing the yield. The process of seed germination may be regulated by external factors, including temperature, light, soil moisture and salinity, metal or mineral composition7,8. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Effect of methyl salicylate (MeSA), an elicitor on growth, physiology and pathology of resistant and susceptible rice varieties received: 29 July 2016 accepted: 14 September 2016 Published: 11 October 2016 received: 29 July 2016 accepted: 14 September 2016 Published: 11 October 2016 Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 Kandaswamy Kalaivani1, Marimuthu Maruthi Kalaiselvi1 & Sengottayan Senthil-Nathan2 p Treatments of seed include applications of chemical agents, biological substances, or physical processes, to increase agricultural production. The seed treatment helps to protect seedlings from insects and disease which pose devastating consequences to crop production during critical growth stages. The application of treatments to seeds promotes stronger and healthier roots, leading to uniform seedling emergence, with improved crop performance9,10. p Seed priming is a method of pre-sowing treatments that exposes seeds to a certain solutions that allows par- tial hydration until the germination process begins11–13.Various seed priming techniques have been reported to enhance germination and allow for more uniform seed emergence, increasing nutrient reserves through increased physiological activities and increased yield in the crop14. p y g y p Various common methods utilized for priming seeds includes: hydro priming, (soaking in water); osmo- priming (soaking seed in osmotic solutions); thermopriming (treatment of seed with low or high temperatures); 1Post Graduate and Research Centre, Department of Zoology, Sri Parasakthi College for Women, Courtrallam-627 802, Tirunelveli, Tamil Nadu, India. 2Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi – 627 412, Tirunelveli, Tamil Nadu, India. Correspondence and requests for materials should be addressed to S.S.-N. (email: senthil@msuniv.ac.in) Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 1 www.nature.com/scientificreports/ solid matrix priming (treatment of seed with solid matrices); and bio priming (hydration using biological com- pounds)15. Halo priming (soaking seed in salt solutions), and priming with plant growth hormones are also performed along with thermo priming, and on-farm priming16.h p g p g p g The mechanism of rice seed priming techniques which uses plant growth regulators may enhance rice perfor- mance. Plant hormones or bio-stimulants are also known to improve plant population and increase productiv- ity. Presoaking seeds with optimal concentrations of plant growth hormones, such as auxin (IAA), gibberellins (GA), abscisic acid, ethylene (ET), Salicylic acid (SA) and Jasmonic acid (JA) have been shown to benefit germi- nation performance and yield of some crop species growth under both normal and stress conditions17–19. The effects of seed priming by soaking seeds of sorghum, rice, or wheat in water, are reported to enhance germina- tion rates. Some strategies for more vigorous plants report improved seedling emergence under induced stress conditions20,21. SA is a natural phenolic compound that plays a role in the regulation of physiological and biochemical pro- cesses. Results G i Germination responses after seed treatment with MeSA on rice seed in greenhouse condition. The seed pretreatment of different concentration of MeSA (i.e. 0, 25, 50, 75 and 100 mg/L) had significant (P < 0.05) effects on the final germination percentage and germination rates (Figs 1 and 2). Treated seeds with MeSA had significantly higher percentage of germination than those in control. Treatment of ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46’ rice varieties with MeSA increased the rate of seedling emergence (Fig. 1A–F). Emergence of rice varieties were more pronounced in all MeSA treatment with 100 mg/L concentration being statistically different with all other treatments. For example 80% germination was observed within 8 days in IR 20 rice varieties after seed treated with 100 mg/L. All the rice varieties treated with MeSA, had greater emer- gence values compared with control rice plant. In ‘IR 20’ 20% emergence, 40% emergence, 60% emergence and 80% emergence occurred earlier in MeSA treated plants than in control plants (20%-F4,30 = 3.08; P < 0.0031; 40%- F4,30 = 2.75; P < 0.046, 60%- F4,30 = 3.56; P < 0.08 and 80%- F4,25 = 5.10; P < 0.004) (Fig. 2A) there was a trend towards faster emergence with increased concentration seed treatment rates. The same developments were observed in rice variety ‘IR 50’ seedling emergence (20%-F4,30 = 2.62; P < 0.055; 40%- F4,30 = 3.24; P < 0.025, 60%- F4,30 = 2.65; P < 0.053 and 80%- F4,29 = 4.24; P < 0.008) (Fig. 2B).ll , , Germination percentage was influenced by MeSA seed treatment, but influence was more pronounced in 100 mg/L than other treatments. Control seeds emerged at lower than 20% at 4 days. However most of the treated rice seeds germinated at 20%, except (IR 20 and ASD 19), at 4 days, being statistically significant. The same observation was noticed in rice varieties ‘IR 64’ (20%-F4,30 = 3.28; P < 0.024; 40%- F4,30 = 2.48; P < 0.065, 60%- F4,30 = 2.84; P < 0.041 and 80%- F4,26 = 2.33; P < 0.082) (Fig. 2C), ‘ASD 16’ (20%-F4,30 = 3.05; P < 0.032; 40%- F4,30 = 2.31; P < 0.081, 60%- F4,28 = 5.49; P < 0.002 and 80%- F4,13 = 2.95; P < 0.061) (Fig. Kandaswamy Kalaivani1, Marimuthu Maruthi Kalaiselvi1 & Sengottayan Senthil-Nathan2 SA is chemically known as 2-hydroxy benzoic acid is a member of phenolic compounds, containing of an aromatic ring bearing a hydroxyl group which is synthesized by the plant22. These hydroxyl groups are involved in lignin biosynthesis; others serve as allelopathic compounds, regulate plant responses to abiotic stress and disease resistance by inducing antimicrobial defense compounds termed phytoalexins or by signaling defense activation23–26. Exogenous application of SA act as potential antioxidant capacity inducers, also increase the levels of hydro- gen peroxide in plants. These pathways also regulate many physiological processes in plants, such as increased germination rate, cell growth, stomatal opening, photosynthesis and ion uptake27. Methyl salicylate (MeSA) is a volatile organic compound which is synthesized from SA. MeSA is associated with induced resistance plant defense which has been used in effective disease control methods28–30. f Seed treatment with MeSA can be used to enhance rice seed germination and seedling growth. We studied the performance of rice seed priming with different concentrations of MeSA (0, 25, 50, 75 and 100 mg/L). Variables assessed include: germination rate, growth of root and shoot heights (cm), biomass of fresh and dry weights of the shoots and roots (mg) disease incidence, mean lesion lengths, disease index and disease incidence of different rice varieties under greenhouse conditions. Results G i 2D).ht The germination rate of seeds was increased after treatment with MeSA in resistant rice varieties ASD 19 (20%-F4,30 = 3.06; P < 0.031; 40%- F4,30 = 3.80; P < 0.013, 60%- F4,28 = 5.61; P < 0.002 and 80%- F4,13 = 3.19; P < 0.049) (Fig. 2E) and ADT 46 (20%-F4,30 = 2.88; P < 0.040; 40%- F4,30 = 2.78; P < 0.045, 60%- F4,27 = 5.46; P < 0.002 and 80%- F4,11 = 3.17; P < 0.058) (Fig. 2F). Compared with control, the seeds showed greater germi- nation rates when treated with MeSA. The germination rates were significantly greater when treated with MeSA at 100 mg/L. g Seed-priming treatments with MeSA significantly enhanced the seedling growth of rice compared with non-treated control. The greatest effect was observed from seed-coating treatments of 100 mg/L MeSA (Fig. 3). On average, seed priming treatments significantly increased the shoot length by 40.78% (F4,30 = 14.88; P < 0.0001), 24.31% (F4,30 = 17.05; P < 0.0001), 20.90%(F4,30 = 10.73; P < 0.0001), 35.34% (F4,30 = 7.30; P < 0.0001), 25.90% (F4,30 = 7.88; P < 0.0001) and 34.82% (F4,30 = 11.10; P < 0.001) for IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46 respectively compared with control. The same trend was also noticed in root length after treatment with 100 mg/L MeSA. For example 54.46% increased root length was observed in ASD 16 after treatment with 100 mg/L MeSA which was significantly different from control (F4, 30 = 11.25; P < 0.0001). Insignificant difference was observed between treatments of 75 and 100 mg/L MeSA. Treatment of seeds with MeSA also improved root: shoot ratio of the rice seedlings under greenhouse condi- tion. For ‘ASD 16’, the effect of MeSA seed treatment was significant for root: shoot ratio (F4,30 = 4.44; P < 0.006) (Fig. 4A). Results G i Interestingly, there was a non-linear response for IR 50 and IR 64, root: shoot ratios and they were not significant between 25–100 mg/L treatments (F4, 30 = 3.73; P < 1.00 and F4, 30 = 3.98; P < 1.00 for IR 50 and IR 64 Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 2 www.nature.com/scientificreports/ 1 2 3 4 5 6 7 8 9 10 11 12 13 0 20 40 60 80 0 25 50 75 100 A 1 2 3 4 5 6 7 8 9 10 11 12 13 0 20 40 60 80 100 B 1 2 3 4 5 6 7 8 9 10 11 12 13 Percentage of emergecne 0 20 40 60 80 C 1 2 3 4 5 6 7 8 9 10 11 12 13 0 20 40 60 80 D Days 1 2 3 4 5 6 7 8 9 10 11 12 13 0 20 40 60 80 E 1 2 3 4 5 6 7 8 9 10 11 12 13 Percentage of emergecne 0 20 40 60 80 F Figure 1. Percentage of emergence of rice varieties treatment with MeSA across 12 days post treatment. (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-ASD 19; F-ADT 46). A 0 20 40 60 80 100 B A B 13 Percentage of emergecne C D Percentage of emergecne F Days Figure 1. Percentage of emergence of rice varieties treatment with MeSA across 12 days post treatment. (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-ASD 19; F-ADT 46). espectively) (Fig. 4A). Further we have observed significant growth of root and shoot morphologically after seed were treated with MeSA (Fig. 4B).if respectively) (Fig. 4A). Further we have observed significant growth of root and shoot morphologically after seed were treated with MeSA (Fig. 4B). All th d t t t h d i ifi t ff t f h d d i ht f h t d t (Fi 5) Sh t f h were treated with MeSA (Fig. 4B). All the seed treatments had significant effects on fresh and dry weight of shoot and root (Fig. 5). Shoot fresh weight significantly increased in seedlings raised from seeds treated with 75 and 100 mg/L MeSA. Results G i Maximum shoot fresh weight of 457.4 mg was attained in ASD 16 seeds treated with 100 mg/L MeSA after 20 days as com- pare to control (361.8 mg) (Fig. 5). However minimum shoot fresh weight of 270.66 mg was observed in con- trol seeds of IR 50 after 20 days. Shoot and root fresh and dry weights of ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46 treated and untreated plants are shown in Fig. 5A–X. Seed treatment with 100 mg/L MeSA signif- icantly increased shoot fresh weights (F4,20 = 14.69, P < 0.0001-IR 20; F4,20 = 5.75, P < 0.003-IR 50; F4,20 = 7.69, P < 0.001-IR 64; F4,20 = 7.18, P < 0.001-ASD 16; F4,20 = 3.22, P < 0.035-ASD 19; F4,20 = 3.91, P < 0.017-ADT 46) and dry weights (F4,20 = 24.08, P < 0.0001-IR 20; F4,20 = 69.90, P < 0.0001-IR 50; F4,20 = 7.88, P < 0.001-IR 64; F4,20 = 29.06, P < 0.001-ASD 16; F4,20 = 7.60, P < 0.001-ASD 19; F4,20 = 13.31, P < 0.0001-ADT 46) at 22 days post treatment. The same linear growth pattern was observed both in fresh and dry weight of root after seed treatment with MeSA. In general, a positive relationship was observed between seed treatment concentration rate and root and shoot weights (Fig. 5). g All the seed treatments had significant effects on fresh and dry weight of shoot and root (Fig. 5). Shoot fresh weight significantly increased in seedlings raised from seeds treated with 75 and 100 mg/L MeSA. Maximum shoot fresh weight of 457.4 mg was attained in ASD 16 seeds treated with 100 mg/L MeSA after 20 days as com- pare to control (361.8 mg) (Fig. 5). However minimum shoot fresh weight of 270.66 mg was observed in con- trol seeds of IR 50 after 20 days. Shoot and root fresh and dry weights of ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46 treated and untreated plants are shown in Fig. 5A–X. Results G i Seed treatment with 100 mg/L MeSA signif- icantly increased shoot fresh weights (F4,20 = 14.69, P < 0.0001-IR 20; F4,20 = 5.75, P < 0.003-IR 50; F4,20 = 7.69, P < 0.001-IR 64; F4,20 = 7.18, P < 0.001-ASD 16; F4,20 = 3.22, P < 0.035-ASD 19; F4,20 = 3.91, P < 0.017-ADT 46) and dry weights (F4,20 = 24.08, P < 0.0001-IR 20; F4,20 = 69.90, P < 0.0001-IR 50; F4,20 = 7.88, P < 0.001-IR 64; F4,20 = 29.06, P < 0.001-ASD 16; F4,20 = 7.60, P < 0.001-ASD 19; F4,20 = 13.31, P < 0.0001-ADT 46) at 22 days post treatment. The same linear growth pattern was observed both in fresh and dry weight of root after seed treatment with MeSA. In general, a positive relationship was observed between seed treatment concentration rate and root and shoot weights (Fig. 5). Control of bacterial blight under greenhouse condition. The effect of MeSA seed treatment in sup- pressing bacterial blight disease was analyzed under greenhouse condition. Bacterial blight disease lesion length (mm) (Fig. 6) and Disease index (Fig. 7) was found to be significantly lower in 100 mg/L MeSA seed treated rice Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 3 www.nature.com/scientificreports/ Figure 2. Days until 20%,40%, 60% and 80% emergence of rice varieties IR 20 (A), IR 50 (B), IR 64 (C), ASD 16 (D), ASD 19 (E), ADT 46 (F) under greenhouse condition after seed treatment with Me SA. Mean (±SEM) followed by the same letter in an individual experiment indicate no significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). Figure 2. Days until 20%,40%, 60% and 80% emergence of rice varieties IR 20 (A), IR 50 (B), IR 64 (C), ASD 16 (D), ASD 19 (E), ADT 46 (F) under greenhouse condition after seed treatment with Me SA. Mean (±SEM) followed by the same letter in an individual experiment indicate no significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). plant. However other lower seed treatments also found to be effective than control. The bacterial blight disease lesion length (mm) was 2.94 mm in control but the ADT 46 rice seed treated with 100 mg/L was inoculated with X. oryzae lesion length (mm) measured as 1.92 mm (F4,20 = 4.39, P < 0.01) (Fig. 6). Results G i However we did not observe any significant difference between control and 25 mg/L MeSA seed treated rice ADT 46 plant (F4,20 = 4.39, P < 0.93).hh The same trend was observed in disease index. The disease index (DI) was higher in (39.2%) control IR 20 plant and the DI was significantly lower in IR 20 rice plant in which the seed was treated with 100 mg/L MeSA (26.40%) (F4,20 = 6.33, P < 0.002) (Fig. 7). Even though three rice varieties (i.e. ASD 16, ASD 19 and ADT 46) are resistant to bacterial blight disease, seed treatment with MeSA further enhance the resistant against the bacterial blight disease and it was proved by the disease index experiment. Discussion T f Treatment of seeds with MeSA increased rate of germination and rice growth. The amount of effect differed among rice varieties and effects were dose rate dependent. Also, effects appeared to be more pronounced at 75 and 100 mg/L seed treatments under normal greenhouse conditions. MeSA influences seed germination, seedling establishment, cell growth, respiration, stomatal closure, senescence-associated gene expression, basal thermotol- erance, nodulation in legumes, and fruit yield25,31. Most consistent response to MeSA seed treatment in this research was an increase in rate of emergence of seeds and biomass of the rice plants after sowing. The effect of MeSA was stronger both in ‘IR 20 and IR 50’ than other rice varieties, which produced higher seedling emergence rates (Kalaivani and Senthil-Nathan, unpublished data).h This study shows that rice plant could be induced by exogenous application of the signaling compounds MeSA. The allelopathic potential of the root and shoot of rice thus increased after treatment with MeSA (Figs 1–5). The same effect was observed in the rice strains IAC165 and Huajingxian32. In the present study MeSA treatment with 100 mg/L of rice seed showed enhanced effects on emergence and biomass of rice seedlings. Discussion T f 15 10 5 0 5 10 15 A c bc ab b a 0 25 50 75 100 Root length in cm Shoot length in cm c bc ab b a 15 10 5 0 5 10 15 20 0 25 50 75 100 0 25 50 75 100 B ab ab c c bc ab a Root length in cm Shoot length in cm b a a 0 25 50 75 100 15 0 25 50 75 100 B 15 10 5 0 5 10 15 20 0 25 50 75 100 0 25 50 75 100 D bc bc c bc abc ab a Root length in cm Shoot length in cm c ab a 5 B 15 A 5 A 15 D 15 0 25 50 75 100 D 15 10 5 0 5 10 15 0 25 50 75 100 0 25 50 75 100 F c bc bc bc c ab a Root length in cm Shoot length in cm c ab a 0 25 50 75 10 C 15 10 5 0 5 10 15 0 25 50 75 100 0 25 50 75 100 E ab ab bc abc b ab a Root length in cm Shoot length in cm c ab a 15E Figure 3. Root and shoot length in cm of rice varieties 22 days post treatment with MeSA (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-ASD 19; F-ADT 46). Mean (±SEM) followed by the same letter in an individual experiment indicate no significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). also shown that natural plant products can enhance defense system of plant along with protection from pest and diseases33–35. Further plant hormonal and other treatments enhanced seedling vigor as measured in longer shoot and root lengths, with increased fresh and dry weights. Accelerated cell division and enlargement of cell explain the production of vigorous seedlings by pre-sowing seed treatment with plant growth regulators16. Several studies have shown impact of MeSA seed treatment on germination and early seedling growth. Enhanced germination, emergence and growth were recorded in wheat, Triticum aestivum L. when the seeds were subjected to pre-sowing seed-soaking treatment in salicylic acid36. Senthil-Nathan et al.37 reported that neem treatment on rice resulted in increased production and diseases tolerance than control. Hayat et al.38 observed the grains of wheat (Triticum aestivum L. cv. Discussion T f Other studies have Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 4 www.nature.com/scientificreports/ 15 10 5 0 5 10 15 0 25 50 75 100 A c bc ab b a 0 25 50 75 100 Root length in cm Shoot length in cm c bc ab b a 15 10 5 0 5 10 15 20 0 25 50 75 100 0 25 50 75 100 B ab ab c c bc ab a Root length in cm Shoot length in cm b a a 15 10 5 0 5 10 15 20 0 25 50 75 100 0 25 50 75 100 C ab ab c c bc ab a Root length in cm Shoot length in cm b a a 15 10 5 0 5 10 15 20 0 25 50 75 100 0 25 50 75 100 D bc bc c bc abc ab a Root length in cm Shoot length in cm c ab a 15 10 5 0 5 10 15 0 25 50 75 100 0 25 50 75 100 E ab ab bc abc b ab a Root length in cm Shoot length in cm c ab a 15 10 5 0 5 10 15 0 25 50 75 100 0 25 50 75 100 F c bc bc bc c ab a Root length in cm Shoot length in cm c ab a Figure 3. Root and shoot length in cm of rice varieties 22 days post treatment with MeSA (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-ASD 19; F-ADT 46). Mean (±SEM) followed by the same letter in an individual experiment indicate no significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 Discussion T f Root and shoot ratio of rice varieties (A) (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-AS after treatment with MeSA. Mean (±SEM) followed by the same letter in an individual experim significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). (B) Root a after treatment with MeSA of ASD 16 rice cultivar. IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 Root shoot ratio 0.0 0.5 1.0 1.5 2.0 2.5 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a ab ab ab b ab a b a a a a b a a b b b ab a a ab b ab ab a ab b ab ab A A Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 A Figure 4 Root and shoot ratio of rice varieties (A) (A IR 20; B IR 50; C IR 64; D ASD 16; E ASD 19; F ADT 46) Rice varieties A Figure 4. Root and shoot ratio of rice varieties (A) (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-ASD 19; F-ADT 46) after treatment with MeSA. Mean (±SEM) followed by the same letter in an individual experiment indicate no significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). (B) Root and shoot growth after treatment with MeSA of ASD 16 rice cultivar. linear with treatment doses. The percentage of emergence (20, 40, 60 and 80%) at the different seed treatment rates indicated that emergence increased with seed treatment concentration rate, with faster emergence from seeds treated with 100 mg/L MeSA. The increased percentage of emergence and biomass with increasing dose is probably due to increased SA in plant cells, as reported in other crops. For example, increased percentage of emergence and growth was observed by Singh and Usha40 and Dolatabadian et al.41 in wheat seeds, Tavares et al.28 in rice, Khodary42 in maize, Jadhav and Bhamburdekar43 and Szepesi et al.44 in tomato, Martin-Mex et al.45 in African violet, McCue et al.46 in pea seeds, Almeida et al.47 in wheat and barley seeds which revealed that MeSA accelerates germination greatly. Seed treatment with MeSA may help in the metabolic pentose phosphate path- way, benefiting the hydrolysis of reserves and increasing the availability of energy to the germination process and seedling emergence25. linear with treatment doses. Discussion T f Raj-3077) were soaked in 0, 10−5, 10−4 and 10−3 M aqueous solutions of seedlings possessed significantly greater leaf numbers, fresh and dry mass. The present finding gained support from these previous reports. p Shakirova et al.39 conclude that pre-sowing T. aestivum seed treatment with SA leads to an activation of ger- mination and seedling growth. The enhancement results in the division of root apical cells which is an important contribution to the growth stimulating effect of SA. Shakirova36 has confirmed with field experiments. In the field, wheat plants pretreated with SA were characterized with increased size of ears, grain yield, indicating pro- longed effect of pre-sowing treatment of seeds, which produced stimultative effect on the productivity of wheat, at harvest. Rate of seedling emergence was increased approximately 25% at seed treatment with 75 and 100 mg/L MeSA. Emergence of ‘IR 20, IR 50 and ASD 16’ seedlings was significantly faster in MeSA treated seeds, but it is not Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 5 www.nature.com/scientificreports/ Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 Root shoot ratio 0.0 0.5 1.0 1.5 2.0 2.5 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a ab ab ab b ab a b a a a a b a a b b b ab a a ab b ab ab a ab b ab ab A Figure 4. Root and shoot ratio of rice varieties (A) (A-IR 20; B-IR 50; C- IR 64; D-ASD 16; E-ASD 19; F-ADT 46) after treatment with MeSA. Mean (±SEM) followed by the same letter in an individual experiment indicate no significant difference (P < 0.05) in a Tukey’s test (Treatment concentration in mg/L). (B) Root and shoot growth after treatment with MeSA of ASD 16 rice cultivar. linear with treatment doses. The percentage of emergence (20, 40, 60 and 80%) at the differen rates indicated that emergence increased with seed treatment concentration rate with faster Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 Root shoot ratio 0.0 0.5 1.0 1.5 2.0 2.5 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a ab ab ab b ab a b a a a a b a a b b b ab a a ab b ab ab a ab b ab ab A Figure 4. Discussion T f The percentage of emergence (20, 40, 60 and 80%) at the different seed treatment rates indicated that emergence increased with seed treatment concentration rate, with faster emergence from seeds treated with 100 mg/L MeSA. The increased percentage of emergence and biomass with increasing dose is probably due to increased SA in plant cells, as reported in other crops. For example, increased percentage of emergence and growth was observed by Singh and Usha40 and Dolatabadian et al.41 in wheat seeds, Tavares et al.28 in rice, Khodary42 in maize, Jadhav and Bhamburdekar43 and Szepesi et al.44 in tomato, Martin-Mex et al.45 in African violet, McCue et al.46 in pea seeds, Almeida et al.47 in wheat and barley seeds which revealed that MeSA accelerates germination greatly. Seed treatment with MeSA may help in the metabolic pentose phosphate path- way, benefiting the hydrolysis of reserves and increasing the availability of energy to the germination process and seedling emergence25. g g Several reports were published in the last decade demonstrating the role of SA applied as seed soaking treat- ment on various physiological processes44–47. It was shown that the pre-soaking of pea seeds in SA had a beneficial effect on growth and photosynthesis48. f MeSA treatments produced measurable effects on root and shoot growth of rice seedling varieties. Gutierrez-Coronado et al.49 reported that foliar application of SA to the shoots of soybean plants positively affected the size of the root. Furthermore Sandoval-Yepiz50 reported similar findings with Tagetus erecta, using low concentrations (10−10 M) of SA. Increased shoot growth was observed in Clitoria sp. after application of Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 6 www.nature.com/scientificreports/ Figure 5. Fresh (A,E,I,M,Q,U) and dry (B,F,J,N,R,V) weight (mg) of shoot; fresh (C,G,K,O,S,W) and dry (D,H,L,P,T,X) weight (mg) of root of IR 20,IR 50, IR 64, ASD 16, ASD 19 and ADT 46 rice varieties after treatment with MeSA in greenhouse condition (-●-10th day -○- 12th day -▼- 14th day-△- 16th day -□- 18th day -■- 20th day -◆-22nd day) (MeSA treatment doses are mg/L). Figure 5. Fresh (A,E,I,M,Q,U) and dry (B,F,J,N,R,V) weight (mg) of shoot; fresh (C,G,K,O,S,W) and dry (D,H,L,P,T,X) weight (mg) of root of IR 20,IR 50, IR 64, ASD 16, ASD 19 and ADT 46 rice varieties after treatment with MeSA in greenhouse condition (-●-10th day -○- 12th day -▼- 14th day-△- 16th day -□- 18th day -■- 20th day -◆-22nd day) (MeSA treatment doses are mg/L). Discussion T f Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 Lesion length(mm) 1 2 3 4 5 6 7 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a ab ab b b ab a bc ab bc a a b c ab bc ababc ab c a ab b ab ab a ab b ab a Figure 6. Mean lesion length (mm) caused by X. oryzae after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L). Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 DI % 10 20 30 40 50 60 70 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a abc ab bc c ab a ab ab b a a b b ab bc ab abc ab c a ab b ab ab a ab b b ab Figure 7. Disease index (DI in percentage) of rice after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L) Fig. 1. Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 Lesion length(mm) 1 2 3 4 5 6 7 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a ab ab b b ab a bc ab bc a a b c ab bc ababc ab c a ab b ab ab a ab b ab a Figure 6. Mean lesion length (mm) caused by X. oryzae after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L). Figure 6. Mean lesion length (mm) caused by X. oryzae after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L). Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 DI % 10 20 30 40 50 60 70 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a abc ab bc c ab a ab ab b a a b b ab bc ab abc ab c a ab b ab ab a ab b b ab Figure 7. Disease index (DI in percentage) of rice after treatment with MeSA. Discussion T f Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L) Fig. 1. Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 DI % 10 20 30 40 50 60 70 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a abc ab bc c ab a ab ab b a a b b ab bc ab abc ab c a ab b ab ab a ab b b ab Figure 7. Disease index (DI in percentage) of rice after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L) Fig. 1. Figure 7. Disease index (DI in percentage) of rice after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L) Fig. 1. responses to these proteins and these events provide significant increases in production and reduce time for the establishment of the crop field, being more tolerant against abiotic stresses. The data of the present study support the above hypothesis. yp For more than two decades the plant hormone salicylic acid (SA) and its derivatives (MeSA) has been stud- ied for its use to protect the plant. However, its systemic acquired signaling act in plants, particularly in defense against plant disease causing microbes, has only become more interest during last two decades31,57. Our research has clearly pointed out the MeSA has a potential to control the bacterial blight disease. Also seed treated with 100 mg/L has less blight lesion and diseases index. It was evident that SA and its derivatives act as a critical defense signal has come from analyses of plants in which endogenous SA levels were triggered or altered. It was proved in series of experiments in Arabidopsis (Transgenic tobacoo) expressing the bacterial nahG gene, encoding the SA-metabolizing enzyme salicylate hydroxylase31,58,59. In addition, endogenous SA levels increase in barley resist- ing infection by the bacterial pathogen P. syringae pv. syringae60. Also in rice homolog enhances resistance to the bacterial blight-causing X. oryzae61–63.i MeSA seed treatments (75 and 100 mg/L) significantly increased the emergence and early growth of rice plants. But the effect was non-linear across treatment concentrations and rice varieties. Discussion T f salicylates51. A more in-depth study would be required to determine the specific modes of action of MeSA on non-linear growth of roots and shoots in different crops. The increase observed in shoot and root weight when treated with MeSA, was dose dependent (75 and h salicylates51. A more in-depth study would be required to determine the specific modes of action of MeSA on non-linear growth of roots and shoots in different crops. f The increase observed in shoot and root weight when treated with MeSA, was dose dependent (75 and 100 mg/L). This response suggests that a greater portion of photosynthates was distributed to the roots than the shoots in seedlings developing from nontreated seeds as compared with seedlings developing from MeSA treated seeds25,36,52. The promoting effect of SA on the leaf area was attributed to its important roles on activating cell division, biosynthesis of organic foods, water uptake and stomatal resistance and tolerating water deficit better. In addition, mentioned that improving effect of SA on the ready availability of nutrients (i.e. N, P, K, Ca, and Mg) to the plant. Increased levels of mineral after treatment with SA were observed in maize53, tomato44 and cucumber54. These results are in support with many other researchers who reported an increased amount of nutrients in roots and shoot after treatments with SA27,55,56. t According to Stout et al.57 and Vazirimehr et al.27 plant seeds treated with SA have higher levels of amino acids, plant growth, ion uptake, transport enzyme activity and synthesis of plant hormones, that increase the plant Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 7 www.nature.com/scientificreports/ Rice varieties IR20 IR50 IR 64 ASD 16 ASD 19 ADT 46 Lesion length(mm) 1 2 3 4 5 6 7 0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L a ab ab b b ab a bc ab bc a a b c ab bc ababc ab c a ab b ab ab a ab b ab a Figure 6. Mean lesion length (mm) caused by X. oryzae after treatment with MeSA. Means followed by the same letter were not significantly different (P < 0.05) according to a Tukey test) (Treatment concentration in mg/L). Plant material and seed treatments Plant material and seed treatments. A commercial formulation of methyl salicylate Plant material and seed treatments. A commercial formulation of methyl salicylate (Methyl salicylate, Sigma-Aldrich) was used for all experiments. The rice varieties ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46’ were used for this study. ‘IR 20, IR 50, IR 64’ are susceptible, conventional, semi-dwarf, long-grain aromatic variety with relatively high seedling vigor, whereas ‘ASD 16, ASD 19 and ADT 46’ are moderate resistance, con- ventional, semi-dwarf long grain variety with low seedling vigor. Rice were surface disinfected by immersing in 2% sodium hypochlorite for about two minutes and washed several times with sterilized water, then dried in sterilized filter paper. i p p Salicylate formulations and treatments consisted of increasing levels of 0, 25, 50, 75 and 100, mg/L of MeSA. A stock solution of MeSA 100 mg/L was used for successive dilutions in distilled water. The seeds were treated by applying 2 ml/kg of seed in each respective MeSA concentration according to methodology described by Tavares et al.28. The solution of MeSA with the different doses was directly placed at the bottom of the zip lock plastic bag. Then 250 g of seeds were added into each bag. They were agitated for 10 minutes. The seeds were spread out on clean paper and left to dry at room temperature for 24 hours. Treated seeds were used for further experiments. Seed treatments under greenhouse conditions. ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46’ rice plants were grown by sowing five seeds of a single treatment rate (0, 25, 50, 75 and 100 mg/L) in 0.5 L pots in a greenhouse. There were 7 pots (replicates) per treatment. The potting soil was composed of two parts of auto- claved silt loam soil with one part each of sand and peat moss. The experiment was conducted in early January and, as temperature regulation in the greenhouse was poor, temperatures fluctuated from 22° to 32 °C. Lighting was ambient, and pots were watered as needed.th Pots were observed daily for emergence of hypocotyls above the surface of the soil after sowing. The experi- ment was terminated after 12 days. For statistical analysis, days until 20%, 40% and 60% seedling emergence were calculated for each pot. At 12th day after sowing, all seedlings were removed from pots, counted, and cleaned with water. Discussion T f Further we conclude that use of MeSA in seed treatment has great potential for increasing uniform emergence, root and shoot length, root and shoot biomass. Thus, treatment of seeds with SA may produce favorable effects on rice growth. Also MeSA seed treatment has better protection against blight diseases. 8 Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 www.nature.com/scientificreports/ = × DI(%) Number of leaves showing symptoms Total number of leaves inoculated 100 (1) (1) mean variance of disease index and disease incidence of five replications containing five plants in each pot were determined by Tukey-family error test (P < 0.05) by using Minitab®17 = + + + + × n n n n n Mean lesion lengths (MLL) 5 100 (2) 1 2 3 4 5 (2) Statistical analysis. Treatment effects on the percentage of emergence, root and shoot weight, and root: shoot ratio, in rice plants were analyzed by analysis of variance (one-way ANOVA), and the treatment means were compared by Tukey-family error test (P < 0.05) by using Minitab®17 software package. The data on above said experiments were arcsine transformed (except percentage of emergence) before undergoing statistical analysis. ffect of MeSA seed treatment on the bacterial blight disease of rice under greenhouse conditions d d h h Th l d f f l d Effect of MeSA seed treatment on the bacterial blight disease of rice under greenhouse conditions. Rice seed treated with MeSA were sown in earthen pots. The potting soil was composed of two parts of autoclaved silt loam soil with one part each of sand and peat moss and maintained under greenhouse conditions. Five rep- licates of five pots, each containing 5 plants, were maintained. X. oryzae inoculum was sub-cultured and grown individually in nutrient broth for 48 h and then centrifuged for 8000 × g for 15 m and then resuspended in sterile distilled water at 2 × 107 CFU/ml. Inoculation was done on twenty-day old plant leaves by scissors-dip method30. In each pot, the number of plants showing symptoms was counted and lesion length was measured at 14 days post-inoculation. The values are represented as percent disease incidence (DI) (Formula 1) and mean lesion length (MLL) (Formula 2). = × DI(%) Number of leaves showing symptoms Total number of leaves inoculated 100 (1) References References 1. Larcher, W. Physiological plant ecology: ecophysiology and stress physiology of functional groups (Springer Science & Business Media, 2003). 2. Fresco, L. Rice is life. Journal of Food Composition and Analysis 18, 249–253 (2005). 3. Senthil-Nathan, S. Biology, Behavioral and Population Dynamics of the Rice Leaffolder Complex: Dynamics of Insect Behavior (Scientific Publishers, Jodphur, pp. 156–167, 2011). 4. Senthil-Nathan, S., Kalaivani, K. & Murugan, K. Behavioural responses and changes in biology of rice leaffolder following treatment with a combination of bacterial toxin and botanical pesticides. Chemosphere 64, 1650–1658 (2006). 5. 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On-farm seed priming: using participatory methods to revive and refine a key technology. Agri. Sys. 69, 151–164 (2001).fl ( ) 1. Moghanibashi, M., Karimmojeni, H., Nikneshan, P. & Behrozi, D. Effect of hydropriming on seed germination indices of sunflowe (Helianthus annuus L.) under salt and drought conditions. Plant Knowl. J. www.nature.com/scientificreports/ Effects of Melia azedarach on nutritional physiology and enzyme activities of the rice leaffolder Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae). Pestic. Biochem. Physiol. 84, 98–108 (2006). f medinalis (Guenée) (Lepidoptera: Pyralidae). Pestic. Biochem. Phy y y 36. Shakirova, F. In Salicylic acid: a plant hormone 69–89 (Springer, 2007).f 36. Shakirova, F. In Salicylic acid: a plant hormone 69–89 (Springer, 2007).f 7. Senthil-Nathan, S. et al. Toxicity and physiological effects of neem pesticides applied to rice on the Nilaparvata lugens Stal, the brown planthopper. Ecotoxicol. Environ. 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Effect of plant compounds on induced activities of defense-related enzymes and pathogenesis related protein in bacterial blight disease susceptible rice plant. Physiol. Mol. Plant Pathol. 80, 1–9 (2012).ff g p p y , ( ) 7. Kuo, Y.-H., Rozan, P., Lambein, F., Frias, J. & Vidal-Valverde, C. Effects of different germination conditions on the contents of free protein and non-protein amino acids of commercial legumes. Food Chem. 86, 537–545 (2004).l p p g , ( ) 8. Ghaderi-Far, F., Gherekhloo, J. & Alimagham, M. Influence of environmental factors on seed germination and seedling emergence of yellow sweet clover (Melilotus officinalis). Planta Daninha 28, 463–469 (2010).f 8. Ghaderi-Far, F., Gherekhloo, J. & Alimagham, M. Influence of environmental factors on seed germination and seedling emergence of yellow sweet clover (Melilotus officinalis). Planta Daninha 28, 463–469 (2010). 9 Li L t l Eff t f ld l t t t d i ti d dli th f b S i R 4 (2014) Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 9 Acknowledgements g Authors would thank authority of Sri Parasakthi College for Women and Sri Paramakalyani Centre for Environmental Sciences for providing facility to conduct the experiments. Authors would grateful to Dr.W.B. Hunter for his comments over the first draft of the manuscript. We thank. Prof. R.K. Murali Baskaran, Prof. S. Suresh and Dr. Ilanchezhian for generously providing seeds of rice varieties. Authors would thank authority of Sri Parasakthi College for Women and Sri Paramakalyani Centre for Environmental Sciences for providing facility to conduct the experiments. Authors would grateful to Dr.W.B. Hunter for his comments over the first draft of the manuscript. We thank. Prof. R.K. Murali Baskaran, Prof. S. Suresh and Dr. Ilanchezhian for generously providing seeds of rice varieties. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5. Shi, Q. & Zhu, Z. Effects of exogenous salicylic acid on manganese toxicity, element contents and antioxidative system in cucumber Environ. Exp. Botany 63, 317–326 (2008).f p y 56. Karlidag, H., Yildirim, E. & Turan, M. Salicylic acid ameliorates the adverse effect of salt stress on strawberry. Sci. Agri. 66, 180 (2009).hh (2009). 57. Stout, M. J., Thaler, J. S. & Thomma, B. P. Plant-mediated interactions between pathogenic microorganisms and herbivorous arthropods. Annu. Rev. Entomol. 51, 663–689 (2006).f p Gaffney, T. et al. Requirement of salicylic acid for the induction of s l k d h l l f y q y y q , ( ) 59. Delaney, T. P., Uknes, S., Vernooij, B. & Friedrich, L. A central role of salicylic acid in plant disease resistance. Science 266, 1247 (1994). f 9. Delaney, T. P., Uknes, S., Vernooij, B. & Friedrich, L. A central role of salicylic acid in plant disease resistance. Science 266, 1247 (1994). 0 Vallélian-Bindschedler, L , Métraux, J -P & Schweizer, P Salicylic acid accumulation in barley is pathogen specific but not required ( ) 0. Vallélian-Bindschedler, L., Métraux, J.-P. & Schweizer, P. Salicylic acid accumulation in barley is pathogen specific but not required for defense-gene activation. Mol. Plant-Microbe Inter. 11, 702–705 (1998). g 61. Chern, M., Fitzgerald, H. A., Canlas, P. E., Navarre, D. A. & Ronald, P. C. Overexpression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light. Mol. Plant-Microbe Inter. 18, 511–520 (2005). p yp y g , ( ) 62. Chern, M. S. et al. Evidence for a disease‐resistance pathway in rice similar to the NPR1‐mediated signaling pathway in Arabidopsis. Plant J. 27, 101–113 (2001). Plant J. 27, 101 113 (2001). 3. Yuan, Y. et al. Functional analysis of rice NPR1‐like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility Plant Biotechnol J 5 313–324 (2007) 63. Yuan, Y. et al. Functional analysis of rice NPR1‐like genes reveals that OsNPR1/NH1 is the rice orthologue conferring di resistance with enhanced herbivore susceptibility. Plant Biotechnol. J. 5, 313–324 (2007). Author Contributions K.K., M.M.K. and S.S.-N. designed the experiments; K.K. planted the rice and conducted the experiments, inoculated BB and sampled; K.K. carried out experiments and M.M.K. analyzed experimental results. K.K. interpreted the data and wrote the manuscript; S.S.-N. revised and approved the final manuscript. K.K., M.M.K. and S.S.-N. designed the experiments; K.K. planted the rice and conducted the experiments, inoculated BB and sampled; K.K. carried out experiments and M.M.K. analyzed experimental results. K.K. interpreted the data and wrote the manuscript; S.S.-N. revised and approved the final manuscript. www.nature.com/scientificreports/ Almeida, A. S. et al. Physiological performance of wheat and barley seeds treated with bioactivator. Am. J. Exp. Agri 2, 90 (2012). 47. Almeida, A. S. et al. Physiological performance of wheat and ba 48. Wang, W. et al. Pre-sowing seed treatments in direct-seeded early rice: consequences for emergence, seedling growth and assoc metabolic events under chilling stress. Sci. Rep. 6 (2016).f 9. Gutiérrez-Coronado, M. A., Trejo-López, C. & Larqué-Saavedra, A. Effects of salicylic acid on the growth of roots and shoots in soybean. Plant Physiol. Biochem. 36, 563–565 (1998). y y 50. Sandoval-Yapiz, M. Reguladores de crecimiento XXIII: efecto del acido salicilico en la biomasa del cempazuchitl (Tagetes erecta), Tesis de Licenciatura Instituto Tecnologico Agropecuario, Conkal, Yucatan, Mexico (2004). 51. Martin-Mex, R. & Larqué-Saavedra, A. In 28th Annual Meeting Plant Growth Regulation Society of America, Miami Beach Flo USA pp. 97–99 (2001). . Larqué-Saavedra, A. & Martin-Mex, R. In Salicylic Acid: A Plant H 52. Larqué-Saavedra, A. & Martin-Mex, R. In Salicylic Acid: A Plant Hormone. 15–23 (Springer, 2007). y g 53. Gunes, A. et al. Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress an nutrition in maize (Zea mays L.) grown under salinity. J. Plant Physiol. 164, 728–736 (2007).f ( y ) g y J y , ( ) 54. Yildirim, E., Turan, M. & Guvenc, I. Effect of foliar salicylic acid applications on growth, chlorophyll, and mineral content of cucumber grown under salt stress. J. Plant Nutr. 31, 593–612 (2008). Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 10 Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Kalaivani, K. et al. Effect of methyl salicylate (MeSA), an elicitor on growth, physiol and pathology of resistant and susceptible rice varieties. Sci. Rep. 6, 34498; doi: 10.1038/srep34498 (2016). How to cite this article: Kalaivani, K. et al. Effect of methyl salicylate (MeSA), an elicitor on growth, physiology and pathology of resistant and susceptible rice varieties. Sci. Rep. 6, 34498; doi: 10.1038/srep34498 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports \| 6:34498 \| DOI: 10.1038/srep34498 11
https://openalex.org/W4226057621	https://www.preprints.org/manuscript/202112.0463/v1/download	English	null	Examine the Association between Metabolic Syndrome and Frailty in an Older Asian Population	Diabetology	2,022	cc-by	5,377	Hiep HH Dao BMedSci 1, Anh Trung Nguyen MD PhD 2, Huyen Thi Thanh Vu MD PhD 2 and Tu Ngoc Nguyen MD PhD 1 1 Westmead Applied Research Centre, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia. 2 Department of Geriatrics, Hanoi Medical University, and Hanoi Medical University Hospital, Hanoi, Viet Nam * Correspondence: Dr. Tu Nguyen, The University of Sydney, Faculty of Medicine and Health, Westmead Applied Research Centre, Australia. Email: n.nguyen@sydney.edu.au. 1 Westmead Applied Research Centre, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia. 2 Department of Geriatrics, Hanoi Medical University, and Hanoi Medical University Hospital, Hanoi, Viet Nam Nam * Correspondence: Dr. Tu Nguyen, The University of Sydney, Faculty of Medicine and Health, Westmead Applied Research Centre, Australia. Email: n.nguyen@sydney.edu.au. Abstract: Background: There has been evidence that metabolic syndrome (MetS) may increase the risk of frailty. However, there is limited evidence on this association in Asian populations. Aims: This study aims to identify the association between MetS and frailty in older people in Vietnam. Methods: This is a cross-sectional analysis of a dataset obtained from an observational study on frailty and sarcopenia in patients aged ≥60 at a geriatric hospital in Vietnam. Frailty was defined by the frailty phenotype. Participants were defined as having MetS if they had ≥3 out of 5 criteria from the definition of the National Cholesterol Education Program (NCEP) Adults Treatment Panel (ATP) III. Multiple logistic regression models were performed to estimate the risk of having frailty in patients with MetS. Results: There were 669 participants (mean age 71, 60.2% female), 62.3% had MetS and 39.0% was frail. The prevalence of frailty was 42.2% in participants with MetS, 33.7% in participants without MetS (p=0.029). On logistic regression models, MetS was associated with in- creased likelihood of being frail (adjusted OR 1.52, 95%CI 1.01-2.28), allowing for age, sex, educa- tion, nutritional status, history of hospitalisation and chronic diseases. Conclusion: There was a significant association between MetS and frailty in this population. Further longitudinal studies are required to confirm this association. Keywords: metabolic syndrome; diabetes; hypertension; obesity; frailty; older people; Asian. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 Type of the Paper Article Type of the Paper Article Examine the association between metabolic syndrome and frailty in an older Asian population ci 1, Anh Trung Nguyen MD PhD 2, Huyen Thi Thanh Vu MD PhD 2 and Tu Ngoc Nguyen 2.1. Participants This study was a secondary analysis, based on a primary study investigating the prevalence of sarcopenia in older patients in Vietnam. The details of this study were de- scribed in a previous publication 19. Consecutive patients aged 60 years or above at a ger- iatric hospital in Vietnam were recruited from 1/2018 to 10/2018. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 characterised by hypertriglyceridaemia, central adiposity, hypercholesterolaemia, insulin resistance and hypertension5. According to the classification framework established by the National Cholesterol Education Program (Adult’s Treatment Panel III), the definition of metabolic syndrome warrants the presence of ≥ 3 of the aforementioned adverse fea- tures.6 Several studies have reported the association between frailty and metabolic syn- drome. However, the majority of relevant literature regarding this topic focuses on Cau- casian population and there is limited evidence on this topic in the context of other ethnic groups, in particular in Asian populations.7-15 g p p p p Therefore, this study aims to explore this discrepancy, by investigating whether there is a significant correlation between MetS and frailty in older people in Vietnam - a country situated in Southeast Asian region. Previous studies in Vietnam showed that the preva- lence of frailty was 21.7% in community-dwelling older people 16, and 32%-55% in older hospitalised patients. 17,18 We hypothesised that in older people, MetS is associated with increased risk of acquiring frailty. 1. Introduction In the recent years, with ageing and considerable changes in population structure worldwide, ranging from higher-income regions to the lesser-developed nations, frailty has emerged as a public health interest due to its debilitating impacts on older people.1,2 Frailty is characterised by a gradual depletion in physiological reserve and homeostatic tolerance following exposure to stressors people.2 A recent systematic review of studies across 62 countries showed that frailty was present in 12% to 24% in community-dwelling populations.1 Such common geriatric condition predisposes older people to various ad- verse health outcomes, including fall, delirium, hospitalisation or even death and thus is regarded as a crucial transition between healthy ageing and disability.2 Even though frailty can be characterised by a plethora of frameworks proposed by different organisa- tions, Fried’s phenotypic classification framework remains prominent owing to its practi- cality. In 2001, Fried and her colleagues proposed phenotypic frailty criteria for frailty, thus enabling classification of older people based on their frailty status, taking into ac- count five adverse health features: unexplained weight loss, exhaustion, reduced physical activity, low grip strength and slow gait speed.3 On the other hand, metabolic syndrome (MetS), or so-called Syndrome X is also a predominant disorder in the elders.4 MetS describes a constellation of metabolic disorders, © © 2021 by the author(s). Distrib Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 All measurements were obtained either through direct measurements by the research staffs (waist circumference, blood pressure) or blood test results (BGL, serum HDL, TG). Frailty definition: Frailty was defined by the frailty phenotype (Fried’s frailty criteria), which was composed of five components: shrinking, weakness, exhaustion, slowness and low physical activity.3 Patients were classified as being frail if they met 3 or more out of the 5 components (1-2: prefrail, 0: robust). However, for the purpose of this study, frailty was treated as a binary variable, with presence of ≥ 3 features serves as a cut-off for acquisition of frailty.3 (1) Shrinking: defined as unintentional weight loss of ≥5% (or 4.5 kg) in the last year or BMI<18.5 (2) Weakness: cut-off points for low grip strength were <28kg in men and <18kg in women, as outlined by the Asian Working Group for Sarcopenia (AWGS) in 2019.21 (3) Self-reported exhaustion: In the primary study, participants were asked to com- plete the Centre for Epidemiological Studies’ Depression scale. Self-reported exhaustion was defined if a participant answered “occasionally or a moderate amount of time (3-4 days)”, or “all of the time” (7 days) after being asked “whether you felt that everything you did was an effort” for the past week. (4) Slowness based on walking speed: Gait speed was deemed “slow” if the partic- ipants mobilised by ≤ 0.8 m/s in the 4-meter walking test. (5) Low physical activity: The International Physical Activity Questionnaire (IPAQ) was used to measure physical activity level of the participants.22 Low physical activity was defined as a total score of <600 MET-minutes per week.22 2.2. Data collection Data in the primary study were collected from patients’ medical records and meas- urements. Information from medical records were extracted by the Vietnamese investiga- tors using a pre-defined data collection form, including demographic characteristics and medical history. Comorbidities were obtained based on a pre-defined list. Nutritional sta- tus was evaluated using the Mini Nutritional Assessment Short Form (MNA-SF) tool (maximum score of 14 points, and a total score of ≤7 points was indicative of a malnour- ished status 20). Participants’ weight (in kg) and height (in m) were measured following standard procedures. Body mass index (BMI) was calculated as weight/height2 (kg/m2) and participants were categorised into three groups: underweight (BMI <18.50), normal (BMI 18.50 – 24.99) and overweight (BMI ≥25.00). Participants’ handgrip strength was measured using a dynamometer (Jamar TM Hydraulic Hand Dynamometer 5030 J1, made in the USA) when participants were sitting with their elbows flexed at a 90-degree angle. Grip strength measurements were taken once on each hand and the highest value of the two readings was recorded and utilised for analysis. Metabolic syndrome definition: Metabolic syndrome (MetS) was defined according to the revised framework by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III6. The definition incorporates five major metabolic disturbances: ab- dominal obesity, hyperglycaemia, hypertriglyceridaemia, low high-density lipoprotein (HDL) cholesterol and hypertension. Presence of at least three out of the five factors con- fers the acquisition of MetS. (1) Abdominal obesity: defined as waist circumference ≥102 cm in men, and ≥88 cm in women. (1) Abdominal obesity: defined as waist circumference ≥102 cm in men, and ≥88 cm in women. (2) Hyperglycaemia: fasting serum blood glucose (BGL) of ≥100 mg/dL or a concur- rent history of diabetes (2) Hyperglycaemia: fasting serum blood glucose (BGL) of ≥100 mg/dL or a concur- rent history of diabetes (3) Hypertriglyceridaemia: defined as serum triglyceride (TG) level of ≥150 mg/dL (3) Hypertriglyceridaemia: defined as serum triglyceride (TG) level of ≥150 mg/dL (4) Low HDL cholesterol: defined as serum HDL cholesterol level of <40 mg/dl in g y g (4) Low HDL cholesterol: defined as serum HDL cholesterol level of <40 mg/dl in men or <50 mg/dl in women. g (5) Hypertension: defined as having systolic blood pressure (SBP) of ≥130 mmHg or diastolic blood pressure (DBP) of ≥85 mmHg or concurrent history of hypertension. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 A total of 996 participants were recruited in the primary study. Among these, data of frailty and metabolic syndrome were available for 669 participants (n = 669) whose char- acteristics were outlined in Table 1. The participants had a mean age of 71.1±8.5. Among the 669 participants, 60.2% were females and 39.0% were classified as being frail. The most common chronic health condi- tions were chronic kidney disease (CKD, 52.9%), followed by hypertension (47.5%), chronic obstructive pulmonary disease (COPD, 37.5%) and diabetes (19.9%). Chronic health conditions were more prevalent in the frail population; however, such associations were only significant in hypertension, heart failure, stroke, CKD, cancer and dementia (p<0.05). Furthermore, the prevalence of low educational status (37.9% in frail versus 16.4% in non-frail) and recent hospitalisations (64.0% in frail versus 33.6% in non-frail) were around two-fold higher in the frail participants compared to their non-frail counter- parts (p<0.001). Frailty was also associated with a significantly higher rate of malnutrition at 18.0% (compared to 2.5% in non-frail participants, p<0.001) within the study popula- tion. Table 1. Participant characteristics. Table 1. Participant characteristics. All (N=669) Nonfrail (N=408) Frail (N=261) P-val- ues Age (years) 71.11 ± 8.55 68.88 ± 7.40 74.65 ± 9.04 <0.001 Female 403 (60.2%) 244 (59.8%) 159 (60.9%) 0.774 Body mass index (kg/m2) 21.77 ± 3.43 22.49 ± 2.86 20.64 ± 3.92 <0.001 Low education 166 (24.8%) 67 (16.4%) 99 (37.9%) <0.001 Malnutrition (MNA <7) 57 (8.5%) 10 (2.5%) 47 (18.0%) <0.001 Having history of hospitaliza- tion in the past year 304 (45.4%) 137 (33.6%) 167 (64.0%) <0.001 Chronic health conditions: Hypertension 318 (47.5%) 177 (43.4%) 141 (54.0%) 0.007 Diabetes 133 (19.9%) 73 (17.9%) 60 (23.0%) 0.107 Myocardial infarction 18 (2.7%) 9 (2.2%) 9 (3.4%) 0.333 Heart failure 10 (1.5%) 3 (0.7%) 7 (2.7%) 0.043 Stroke 25 (3.7%) 10 (2.5%) 15 (5.7%) 0.028 Peripheral vascular disease 30 (4.5%) 16 (3.9%) 14 (5.4%) 0.379 Chronic kidney disease 354 (52.9%) 179 (43.9%) 175 (67.0%) <0.001 Chronic obstructive pulmo- nary disease 251 (37.5%) 143 (35.0%) 108 (41.4%) 0.099 Cancer 10 (1.5%) 2 (0.5%) 8 (3.1%) 0.007 Dementia 4 (0.6%) 0 (0) 4 (1.5%) 0.012 Continuous data are presented as mean ± standard deviation. Categorical data are shown as n (%). MNA, Mini Nutritional Assessment. SBP, systolic blood pressure. DBP, diastolic blood pressure. HDL, high-density lipoprotein. SBP, systolic blood pressure. DBP, diastolic blood pressure. HDL, high-density lipoprotein. 2.3. Statistical analysis Binary variables are presented as frequency and percentage, and continuous varia- bles are presented as mean and standard deviation. Frailty was treated as a binary variable (yes/no) with the presence of ≥3 adverse features confirming the frailty status. Compari- sons between participants with and without frailty were assessed using Chi-square tests for binary variables and Student’s t-tests for continuous variables. Two-tailed P values < 0.05 were deemed statistically significant. To examine the relationship between metabolic syndrome and frailty, univariate lo- gistic regression was conducted with frailty as the dependent variable and metabolic syn- drome as the independent variable of interest. Univariate logistic regressions were also performed for other factors that can be associated with frailty, such as age, sex, education, living condition, comorbidities. The relationship between metabolic syndrome and frailty was then examined further in multivariable logistic regression models controlling for fac- tors that had a p-value <0.05 on the univariate logistic regressions. Results were presented as odds ratios (ORs) and 95% confidence intervals (CIs). All variables were checked for multicollinearity and interactions. Data analysis was conducted using SPSS for Windows 20.0 (IBM Corp., Armonk, NY, USA). Sample size justification: The sample size of this study was estimated based on the Longitudinal Aging Study Amsterdam. Their baseline data showed that the prevalence of frailty in older participants with MetS was 16.7%, and 8.8% in those without MetS. Therefore, we estimated that a sample size of at least 558 participants will enable the detection of a significant difference in frailty prevalence between older people with and without MetS (at 80% power, 5% significance level) 3. Results 3. Results Table 2. The prevalence of metabolic syndrome and its components by gender. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 MetS was present in 62.3% of the participants. The prevalence of MetS was signifi- cantly higher in frail participants (67.4% compared to 59.1% in non-frail participants, p=0.029). The prevalence of MetS and its constitutive components by gender is demon- strated in Table 2. Among the individual components of MetS, elevated blood pressure (67.3%) was the most prevalent, followed by low HDL-C (66.8%), high TG (63.2%). ele- vated fasting plasma glucose (61.3%), and central obesity (25.0%). MetS was more preva- lent in women (71.2% vs. 48.9% in men, p<0.001). Abdominal obesity, elevated fasting plasma glucose and low HDL cholesterol were more common in women than in men. Table 2. The prevalence of metabolic syndrome and its components by gender. doi:10.20944/preprints202112.0463.v1 (N=669) (N=266) (N=403) Having metabolic syndrome 417 (62.3%) 130 (48.9%) 287 (71.2%) <0.001 Abdominal obesity (waist circum- ference ≥102 cm in men, and ≥88 cm in women) 167 (25.0%) 6 (2.3%) 161 (40.0%) <0.001 Fasting blood glucose ≥100 mg/dl or having a history of diabetes 410 (61.3%) 147 (55.3%) 263 (65.3%) 0.009 SBP ≥130 mmHg or DBP ≥85 mmHg or having a history of hy- pertension 450 (67.3%) 174 (65.4%) 276 (68.5%) 0.407 HDL cholesterol <40 mg/dl in men or < 50 mg/dl in women. 447 (66.8%) 147 (55.3%) 300 (74.4%) <0.001 Triglyceride ≥150 mg/dl 423 (63.2%) 165 (62.0%) 258 (64.0%) 0.601 SBP, systolic blood pressure. DBP, diastolic blood pressure. HDL, high-density lipoprotein. Figure 1. presents the prevalence of frailty and its components in participants with and without MetS. Overall, the prevalence of frailty was higher in participants with MetS than in participants without MetS (42.2% versus 33.7%, respectively, p=0.029). Among the five components of frailty, slowness was the most prevalent (86.1%), followed by weak- ness (71.3%) and low physical activity (29.9%). Participants with MetS had significantly higher prevalence of slowness, low physical activity and exhaustion compared to those without MetS. Figure 1. The prevalence of frailty and its components in participants with and without metabolic syndrome (MetS). Figure 1. The prevalence of frailty and its components in participants with and without metabolic syndrome (MetS). On univariate logistic regression, there was a significant relationship between the presence of MetS and an increased risk of frailty (unadjusted OR 1.44, 95%CI 1.04-1.99, p=0.03). Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 This relationship was further explored in multivariate logistic regression, ad- justed to age, sex and the variables that were found to demonstrate significant associations with frailty on univariate analyses (Table 3). doi:10.20944/preprints202112.0463.v1 Table 3. Univariate logistic regression of potentially associated factors on frailty. Variables Unadjusted odds ratios for being frail (95%CI) P-values Having metabolic syndrome 1.44 (1.04 – 1.99) 0.030 Age (per year) 1.10 (1.08 – 1.12) <0.001 Female 1.10 (0.84 – 1.45) 0.499 Recruitment source (inpatients vs. outpa- tients) 2.92 (2.13 – 3.99) <0.001 Low education 2.92 (2.15 – 3.97) <0.001 Malnutrition 9.14 (4.71 – 17.74) <0.001 History of hospitalization in the last year 2.99 (2.26 – 3.95) <0.001 Myocardial infarction 0.88 (0.38 – 2.01) 0.760 Heart failure 1.95 (0.80 – 4.75) 0.143 Stroke 2.07 (1.02 – 4.22) 0.045 Peripheral vascular disease 1.07 (0.59 – 1.94) 0.830 Chronic kidney disease 2.66 (1.98 – 3.58) <0.001 Chronic lung disease 1.05 (0.79 – 1.38) 0.761 Cancer 4.26 (1.12 – 16.15) 0.033 Dementia 3.16 (0.58 – 17.35) 0.185 Table 3. Univariate logistic regression of potentially associated factors on frailty. After adjusting to age, sex, low educational and nutritional statuses, recent hospital- isation, recruitment resources and chronic health conditions (CKD, stroke and cancer), MetS still remained significantly associated with a higher risk frailty (adjusted OR 1.52, 95%CI 1.01 - 2.28, p=0.043) (Table 4). Table 4. Multivariate logistic regression of metabolic syndrome on frailty status. Table 4. Multivariate logistic regression of metabolic syndrome on frailty status. Adjusted odds ratios for being frail (95%CI) P Model 1 1.42 (1.00 – 2.00) 0.049 Model 2 1.42 (1.00 – 2.02) 0.049 Model 3 1.47 (1.01 – 2.14) 0.045 Model 4 1.62 (1.10 – 2.38) 0.015 Model 5 1.52 (1.01 – 2.28) 0.043 Table 4. Multivariate logistic regression of metabolic syndrome on frailty status. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 Adjusted odds ratios for being frail (95%CI) P Model 1 1.42 (1.00 – 2.00) 0.049 Model 2 1.42 (1.00 – 2.02) 0.049 Model 3 1.47 (1.01 – 2.14) 0.045 Model 4 1.62 (1.10 – 2.38) 0.015 Model 5 1.52 (1.01 – 2.28) 0.043 Model 1: adjusted to age Model 2: adjusted to age, education Model 3: adjusted to age, education, nutritional status, hospitalisation in the past year Model 4: adjusted to age, education, nutritional status, hospitalisation in the past year, chronic health conditions (chronic kidney disease, stroke, peripheral vascular disease, cancer) Model 5: adjusted to age, sex, education, nutritional status, hospitalisation in the past year, chronic health conditions (chronic kidney disease, stroke, periph- eral vascular disease, cancer) and recruitment sources. Model 1: adjusted to age Model 2: adjusted to age, education Model 3: adjusted to age, education, nutritional status, hospitalisation in the past year Model 4: adjusted to age, education, nutritional status, hospitalisation in the past year, chronic health conditions (chronic kidney disease, stroke, peripheral vascular disease, cancer) Model 5: adjusted to age, sex, education, nutritional status, hospitalisation in the past year, chronic health conditions (chronic kidney disease, stroke, periph- eral vascular disease, cancer) and recruitment sources. Model 1: adjusted to age Model 2: adjusted to age, education Model 3: adjusted to age, education, nutritional status, hospitalisation in the past year Model 4: adjusted to age, education, nutritional status, hospitalisation in the past year, chronic health conditions (chronic kidney disease, stroke, peripheral vascular disease, cancer) Model 5: adjusted to age, sex, education, nutritional status, hospitalisation in the past year, chronic health conditions (chronic kidney disease, stroke, periph- eral vascular disease, cancer) and recruitment sources. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 cross-sectional study of 118 non-institutionalised older people in Italy (mean age 76.1±5.0 years, 60% women), Viscogliosi and colleagues found that the prevalence of frailty was significantly higher in the participants with MetS compared to those without MetS (60.7% vs. 12.9%, respectively), and the adjusted odds ratio of MetS for frailty acquisition was comparable to our study at 1.53 (95%CI 1.33-1.76).7 Their longitudinal data also showed that baseline MetS increased the risk of reduced handgrip strength and gait speed.23 An analysis from the Salus in Apulia study in Italy also showed that metabolic syndrome was associated with increased risk of physical frailty (OR 1.42, 95%CI 1.00-2.03).13 In another cross-sectional study conducted in Germany in 1,486 elderly participants with a mean age of 68.7 years, the odds of being prefrail/frail was significantly increased with the presence of MetS (adjusted OR 1.5, 95%CI 1.2-1.9).10 Another data analysis from 1247 elderly par- ticipants partaking in the Longitudinal Aging Study Amsterdam also reported a signifi- cantly higher prevalence of frailty in those with MetS (16.7%) compared to their unaf- fected counterpart (8.8%).8 In a longitudinal study by Perez-Tasigchana in Spain, after fol- lowing 1499 community-dwelling participants (aged ≥ 60 years) for 3.5 years, they found that participants with MetS had higher risk of developing frailty than those without MetS (OR 1.85, 95%CI 1.12-3.05), adjusting for participants’ socio-economic factors, healthy be- haviours and comorbidities.9 Although the pathophysiological linkage between MetS and frailty is still an area of active research, several studies suggested low-grade chronic inflammation state, high cir- culating inflammatory markers and neuroendocrine dysfunction as common grounds be- tween the two syndromes. A meta-analysis of 32 cross-sectional studies demonstrated significant associations between increased inflammatory markers, in particular IL-6 and C-reactive protein (CRP) with impaired muscle function, predisposing older people to in- creased risk of frailty.24 Similarly, pro-inflammatory state induced by adipokines and other inflammatory mediators was also postulated to be central to insulin resistance and thus MetS pathogenesis.25 The individual components defining MetS were shown to con- tribute to the presence of frailty in several studies. 4. Discussion In this study of 669 older participants, there was a high prevalence of frailty (39.0%) and MetS (62.3%). The presence of MetS was associated with a significantly higher likeli- hood of being frailty in the participants. Our findings on the significant association between MetS and frailty coincided with findings from several previous studies. Our literature search revealed that most of the studies on this topic were conducted in Caucasian older populations 7-13, with only two studies reported the association between frailty and metabolic syndrome in Asian popu- lations.14,15 Chao and colleagues found that among 2862 community-dwelling older adults, the presence of MetS were associated with a significantly higher risk of combined frailty/prefrailty (OR 2.53, 95%CI 1.78-3.60).14 Lee and colleagues also reported that meta- bolic syndrome were strongly associated with frailty status (OR 3.2, 95%CI 1.7-6.0).15 In a Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 Hypertension, in particular high sys- tolic blood pressure was shown to have a positive correlation with frailty in studies by Newman, Bastos-Barbosa and their respective colleagues.26,27 Abdominal obesity, after ad- justed for BMI, was demonstrated to increase the risk of frailty, on both frailty index (FI) and phenotypic classifications according to Hubbard and colleagues.28 Several other stud- ies showed that insulin resistance was amongst the most commonly-documented compo- nent of MetS, with respect to its association with frailty.9,29 Longer duration of insulin re- sistance or overt diabetes, coupled with poor glycaemic control could have resulted in suboptimal muscle quality and strength, thus directly increasing the odds of falls and frailty.30 To the best of our knowledge, this study is the first of its kind to provide evidence on the association between metabolic syndrome and frailty in Vietnam. However, this study has several limitations. First, this was a secondary analysis, and thus, we were limited by the available data. A total of 327 participants, despite meeting criteria for inclusion from the primary study, was excluded from our analysis because of insufficient data to define frailty and MetS. Second, this study was conducted at only one geriatric hospital in Vi- etnam. Therefore, the studied population may not be representative for all older patients in Vietnam and the results should be interpreted cautiously. Although the ability for gen- eralisation was somewhat limited, owing to its single site nature, our study still remains relevant in a public health point-of-view, serving as a starting point for future studies linking the two prominent risk factors for adverse health outcomes in older people. As metabolic syndrome and its constitutive components are targetable pharmacologically and conservatively through public health directives, a significant relationship between MetS and frailty could aid decision makers, justify the role of MetS management as a pri- mary prevention for frailty syndrome in older people. 5. Conclusion References 1. O'Caoimh R, Sezgin D, O'Donovan MR, et al. Prevalence of frailty in 62 countries across the world: meta-analysis of population-level studies. Age Ageing 2021; 50(1): 96-104. p p g g g ( ) ung J, Iliffe S, Rikkert MO, Rockwood K. Frailty in elderly people. The Lancet 2013; 381(9868): 752-62. 2. Clegg A, Young J, Iliffe S, Rikkert MO, Rockwood K. Frailty in elderly people. The Lancet 2013; 381(9868): 752-62. 3. Fried LP, Tangen CM, Walston J, et al. Frailty in older adults: evidence for a phenotype. J Gerontol A Biol Sci Med Sci 2001; 56(3): M146-56. 4. Pucci G, Alcidi R, Tap L, Battista F, Mattace-Raso F, Schillaci G. Sex- and gender-related prevalence, cardiovascular risk and therapeutic approach in metabolic syndrome: A review of the literature. Pharmacological Research 2017; 120: 34-42. 5. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet 2005; 365(9468): 1415-28. 4. Pucci G, Alcidi R, Tap L, Battista F, Mattace-Raso F, Schillaci G. Sex- and gender-related prevalence therapeutic approach in metabolic syndrome: A review of the literature. Pharmacological Research 2017 p pp y g 5. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet 2005; 365(9468): 1415-28. y y 6. Expert Panel on Detection E, Adults ToHBCi. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001; 285(19): 2486-97. 7. Viscogliosi G. The Metabolic Syndrome: A Risk Factor for the Frailty Syndrome? J Am Med Dir Assoc 2016; 17(4): 364-6. 8. Hoogendijk EO, Huisman M, van Ballegooijen AJ. The role of frailty in explaining the association bet drome and mortality in older adults. Exp Gerontol 2017; 91: 5-8. EO, Huisman M, van Ballegooijen AJ. The role of frailty in explaining the association between the metabo mortality in older adults. Exp Gerontol 2017; 91: 5-8. y p 9. Perez-Tasigchana RF, Leon-Munoz LM, Lopez-Garcia E, et al. Metabolic syndrome and insulin resistance are associated with frailty in older adults: a prospective cohort study. Age Ageing 2017; 46(5): 807-12. y p p y g g g 10. Buchmann N, Spira D, Konig M, Demuth I, Steinhagen-Thiessen E. Frailty and the Metabolic Syndrome - Results of the Berlin Aging Study II (BASE-II). J 2019; 8(4): 169-75. 11. Kane AE, Gregson E, Theou O, Rockwood K, Howlett SE. Competing interests: None Author Contributions: All authors (Hiep HH Dao, Tu Ngoc Nguyen, Huyen TT Vu, Anh Trung Nguyen) contributed to the study conception and design. Participant recruitment and data curation were performed by Huyen TT Vu and Anh Trung Nguyen. Data analysis were performed by Hiep HH Dao and Tu Ngoc Nguyen. Hiep HH Dao and Tu Ngoc Nguyen wrote the various drafts of the manuscript. All authors contributed to interpretation of data and commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the National Geriatric Hospital in Hanoi, Vietnam (protocol code 1235/IRB, date of approval 28/11/2017). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The study data is available from the corresponding author upon rea- sonable request. Acknowledgments: We thanks all participants for their participation in the study. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 December 2021 doi:10.20944/preprints202112.0463.v1 This study found that metabolic syndrome was present in around two-thirds of the participants and was associated with increased risk of frailty. Further longitudinal studies are required to confirm this association. These findings support routine assessment for frailty in older people with cardio-metabolic disorders. 5. Conclusion 5. Conclusion References The association between frailty, the metabolic syndrome, and mor- tality over the lifespan. Geroscience 2017; 39(2): 221-9. 12. Veronese N, Sigeirsdottir K, Eiriksdottir G, et al. Frailty and Risk of Cardiovascular Diseases in Older Persons: The Age, Gene/Environment Susceptibility-Reykjavik Study. Rejuvenation research 2017; 20(6): 517-24. 13. Castellana F, Lampignano L, Bortone I, et al. Physical Frailty, Multimorbidity, and All-Cause Mortality in an Older Population From Southern Italy: Results from the Salus in Apulia Study. Journal of the American Medical Directors Association 2021; 22(3): 598- 605. 14. Chao CT, Lee YH, Li CM, Han DS, Huang JW, Huang KC. Advanced Age and Chronic Kidney Disease Modify the Association Between Metabolic Syndrome and Frailty Among Community-Dwelling Elderly. Rejuvenation Res 2019. 15. Lee WJ, Peng LN, Chen LK. Metabolic Syndrome and Its Components Are Associated with Frailty: A Nationwide Population- Based Study in Taiwan. Aging Med Healthc 2020; 11(2): 47-52. y g g ( ) 16. Nguyen AT, Nguyen LH, Nguyen TX, et al. Frailty Prevalence and Association with Health-Related Quality of Life Impairment among Rural Community-Dwelling Older Adults in Vietnam. Int J Environ Res Public Health 2019; 16(20). doi:10.20944/preprints202112.0463.v1 17. Nguyen AT, Nguyen TX, Nguyen TN, et al. The impact of frailty on prolonged hospitalization and mortality in elderly inpa- tients in Vietnam: a comparison between the frailty phenotype and the Reported Edmonton Frail Scale. Clin Interv Aging 2019; 14: 381-8. 18. Nguyen TV, Ly TT, Nguyen TN. A Pilot Study of the Clinical Frailty Scale to Predict Frailty Transition and Readmission in Older Patients in Vietnam. Int J Environ Res Public Health 2020; 17(5). 19. Nguyen TN, Nguyen TN, Nguyen AT, et al. Prevalence of sarcopenia and its associated factors in patients attending geriatric clinics in Vietnam: a cross-sectional study. BMJ Open 2020; 10(9): e037630. y 20. Kaiser MJ, Bauer JM, Ramsch C, et al. Validation of the Mini Nutritional Assessment short-form (MNA-SF): a practical tool for identification of nutritional status. J Nutr Health Aging 2009; 13(9): 782-8. santachai P, et al. Asian Working Group for Sarcopenia: 2019 Consensus Update on Sarcopenia Diagnosis m Med Dir Assoc 2020; 21(3): 300-7.e2. 21. Chen LK, Woo J, Assantachai P, et al. Asian Working Group for Sarcopenia: 2019 Consensus Update and Treatment. J Am Med Dir Assoc 2020; 21(3): 300-7.e2. J ( ) 22. Tran DV, Lee AH, Au TB, Nguyen CT, Hoang DV. References Reliability and validity of the International Physical Activity Questionnaire- Short Form for older adults in Vietnam. Health Promot J Austr 2013; 24(2): 126-31. J ( ) 23. Viscogliosi G, Andreozzi P, Ettorre E, Chiriac IM. The Metabolic Syndrome and the Phenotype of Frailty: A Causal Link? J Am Med Dir Assoc 2016; 17(10): 956-7. 24. Soysal P, Stubbs B, Lucato P, et al. Inflammation and frailty in the elderly: A systematic review and meta-analysis. Ageing Research Reviews 2016; 31: 1-8. 25. Welty FK, Alfaddagh A, Elajami TK. Targeting inflammation in metabolic syndrome. Translational Res 25. Welty FK, Alfaddagh A, Elajami TK. Targeting inflammation in metabolic syndrome. Translational Research 2016; 167(1): 257-80. 26. Newman AB, Gottdiener JS, McBurnie MA, et al. Associations of subclinical cardiovascular disease with frailty. J Gerontol A Biol Sci Med Sci 2001; 56(3): M158-66. y g j g g y ( ) 26. Newman AB, Gottdiener JS, McBurnie MA, et al. Associations of subclinical cardiovascular disease with frailty. J Gerontol A Biol Sci Med Sci 2001; 56(3): M158-66. ( ) 27. Bastos-Barbosa RG, Ferriolli E, Coelho EB, Moriguti JC, Nobre F, Lima NK. Association of frailty syndrome in the elderly with higher blood pressure and other cardiovascular risk factors. Am J Hypertens 2012; 25(11): 1156-61. 28. Hubbard RE, Lang IA, Llewellyn DJ, Rockwood K. Frailty, Body Mass Index, and Abdominal Obesity in Older People. The Journals of Gerontology: Series A 2009; 65A(4): 377-81. f gy 29. Barzilay JI, Blaum C, Moore T, et al. Insulin resistance and inflammation as precursors of frailty: the Cardiovascular Health Study. Arch Intern Med 2007; 167(7): 635-41. 30. Park SW, Goodpaster BH, Strotmeyer ES, et al. Decreased muscle strength and quality in older adults with type 2 diabetes: the health, aging, and body composition study. Diabetes 2006; 55(6): 1813-8.
https://openalex.org/W3131695410	https://ecommons.aku.edu/cgi/viewcontent.cgi?article=1035&context=coe-wch	English	null	Delivering maternal and neonatal health interventions in conflict settings: a systematic review	BMJ global health	2,021	cc-by	12,224	eCommons@AKU eCommons@AKU Centre of Excellence in Women and Child Health Centres of Excellence 2-1-2021 Delivering maternal and neonatal health interventions in conflict Delivering maternal and neonatal health interventions in conflict settings: A systematic review settings: A systematic review Mariella Munyuzangabo Michelle F. Gaffey Dina S. Khalifa Daina Als Anushka Ataullahjan See next page for additional authors Follow this and additional works at: https://ecommons.aku.edu/coe-wch Part of the Maternal and Child Health Commons, and the Women's Health Commons eCommons@AKU eCommons@AKU eCommons@AKU eCommons@AKU Centres of Excellence Authors Authors Mariella Munyuzangabo, Michelle F. Gaffey, Dina S. Khalifa, Daina Als, Anushka Ataullahjan, Mahdis Kamali, Reena P. Jain, Sarah Meteke, Amruta Radhakrishnan, Shailja Shah, Fahad J. Siddiqui, and Zulfiqar Ahmed Bhutta Delivering maternal and neonatal health interventions in conflict Delivering maternal and neonatal health interventions in conflict settings: A systematic review settings: A systematic review See next page for additional authors Follow this and additional works at: https://ecommons.aku.edu/coe-wch Part of the Maternal and Child Health Commons, and the Women's Health Commons To cite: Munyuzangabo M, Gaffey MF, Khalifa DS, et al. Delivering maternal and neonatal health interventions in conflict settings: a systematic review. BMJ Global Health 2021;5:e003750. doi:10.1136/ bmjgh-2020-003750 What is already known? ► ►The insecurity, population displacement and health service disruption that are associated with armed conflict may increase both maternal and neonatal mortality. ► ►While there is guidance available on the essential interventions for mothers and newborns in humani- tarian crises, there is still a lack of guidance on how best to deliver these interventions, especially in con- flict settings where there are even more barriers to the access of services. Handling editor Seye Abimbola ► ►Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ bmjgh-2020-003750). Methods We searched MEDLINE, Embase, CINAHL and PsycINFO databases using terms related to conflict, women and children, and maternal and neonatal health. We searched websites of 10 humanitarian organisations for relevant grey literature. Publications reporting on conflict-affected populations in low-income and middle- income countries and describing a maternal or neonatal health intervention delivered during or within 5 years after the end of a conflict were included. Information on population, intervention, and delivery characteristics were extracted and narratively synthesised. Quantitative data on intervention coverage and effectiveness were tabulated but no meta-analysis was undertaken. Received 18 August 2020 Revised 15 January 2021 Accepted 26 January 2021 Key questions ABSTRACT Background While much progress was made throughout the Millennium Development Goals era in reducing maternal and neonatal mortality, both remain unacceptably high, especially in areas affected by humanitarian crises. While valuable guidance on interventions to improve maternal and neonatal health in both non-crisis and crisis settings exists, guidance on how best to deliver these interventions in crisis settings, and especially in conflict settings, is still limited. This systematic review aimed to synthesise the available literature on the delivery on maternal and neonatal health interventions in conflict settings. ► ►Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ bmjgh-2020-003750). What are the new findings? ► ►Maternal health interventions were mostly deliv- ered by skilled health workers including doctors and nurses and in hospitals and clinics; the majority of reported interventions focused on antenatal care and its different components. ► ►Although few, the reported newborn interventions focused on essential newborn care such as neonatal resuscitation and kangaroo mother care, and were also mostly delivered by skilled health workers in clinics and hospitals. ► ►Among the reported barriers to intervention delivery were the lack of safety, population displacement, and the lack of resources and skilled health workers; however, facilitators included the availability of fund- ing, multisectoral collaboration and the availability of training for staff. Results 115 publications met our eligibility criteria. Intervention delivery was most frequently reported in the sub-Saharan Africa region, and most publications focused on displaced populations based in camps. Reported maternal interventions targeted antenatal, obstetric and postnatal care; neonatal interventions focused mostly on essential newborn care. Most interventions were delivered in hospitals and clinics, by doctors and nurses, and were mostly delivered through non-governmental organisations or the existing healthcare system. Delivery barriers included insecurity, lack of resources and lack of skilled health staff. Multi-stakeholder collaboration, the introduction of new technology or systems innovations, and staff training were delivery facilitators. Reporting of intervention coverage or effectiveness data was limited. Discussion The relevant existing literature focuses mostly on maternal health especially around the antenatal period. There is still limited literature on postnatal care in conflict settings and even less on newborn care. In crisis settings, as much as in non-crisis settings, there is a need to focus Delivering maternal and neonatal health interventions in conflict settings: a systematic review copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ hed as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Mariella Munyuzangabo,1 Michelle F Gaffey,1 Dina S Khalifa,1 Daina Als,1 Mariella Munyuzangabo,1 Michelle F Gaffey,1 Dina S Khalifa,1 Daina Als,1 Anushka Ataullahjan,1 Mahdis Kamali,1 Reena P Jain ‍ ‍ ,1 Sarah Meteke,1 Amruta Radhakrishnan,1 Shailja Shah ‍ ‍ ,1 Fahad J Siddiqui,1,2 Zulfiqar A Bhutta1 Mariella Munyuzangabo,1 Michelle F Gaffey,1 Dina S Khalifa,1 Daina Als,1 Anushka Ataullahjan,1 Mahdis Kamali,1 Reena P Jain ‍ ‍ ,1 Sarah Meteke,1 Anushka Ataullahjan, Mahdis Kamali, Reena P Jain ‍ ‍ , Sarah Meteke, Amruta Radhakrishnan,1 Shailja Shah ‍ ‍ ,1 Fahad J Siddiqui,1,2 Zulfiqar A Bhutta1 Amruta Radhakrishnan,1 Shailja Shah ‍ ‍ ,1 Fahad J Siddiqui,1,2 Zulfiqar A Bhutta1 © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Authors Authors copyright. on October 27, 2021 at Aga Khan University. Pr http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Original research 1Centre for Global Child Health, The Hospital for Sick Children, Toronto, Ontario, Canada 2Health Services and Systems Research, Duke-NUS Graduate Medical School, Singapore 3Centre of Excellence in Women and Child Health, Aga Khan University, Karachi, Pakistan © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Literature search A systematic search of published literature from 1st January 1990 to 31st March 2018 was conducted; records were retrieved in MEDLINE, Embase, CINAHL and PsycINFO using OVID and EBSCO interfaces. We used sets of terms related to three concepts: (a) conflict; (b) women and children; and (c) MNH. Conflict-related terms included war, crisis, refugees and internally displaced persons (IDPs). Population-related terms included: women, children, pregnant, adolescents and newborn. MNH- related terms included: pregnancy, obstetrics, labour complications and neonatal care, among others. The full MEDLINE search syntax is provided in the online supplemental appendix A. Relevant publications from key systematic reviews11 13–15 were also hand searched for potentially relevant publications. Although much progress was made throughout the Millennium Development Goals era, about 810 women still die from pregnancy-related or childbirth-related complications every day. This is especially true in low/ middle-income countries (LMICs) where 94% of these deaths occur.1 Approximately 7000 newborns also die every day,2 accounting for 47% of all under-5 child deaths, and with 36% of them occurring during the first 24 hours after birth and 73% within the first week.3 Although it has been shown that increasing coverage of antenatal care (ANC), skilled birth attendance and postnatal care for both the mother and the baby can prevent high maternal and neonatal mortality rates,4 data from existing surveys still show that many women and newborns are not reached with these essential interventions.1 p y p We searched the websites of 10 major humanitarian agencies and organisations that are actively involved in responding to or researchin conflict situations for grey literature on the implementation of health interven- tions in pregnant and postpartum women or newborns. These websites included: Emergency Nutrition Network, International Committee of the Red Cross, International Rescue Committee, Médecins Sans Frontières, Save the Children, United Nations (UN) Population Fund, UN High Commissioner for Refugees, UNICEF, Women’s Refugee Commission and World Vision. We used broad terms for conflict and health interventions tailored to the search functionality of each website. Documents published from 1 January 2013 to 30 November 2018 were reviewed. Literature search Within humanitarian settings and especially in the context of armed conflict, insecurity coupled with displacement and service disruption produce an insta- bility that may lead to a higher risk of maternal mortality.5 While increased maternal mortality may be a direct effect of armed conflict, it has also been shown that maternal mortality may remain elevated even 1–3 years post- conflict, indicating the longer terms effects of conflict on the disruption of essential health services for preg- nant women.5 Newborn mortality has also been shown to increase in the context of armed conflict,6 and is highest in fragile settings and in countries that have recently experienced a humanitarian crisis.7 Comprehensive global guidance exists for addressing maternal and neonatal health (MNH) in non-crisis settings, including the interventions and services outlined in the Every Newborn Action Plan launched in 2014.8 Guidance is also available to support MNH in humanitarian crises, including the latest version of the Inter-Agency Field Manual on Reproductive Health in Humanitarian Settings and the Newborn Health in Humanitarian Settings–Field Guide, both released in 2018.9 10 However, guidance on how best to deliver recommended interventions for pregnant women and newborns in humanitarian settings, and especially in conflict settings, is still very limited. A review on public health interventions that was included in the 2017 Lancet Series on Health in Humanitarian Crises highlighted the What do the new findings imply? What do the new findings imply? The present review aimed to systematically synthesise the available indexed and grey literature reporting on the delivery of maternal and newborn interventions in conflict settings. We sought to characterise how and by whom such interventions have been and are being deliv- ered to conflict-affected women and newborns and, if possible, to what extent and with what effectiveness. ► ►There is a need to do more evaluations on the use of lower-cadre health workers and community-based sites in the delivery of ma- ternal health interventions. ► ►It is also important to assess the feasibility and importance of de- ploying people from the affected communities, especially in out-of- camp settings where there may not be as many services/resources available. copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ lished as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from ► ►There is an urgent need to prioritise newborn health interventions. More research is needed on how best to deliver newborn inter- ventions using lower-skilled health workers and at the community level. g ► ►There is still a wide gap in reporting on newborn health, with very few eligible publications reporting on interventions targeting newborns. ► ►There is still a wide gap in reporting on newborn health, with very few eligible publications reporting on interventions targeting newborns. © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. 1Centre for Global Child Health, The Hospital for Sick Children, Toronto, Ontario, Canada 2Health Services and Systems Research, Duke-NUS Graduate Medical School, Singapore 3Centre of Excellence in Women and Child Health, Aga Khan University, Karachi, Pakistan on the first day of birth for both maternal and neonatal health. There is also a need to do more research on how best to involve community members in the delivery of maternal and neonatal health interventions. on the first day of birth for both maternal and neonatal health. There is also a need to do more research on how best to involve community members in the delivery of maternal and neonatal health interventions. PROSPERO registration number CRD42019125221. INTRODUCTION Correspondence to Dr Zulfiqar A Bhutta; zulfiqar.bhutta@sickkids.ca Correspondence to Dr Zulfiqar A Bhutta; zulfiqar.bhutta@sickkids.ca Maternal and newborn mortality remain two of the biggest challenges in global health. Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 1 copyright. on October 27, 2021 at Aga Khan University. Protected http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from copyright. on October 27, 2021 http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from BMJ Global Health BMJ Global Health copyright. on October 27, 2021 at Aga Khan Univer http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from need for higher quality studies on MNH that focus on the most effective means of delivering those interventions in crisis contexts.11 12 Data extraction and analysis Figure 1 Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow diagram: publication selection process for systematic review on the delivery of maternal and neonatal health interventions in conflict settings. All identified indexed records were downloaded into EndNote software17 and duplicates were removed. Unique records were subsequently added into Covi- dence software for screening.18 Two reviewers inde- pendently screened titles and abstracts for relevance, and any discrepancies were resolved through discussion or by a third reviewer, if necessary. A single reviewer then screened the full texts of potentially relevant publications to determine their eligibility for inclusion in this review. The same approach was applied to the grey literature, with two reviewers screening titles of retrieved publica- tions for relevance and one reviewer assessing the full text to determine eligibility. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting or dissemination plans of this systematic review of the literature. Eligibility criteria Our eligibility criteria included publications reporting on populations affected by conflict in LMICs, as classi- fied by the World Bank.16 Eligible publications must have described an MNH intervention targeting or including pregnant or postpartum women or neonates, and deliv- ered during or within 5 years of cessation of a conflict. Where needed, we consulted online encyclopaedic sources as well as the UN Office for the Coordination of Humanitarian Affairs website for information on the duration of a specific conflict, to assess whether the time period of intervention delivery reported in a candidate publication was eligible. In order to identify the most Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 2 copyright. on October 27, 2021 at Aga Khan Unive http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from BMJ Global Health Figure 1 Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow diagram: publication selection process for systematic review on the delivery of maternal and neonatal health interventions in conflict settings. informative resources from the large volume of grey liter- ature available, the same inclusion criteria set for indexed literature was applied, with the additional requirement of explicit reporting on the delivery site and personnel for each intervention. informative resources from the large volume of grey liter- ature available, the same inclusion criteria set for indexed literature was applied, with the additional requirement of explicit reporting on the delivery site and personnel for each intervention. Non-English publications; case reports of a single patient; or publications reporting on military personnel, refugee populations in high-income countries, surgical techniques, or economic or mathematical modelling studies were excluded from our review. We also excluded systematic and other literature reviews, editorials, commentaries, first-person narratives, newspaper and magazine articles, opinion pieces, guidelines and studies where no specific health intervention was described (eg, prevalence studies). copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ ublished as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 BMJ Global Health Figure 3 Maternal health interventions in pregnant and postpartum women. ANC, antenatal care; MH, maternal health; STI, sexually transmitted infection. Figure 3 Maternal health interventions in pregnant and postpartum women ANC antenatal care; MH maternal di i i i d li d i hi Table 1 Characteristics of included publications and included interventions Study and population characteristics (N=115) Geographical region* n East Asia and Pacific 31 Europe and Central Asia 3 Latin America and the Caribbean 0 Middle East and North Africa 22 South Asia 14 Sub-Saharan Africa 50 Publication type n Non-research report 19 Mixed methods 11 Observational study 78 Qualitative study 4 Quasi-experimental study 0 Randomised controlled trial 3 Displacement status of beneficiary population* n Refugees 75 IDPs 36 Non-displaced 20 Returning refugees 2 Host 11 Unreported 12 Setting of displaced populations† n Camp 52 Dispersed 16 Mixed 14 Unreported 33 Intervention delivery characteristics (N=313) Target population type n Neonates (<28 days) 32 Pregnant women 253 Postpartum women 51 Implementation platform* n Existing health system 148 NGO/UN agencies 234 Military based 3 Research based 38 *Publications and interventions can be included in more than one category. †Only reflects publications that reported on displaced populations (refugees, IDPs or returning refugees). IDPs, internally displaced persons; NGO, non-governmental organisations; UN, United Nations. copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Figure 3 Maternal health interventions in pregnant and postpartum women. ANC, antenatal care; MH, maternal health; STI, sexually transmitted infection. also a few publications reporting on interventions deliv- ered to non-displaced but conflict-affected populations and to host communities in countries with refugees. Among those publications reporting population displace- ment settings, intervention delivery to refugees and IDPs was reported most frequently in camp settings (45%). Pregnant women were the most frequently reported inter- vention target populations, and most reported interven- tions were delivered by non-governmental organisations (NGOs) (including community-based organisations) or UN agencies, either through the existing healthcare system or in parallel. Characteristics of the included literature From 37 714 indexed publications retrieved through our database search, 101 met our eligibility criteria. An additional 14 eligible publications were identified from our grey literature search, and from the reference lists of other reviews. A total of 115 publications were therefore included in this review (figure 1).21–135 Of these, only 26 reported on neonatal interventions.24 26 28 29 33 38 42 44 55 60 We used a customised form on the REDCap platform19 to extract relevant quantitative and qualitative informa- tion from all included publications. Data and information on key variables including setting, population charac- teristics, study design, intervention details and delivery characteristics including reported delivery barriers and facilitators, as well as quantitative data on coverage and effectiveness (when available), were extracted. Two reviewers entered the data in duplicate independently and any inconsistencies identified were resolved through a discussion or by a third reviewer. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ p 63 71 75 97 106 110–113 116 118 120 123 124 133 135 The characteristics of all included publications are presented in the online supplemental appendix B. of all included publications are presented in the online supplemental appendix B. of all included publications are presented in the online supplemental appendix B. Of the 115 included publications, MNH intervention delivery was most frequently reported in the sub-Saharan Africa region (43%), followed by the East Asia and Pacific region (27%), mainly in Thailand (figure 2). There were y Descriptive statistics were performed to summarise key characteristics of the intervention, including displace- ment status of the target population, delivery characteris- tics, and reported factors impeding or facilitating delivery. We plotted intervention delivery frequencies stratified by population displacement status, and we mapped reported interventions against reported delivery personnel and site, stratified by intervention type. We tabulated coverage and effectiveness estimates that were reported for preg- nant or postpartum women or newborns, but meta- analysis was not conducted due to extensive variability in interventions, outcomes, study settings and populations. All descriptive analyses were conducted using Stata V.14.0 statistical software.20 Finally, we narratively synthesised reported information on delivery barriers and facilitators by classifying related information into subgroups and then into broader themes. Figure 2 Geographical distribution of included publications. Figure 2 Geographical distribution of included publications. Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 3 copyright. Characteristics of the included literature on October 27, 202 http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Maternal health interventions The reported interventions targeting pregnant or postpartum women covered different components of maternal health such as antenatal, obstetric and postnatal care, as well as general maternal health (figure 3). Within ANC, the most commonly reported interventions were screening interventions for HIV, sexually transmitted infections (STIs), malaria and anaemia; micronutrient supplementation; malaria prevention and treatment; and behavioural education activities. Of the publications that reported on the delivery of ANC, about 35% of them did not specify the components provided. Reported obstetric care interventions were mostly safe delivery care inter- ventions and the provision of basic and comprehensive emergency obstetric care (EmOC), including caesarean sections. Other obstetric care interventions included 27, 2021 at Aga Khan University. Protected by g y †Only reflects publications that reported on displaced populations (refugees, IDPs or returning refugees). IDPs, internally displaced persons; NGO, non-governmental organisations; UN, United Nations. no studies reporting on interventions delivered within the Latin America and Caribbean region. The majority of included publications reported on observational studies (68%), and 17% were non-research reports (table 1). p More than half of the included publications reported on interventions delivered to refugees (62%), and about one-third of those delivered to IDPs (27%). There were Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 4 Figure 4 Reported maternal and neonatal health interventions by delivery personnel and intervention type. ANC, antenatal care; CHWs, community health workers; NGO, non-governmental organisation; STI, sexually transmitted infection; TBAs, traditional birth attendants. BMJ Global Health copyright. on October 27, 2021 at Aga Khan Univers http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from When we compare in-camp and out-of-camp popula- tions, in-camp mothers were reported to have access to a wider range of personnel compared with out-of-camp populations (data not shown). In-camp interventions were delivered by health workers, nurses, doctors and OB/GYN specialists, while for out-of-camp populations, health workers were the most reported delivery personnel, but with fewer specified professionals reported such as OB/GYN specialists, doctors and nurses. There was also only one reported instance of a TBA being used in an out-of-camp population and very few interventions were reported to be delivered by SBAs or NGO staff. Sites Maternal health interventions were mostly delivered using the inpatient and outpatient platforms, with most interventions reported to be delivered within either hospitals or clinics (figure 5). Other common delivery sites included health posts and mobile clinics, mostly for the delivery of ANC interventions. Community-based sites such as homes and communal spaces were also used for ANC and postnatal care interventions. There were few differences between in-camp and out-of-camp popu- lations in terms of delivery sites (data not shown). There were no MNH interventions reported to be delivered in a hospital within out-of-camp populations, and there was also more use of communal spaces for delivery reported within these populations. Interventions targeted towards refugees were delivered more often in clinics and hospi- tals, with no reports of delivery using outreach sites such as health posts and mobile clinics and very few reporting delivery at the household (data not shown). Interven- tions targeted towards IDPs used outreach approaches more frequently, with interventions delivered at health posts and through mobile clinics. When comparing mothers living in camps with those living outside of camps, more interventions were reported to be delivered to mothers based in camps (figure 3). Some interventions were not reported at all for women living outside of camps, such as training of health workers or interventions targeting the prevention of HIV and STIs during ANC. There were also very few postnatal care interventions and no general maternal health interventions reported to be delivered to out-of- camp mothers. Maternal health interventions There were few differences in delivery personnel between refu- gees and IDPs (data not shown), except that while CHWs are mainly used for behavioural education and referral in refugee populations, they were also used for the ANC interventions such as micronutrient supplementation or malaria prevention in IDP populations. copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ b Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Figure 4 Reported maternal and neonatal health interventions by delivery personnel and intervention type. ANC, antenatal care; CHWs, community health workers; NGO, non-governmental organisation; STI, sexually transmitted infection; TBAs, traditional birth attendants. training of healthcare workers and referral for care. Reported postnatal care interventions included postnatal check-ups within 48 hours of delivery, education activi- ties and micronutrient supplementation among others. Reported general maternal health interventions mainly included training healthcare workers and counselling interventions. Delivery characteristics of maternal health interventions Personnel Trained health professionals were commonly reported personnel for maternal health interventions. These included doctors, nurses, health workers, skilled birth attendants (SBAs) and OB/GYN specialists (figure 4). NGO staff and researchers were also reported to have delivered most maternal health interventions such as ANC, behavioural education, postnatal care and training. Community health workers (CHWs) were reported to deliver ANC interventions such as malaria treatment and prevention, micronutrient supplementation, behav- ioural education and counselling among others. Tradi- tional birth attendants (TBAs) were not only involved in delivery care, but were also able to deliver some ANC interventions such as malaria prevention, medication and postnatal check-ups. There were no interventions delivered by community members such as civic leaders or trained civilians and there was only one training interven- tion where teachers were involved, which was a literacy programme for women. Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 Reported coverage and effectiveness of MNH interventions Reported coverage and effectiveness of MNH interventions Full details of the retrieved intervention coverage and effectiveness data are presented in the online supple- mental appendices C and D. Most estimates were derived from camp-based refugee populations. The majority of reported outcomes in pregnant women assessed coverage of ANC interventions such as the proportion of women accessing four or more ANC visits or the proportion of women receiving different components of ANC such as behavioural education, HIV/STI testing or malaria treat- ment. Other commonly reported coverage outcomes were on obstetric care such as the rate of caesarean sections and other major obstetric interventions. There were very few outcomes on postnatal care, with only two publications reporting on the proportions of postpartum women attending a postnatal consultation. Figure 5 Reported maternal and neonatal health interventions by delivery site and intervention type. ANC, antenatal care; STI, sexually transmitted infection. HIV or malaria or delivering vaccinations or micronutrient supplementation among newborns outside of camps. Delivery characteristics of newborn health interventions Personnel Newborn interventions were delivered by SBAs, doctors, nurses, health workers and NGO staff (figure 4). NGO staff were reported to deliver essential newborn care and training interventions. Trained TBAs were also reported to deliver essential newborn care. There were no reports of interventions delivered by CHWs or trained community members. There were no major differences between delivery personnel used for refugee compared with IDP populations. However, there were no reports of OB/GYN specialists being used to deliver newborn interventions among IDP populations. There were also no reports of nurses delivering newborn interventions in refugee populations. There were very few observa- tions of interventions delivery among non-displaced newborns. The majority of maternal intervention effectiveness estimates reported on the odds of receiving all ANC medical tests or visits, or of delivering at home in women who had received a maternal and child health handbook or maternal education compared with those who had not. There were also a small number of estimates reported regarding the effect of having a CHW in a village on the likelihood of a pregnant women receiving ANC or of having SBA at birth. g The included publications reported very few coverage or effectiveness estimates for newborn interventions. Coverage estimates included those for initiation of breast feeding, kangaroo mother care, antiretroviral (ARV) prophylaxis and healthcare worker training. Effective- ness estimates reported on the rate of exclusive breast feeding after an educational intervention and the rate of death in infants in a special care baby unit after training health workers. Figure 6 Neonatal health interventions. STI, sexually transmitted infection. No meta-analysis of intervention coverage or effec- tiveness data was undertaken given the wide variation in outcome definitions and measures, population charac- teristics and study designs. copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ lob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ ublished as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Newborn health interventions Very few studies reported on newborn care, and only about 10% of all interventions reported were targeting newborns. These interventions were focused mostly on essential newborn care such as neonatal resuscitation, kangaroo mother care and infection prevention. Other common interventions were postnatal check-ups, training of health workers and HIV prevention or follow-up through preven- tion of mother-to-child transmission (figure 6). 27, 2021 at Aga Khan University. Protected by There were more reported interventions on newborns living in camps (69%) compared with those living out of camps (16%). The three most common newborn inter- ventions in camps were essential newborn care, postnatal check-ups and training, while the most common interven- tion outside of camps was health worker training (figure 6). There were no reported instances of interventions targeting 5 Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 copyright. on October 27, 2021 at Aga Khan Unive http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from BMJ Global Health Figure 5 Reported maternal and neonatal health interventions by delivery site and intervention type. ANC, antenatal care; STI, sexually transmitted infection. Sites All newborn interventions were delivered in hospitals and clinics (figure 5) There were no reported newborn interventions delivered from an outreach or community- based platform. With very limited reporting on newborn interventions delivered outside of camps, it is not possible to compare delivery sites by camp status. Barriers to and facilitators of intervention delivery There were multiple reported barriers to delivering MNH interventions in conflict settings; key barriers and examples are presented in table 2. The most commonly reported barriers were security constraints and displace- ment. Populations are often not able to access health- care facilities due to security issues, and in some cases, because facilities have been destroyed. Continuous popu- lation movement also limits women’s access to services, and as health workers are likely also affected by the conflict, there may be staff shortages and high turnover. Figure 6 Neonatal health interventions. STI, sexually transmitted infection. Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 6 copyright. on October 27, 2021 at Aga Khan University. Pro http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from BMJ Global Healt Table 2 Reported barriers and facilitators in the implementation of MNH interventions Barriers Security Being in an insecure environment was often mentioned as a hindrance to the delivery of interventions. Health facilities are destroyed, patients were also unable to access clinics due to security issues. This was especially relevant for services such as emergency obstetric car when women may need to be referred to hospitals. Lack of resources Shortages of supplies/resources (medicine, diagnostic tests) during a period of conflict were also noted as barriers and hampered both maternal and newborn care. Materials such as gloves for delivery, obstetric equipment or beds for premature babies were some of the ones noted as missing.43 111 Population movement The continuous population movement limits both delivery and access to health services. Lack of skilled health workers The limited training of health workers was a major barrier in the delivery of interventions such as obstetric and newborn care. A few studies mentioned the lack of obstetric specialists as a barrier.43 104 A short training although helping to increase knowledge was also noted as not being enough. Social norms/stigma This was noted as a barrier for both patients as well as healthcare workers. Refugees may also be stigmatised by their hosts.63 Lack of funding Limited funding was also noted as a barrier as it limited to range of services and materials available. Limited movement for the women/cost barriers Conflict reduces means of generating income, especially during displacement. Therefore, the cost of getting health services or of transport might be weighed against other priorities. Barriers to and facilitators of intervention delivery In some instances where healthcare is not subsidised, cost of care also influenced where women delivered.49 Staff affected by conflict Health services were also limited as staff are also affected by displacement and security concerns. Shortage of staff was a big concern and in some areas was caused by the warring party. In one study, the Taliban were preventing female health providers from working.106 Some areas also experienced a high staff turnover, especially in a prolonged conflict.134 Limited services Conflict reduces the range of available services. For maternal health, this was especially dangerous in cases where there is limited EmOC services as needed surgical facilities were not available or there was no training in the management of postpartum complications,33 64 emergency referral services were also not always available. Quality of care There were some differences noted between the quality of care delivered at hospitals, compared with clinics; hospitals having a higher quality.111 ANC and postnatal care were als not always delivered to their full extent. Lack of guidelines A few studies mentioned lack of guidelines for STI prevention or lack of newborn-specific clinical and referral protocols as barriers to implementation.112 Facilitators Collaboration Multisectoral collaboration between international NGOs, the Ministry of Health and existing district health offices/public sector was noted as facilitators. Working with local NGOs was also a facilitator as they are already connected to the community. Staff training Training improved the skills of health workers and increased motivation. Continuous supervision/refresher training was encouraged, especially if provided by trained paediatrician or obstetricians.122 S-CORT modules are an innovative approach that focuses on training on the clinical services included in the MISP.122 One NGO developed a simple and low-cost 38- hour training course to upgrade the skills of TBAs.113 Availability of funding/ resources Having adequate funding allowed for more resources. CHW involvement/outreach workers Having maternal CHWs from the same community or refugee population was noted as a facilitator in educating women about maternal health. Use of existing infrastructure Using the existing infrastructure facilitated the delivery of interventions.71 110 Technological/systems innovations Improving systems such as introducing an ultrasound in outreach settings was noted as a facilitator,23 or introducing ANC into family medicine clinics as shown by Homan et al.57 Usin GIS technology with a mobile clinic was shown as being effective in delivering SRH services 114 BMJ Global Health Continued copyright. on October 27, 2021 at Aga http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from doi:10.1136/bmjgh-2020-003750 BMJ Global Health Barriers Refugee participation Refugee participation was noted as a facilitator as it provided manpower and community leadership.90 Refugee services run by refugees were shown to be feasible if there is sustained funding and technical assistance.130 Dedicated staff Having dedicated healthcare workers was noted multiple times as being a facilitator.86 106 121 ANC, antenatal care; CHWs, community health workers; EmOC, emergency obstetric care; GIS, geographic information system; IDPs, internally displaced persons; MISP, Minimum Initial Service Package; MNH, maternal and neonatal health; NGOs, non-governmental organisations; SRH, sexual and reproductive health; STI, sexually transmitted infection; TBAs, traditional birth attendants. Table 2 Continued copyright. on Octobe http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from copyright. on October 27, 2021 at Aga Khan Univers http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from copyright. on October 27, 2021 at Aga Khan University. Protected by http://gh.bmj.com/ st published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from ANC, antenatal care; CHWs, community health workers; EmOC, emergency obstetric care; GIS, geographic in internally displaced persons; MISP, Minimum Initial Service Package; MNH, maternal and neonatal health; NG organisations; SRH, sexual and reproductive health; STI, sexually transmitted infection; TBAs, traditional birth of interventions for neonates born in camps with those born outside of camps. Other reported barriers include lack of infrastructure and resources to provide EmOC services, lack of special- ised health workers and stigma towards refugees. A few publications also mentioned a lack of guidelines, such as newborn-specific protocols. Other reported barriers include lack of infrastructure and resources to provide EmOC services, lack of special- ised health workers and stigma towards refugees. A few publications also mentioned a lack of guidelines, such as newborn-specific protocols. Conflict hinders the delivery of MNH interventions and common barriers reported include the lack of safety, population displacement, limited resources and services, and a lack of skilled health workforce. Where the work- force is available, there are other barriers such as lack of clear guidelines. However, other factors such as avail- ability of funding, multisectoral collaboration, training and supervision of staff, as well as involving CHWs and refugees, were all reported to have facilitated the delivery of MNH interventions. p p Despite the barriers, there were other factors that were reported to facilitate the delivery of interventions. copyright. on October 27, 2021 at Aga http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from Being in an insecure environment was often mentioned as a hindrance to the delivery of interventions. Health facilities are destroyed, patients were also unable to access clinics due to security issues. This was especially relevant for services such as emergency obstetric care when women may need to be referred to hospitals. Shortages of supplies/resources (medicine, diagnostic tests) during a period of conflict were also noted as barriers and hampered both maternal and newborn care. Materials such as gloves for delivery, obstetric equipment or beds for premature babies were some of the ones noted as missing.43 111 This was noted as a barrier for both patients as well as healthcare workers. Refugees may also be stigmatised by their hosts.63 Limited funding was also noted as a barrier as it limited to range of services and materials available. Conflict reduces the range of available services. For maternal health, this was especially dangerous in cases where there is limited EmOC services as needed surgical facilities were not available or there was no training in the management of postpartum complications,33 64 emergency referral services were also not always available. There were some differences noted between the quality of care delivered at hospitals, compared with clinics; hospitals having a higher quality.111 ANC and postnatal care were also not always delivered to their full extent. A few studies mentioned lack of guidelines for STI prevention or lack of newborn-specific clinical and referral protocols as barriers to implementation.112 Multisectoral collaboration between international NGOs, the Ministry of Health and existing district health offices/public sector was noted as facilitators. Working with local NGOs was also a facilitator as they are already connected to the community. Training improved the skills of health workers and increased motivation. Continuous supervision/refresher training was encouraged, especially if provided by trained paediatricians or obstetricians.122 S-CORT modules are an innovative approach that focuses on training on the clinical services included in the MISP.122 One NGO developed a simple and low-cost 38- hour training course to upgrade the skills of TBAs.113 Continued 7 Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. Principal findings The majority of reported maternal health interventions delivered in conflict settings focused on ANC and its different components. Caesarean sections and the avail- ability of EmOC more broadly were also reported rela- tively commonly, but with little detail on EmOC compo- nents. Postnatal care was reported relatively infrequently, also with little detail about what such care was composed of. Maternal health interventions were reported to be delivered mainly by skilled health workers and within hospitals and clinics, but health posts and mobile clinics were also cited, as well as homes and communal spaces at the community level. We did note some differences in delivery personnel and sites between in-camp and out- of-camp mothers, where out-of-camp mothers are mostly attended to by lower cadres of health workers and where communal spaces are used more often. copyright. on October 27, 2021 at Aga http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from The presence of skilled and dedicated staff, as well as having a trained doctor/midwife present to provide mentor- ship for other staff, were noted as facilitators. Another important facilitator reported was the further training of existing healthcare workers. Involving CHWs and training TBAs from the same populations as those being served were also noted as facilitators; these are usually trusted members of the community with whom local women may feel more comfortable. Other reported facilitators include multisectoral collaboration between NGOs and the existing health system, having adequate funding, and refugee participation, among others (table 2). Most publications that included quantitative data reported on intervention coverage, and the majority on ANC coverage specifically. There were very few publica- tions that reported on effectiveness of MNH interven- tions; some studies did compare the rates of caesarean sections or other major obstetric interventions before and after the introduction of an intervention such as the training of health workers. Reported quantitative data on newborn health were mainly coverage estimates of the initiation of ARV prophylaxis in infants exposed to HIV and on initiation of breast feeding. It was not possible to infer differences in intervention coverage or effective- ness by delivery characteristics because of the large vari- ability in outcome measures and populations. Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 Evidence gaps N bli i No publications reported on the use of lay community members in the delivery of MNH interventions in conflict settings. It has already been shown that involving commu- nity members, including men and community leaders, in the promotion and delivery of maternal health inter- ventions such education/campaigns can be highly effec- tive,136 and refugee participation was also noted as a facil- itator in one of the studies. 21 at Aga Khan University. Protected by We found very limited literature on newborn care, perhaps reflecting the relative neglect that this area continues to endure in LMICs more generally. However, this may also simply reflect relatively limited reporting of newborn care interventions in the literature to date, rather than actual intervention priorities in the field. While it is encouraging that most reported neonatal interventions were able to be delivered in hospitals and clinics, there may be missed opportunities to use outreach and community-based sites such as health posts There were very few reported newborn health inter- ventions, focusing mostly on essential newborn care delivered by trained health workers, or in some cases, by TBAs. Nearly all reported newborn interventions were delivered within hospitals or clinics, with only one (post- natal check-up) delivered in a health post, and none were delivered using a community-based approach. Due to the limited literature, it is difficult to compare the delivery 8 Munyuzangabo M, et al. BMJ Global Health 2021;5:e003750. doi:10.1136/bmjgh-2020-003750 copyright. on October 27, 2021 at Aga Khan University. Protected http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from BMJ Global Health copyright. on October 27, 2021 at Aga Khan Unive http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-003750 on 19 February 2021. Downloaded from or homes, especially when it comes to postnatal care and follow-up for diseases such as HIV. Lower cadre health workers such as CHWs, who seem to be underused when it comes to newborn health, could also be used to deliver these interventions. by whom essential interventions are delivered in conflict settings, and more and better evaluation of the effective- ness of the delivery platforms used. In particular, robust evaluation of the wider use of lower level health workers, including those recruited from affected communities, to deliver a range of MNH interventions outside of camp settings would be especially useful. Evidence gaps N bli i on October 27, 2021 at Aga Khan Univ http://gh.bmj.com/ February 2021. Downloaded from Map disclaimer The depiction of boundaries on this map does not imply the expression of any opinion whatsoever on the part of BMJ (or any member of its group) concerning the legal status of any country, territory, jurisdiction or area or of its authorities. This map is provided without any warranty of any kind, either express or implied. Competing interests None declared. Patient consent for publication Not required. CONCLUSION While the challenges of research in humanitarian crises make it difficult to determine how well the existing literature on MNH intervention delivery in conflict settings reflects what is actually happening in the field, our results indicate a number of potential implications for research, programming and policy. There is a clear need for more and better reporting on how, where and Study strengths and weaknesses Provenance and peer review Not commissioned; externally peer reviewed. While we sought to retrieve a comprehensive selection of the grey literature reporting on MNH intervention delivery in conflict settings, the very large volume of potentially relevant reports meant that we had to restrict our search, and we may not have captured the full range of relevant interventions and their delivery character- istics. Moreover, at least some gaps in the existing liter- ature likely reflect reporting gaps rather than actual programmatic gaps, given the many constraints on data collection in conflict settings and on humanitarian health responders’ time and research capacities. Nonetheless, this is the first review, to our knowledge, to systematically capture and synthesise reported information in both the indexed and grey literature on how MNH interventions targeting conflict-affected populations have been and are being delivered in the field. Data availability statement All data relevant to the study are included in the article or uploaded as supplemental information. 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. October 27, 2021 at Aga Khan University. Protected by 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/. ORCID iDs Reena P Jain http://orcid.org/0000-0001-7004-5093 Shailja Shah http://orcid.org/0000-0003-2329-5027 Evidence gaps N bli i Further, despite the continued high burden of neonatal mortality in crisis settings, the delivery of newborn health interventions in conflict settings still does not appear to be sufficiently prioritised. This needs to change, urgently. There was limited literature on the implementation of the Minimal Initial Service Package (MISP), which is considered key, universal guidance for sexual and repro- ductive health at the onset of an emergency, including for reducing maternal and neonatal mortality. While a few articles did mention following the MISP guidelines, these did not provide further details on which components. Few publications reported quantitative data, and of those that did, most reported on intervention coverage, mainly ANC coverage. However, even within this one area, there is much variability between publications with respect to how coverage is defined and reported, including with respect to whether such care was provided by skilled health personnel. There is also very limited evidence reported about the effectiveness of these interventions. Twitter Fahad J Siddiqui @fjsepi Contributors ZAB, MFG and FJS conceptualised the review. FJS created the search strategy and conducted the literature search in electronic databases. DSK conducted the grey literature search. MM, DSK, SM, DA, MK, RPJ, SS and AR screened studies for eligibility and performed data extraction. MM and SM conducted the analysis with support from MFG and FJS. MM drafted the manuscript, and MFG and ZAB critically revised it. All authors approved the final version. Funding As coordinator of the BRANCH Consortium (Bridging Research & Action in Conflict Settings for the Health of Women & Children), the Sick Kids Centre for Global Child Health has received funding for research activities from the International Development Research Centre (IDRC) (108416-002 & 108640-001), the Norwegian Agency for Development Cooperation (Norad) (QZA-16/0395), the Bill & Melinda Gates Foundation (OPP1171560), and UNICEF (PCA 20181204). Aga Khan University has received funding for BRANCH activities from the Family Larsson-Rosenquist Foundation. 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https://openalex.org/W4232154382	https://peerj.com/articles/11886v0.4/submission	English	null	Peer Review #2 of "Distribution and abundance of the land snail Pollicaria elephas (Gastropoda: Pupinidae) in limestone habitats in Perak, Malaysia (v0.3)"	null	2,021	cc-by	15,020	Manuscript to be reviewed 15 Abstract 16 This study aimed to reveal the habitat variables that determine the distribution and abundance of 17 the land snail Pollicaria elephas in limestone habitats in Perak, Malaysia. Seventeen plots were 18 selected on a limestone hill to determine the effect of environmental variables on the abundance 19 of this land snail. The environmental variables we considered included habitat (canopy cover and 20 leaf litter thickness), topography (elevation, aspect, ruggedness, and slope), microclimate (soil 21 temperature, air temperature, and humidity), and vegetation (abundance of respective vascular 22 plant species). The correlation analyses suggested that the snails’ abundance was positively 23 correlated with the abundance of the four vascular plant species: Diospyros toposia var. 24 toposoides, Croton cascarilloides, Kibatalia laurifolia, and Mallotus peltatus. Plots with lower 25 soil temperatures had more snails than plots with higher soil temperatures. Our results show that 26 plots in the southern part of the limestone hill, in which P. elephas were absent, were similar in 27 habitat, topography, microclimate, and vegetation to the plots in the northern part of the limestone 28 hill, where specimens were mostly present. The absence of this species in suitable habitats may be 29 due to their low dispersal ability rather than adverse environmental conditions. 30 31 32 33 34 35 36 37 38 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) 16 This study aimed to reveal the habitat variables that determine the distribution and abundance of 17 the land snail Pollicaria elephas in limestone habitats in Perak, Malaysia. Seventeen plots were 18 selected on a limestone hill to determine the effect of environmental variables on the abundance 19 of this land snail. The environmental variables we considered included habitat (canopy cover and 20 leaf litter thickness), topography (elevation, aspect, ruggedness, and slope), microclimate (soil 21 temperature, air temperature, and humidity), and vegetation (abundance of respective vascular 22 plant species). The correlation analyses suggested that the snails’ abundance was positively 23 correlated with the abundance of the four vascular plant species: Diospyros toposia var. 24 toposoides, Croton cascarilloides, Kibatalia laurifolia, and Mallotus peltatus. Plots with lower 25 soil temperatures had more snails than plots with higher soil temperatures. Our results show that 26 plots in the southern part of the limestone hill, in which P. 1 Distribution and abundance of the land snail Pollicaria 2 elephas (Gastropoda: Pupinidae) in limestone habitats in P k M l i 5 Thor-Seng Liew Corresp.,1, Chee-Chean Phung1, Mohamad Afandi Mat Said2, Pui Kiat Hoo3 6 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Malaysia 7 2 Associated Pan Malaysia Cement, Perak, Malaysia 8 3 Faculty of Science, University of Malaya, Malaysia 9 Distribution and abundance of the land snail Pollicaria elephas (Gastropoda: Pupinidae) in limestone habitats in Perak Malaysia The environmental variables we consid 20 leaf litter thickness), topography (elevation, aspect, rugg 21 temperature, air temperature, and humidity), and vegeta 22 plant species). The correlation analyses suggested that 23 correlated with the abundance of the four vascular 24 toposoides, Croton cascarilloides, Kibatalia laurifolia, a 25 soil temperatures had more snails than plots with higher 26 plots in the southern part of the limestone hill, in which 27 habitat, topography, microclimate, and vegetation to the p 28 hill, where specimens were mostly present. The absence o 29 due to their low dispersal ability rather than adverse envir 30 31 32 33 34 Distribution and abundance of the land snail Pollicaria elephas (Gastropoda: Pupinidae) in limestone habitats in Perak Malaysia Thor-Seng Liew Corresp., 1 , Chee-Chean Phung 1 , Mohamad Afandi Mat Said 2 , Pui Kiat Hoo 1, 3 Thor-Seng Liew Corresp., 1 , Chee-Chean Phung 1 , Mohamad Afandi Mat Said 2 , Pui Kiat Hoo 1, 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Saba 2 Associated Pan Malaysia Cement, Chemor, Perak, Malaysia 3 Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Corresponding Author: Thor-Seng Liew Email address: thorseng@ums.edu.my 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Kota Kinabalu, Malaysia 2 Associated Pan Malaysia Cement, Chemor, Perak, Malaysia 3 Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Corresponding Author: Thor-Seng Liew Email address: thorseng@ums.edu.my 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Kota Kinabalu, Malaysia 2 Associated Pan Malaysia Cement, Chemor, Perak, Malaysia 3 Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia This study aimed to reveal the habitat variables that determine the distribution and abundance of the land snail Pollicaria elephas in limestone habitats in Perak, Malaysia. Seventeen plots were selected on a limestone hill to determine the eﬀect of environmental variables on the abundance of this land snail. The environmental variables we considered included habitat (canopy cover and leaf litter thickness), topography (elevation, aspect, ruggedness, and slope), microclimate (soil temperature, air temperature, and humidity), and vegetation (abundance of respective vascular plant species). The correlation analyses suggested that the snails’ abundance was positively correlated with the abundance of the four vascular plant species : Diospyros toposia var. toposoides, Croton cascarilloides, Kibatalia laurifolia, and Mallotus peltatus. Plots with lower soil temperatures had more snails than plots with higher soil temperatures. Our results show that plots in the southern part of the limestone hill, in which P. elephas were absent, were similar in habitat, topography, microclimate, and vegetation to the plots in the northern part of the limestone hill, where specimens were mostly present. The absence of this species in suitable habitats may be due to their low dispersal ability rather than adverse environmental conditions. Distribution and abundance of the land snail Pollicaria elephas (Gastropoda: Pupinidae) in limestone habitats in Perak Malaysia PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Manuscript to be reviewed 1 Distribution and abundance of the land snail Pollicaria 2 elephas (Gastropoda: Pupinidae) in limestone habitats i 3 Perak, Malaysia 4 5 Thor-Seng Liew Corresp.,1, Chee-Chean Phung1, Mohamad Afandi Mat Said2, Pui Kiat Hoo3 6 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Malaysia 7 2 Associated Pan Malaysia Cement, Perak, Malaysia 8 3 Faculty of Science, University of Malaya, Malaysia 9 10 11 Corresponding Author: Thor-Seng Liew 12 Email address: thorseng@ums.edu.my 13 14 15 Abstract 16 This study aimed to reveal the habitat variables that determine the distribution and abun 17 the land snail Pollicaria elephas in limestone habitats in Perak, Malaysia. Seventeen pl 18 selected on a limestone hill to determine the effect of environmental variables on the ab 19 of this land snail. The environmental variables we considered included habitat (canopy c 20 leaf litter thickness), topography (elevation, aspect, ruggedness, and slope), microclim 21 temperature, air temperature, and humidity), and vegetation (abundance of respective 22 plant species). The correlation analyses suggested that the snails’ abundance was p 23 correlated with the abundance of the four vascular plant species: Diospyros topo 24 toposoides, Croton cascarilloides, Kibatalia laurifolia, and Mallotus peltatus. Plots wi 25 soil temperatures had more snails than plots with higher soil temperatures. Our results s 26 plots in the southern part of the limestone hill, in which P. elephas were absent, were s 27 habitat, topography, microclimate, and vegetation to the plots in the northern part of the l 28 hill, where specimens were mostly present. The absence of this species in suitable habitat 29 due to their low dispersal ability rather than adverse environmental conditions. 30 31 32 33 34 35 1 Distribution and abundance of the la 2 elephas (Gastropoda: Pupinidae) in 3 Perak, Malaysia 4 5 Thor-Seng Liew Corresp.,1, Chee-Chean Phung1, Mohamad Afand 6 1 Institute for Tropical Biology and Conservation, Universiti M 7 2 Associated Pan Malaysia Cement, Perak, Malaysia 8 3 Faculty of Science, University of Malaya, Malaysia 9 10 11 Corresponding Author: Thor-Seng Liew 12 Email address: thorseng@ums.edu.my 13 14 15 Abstract 16 This study aimed to reveal the habitat variables that deter 17 the land snail Pollicaria elephas in limestone habitats in 18 selected on a limestone hill to determine the effect of env 19 of this land snail. Manuscript to be reviewed 39 40 Introduction 41 There are approximately 1,000 recognised land snail species in Malay 42 (http://malaypeninsularsnail.myspecies.info/, http://opisthostoma.myspecies.in 43 http://borneanlandsnails.myspecies.info/). However, the ecology of the land snail species is of 44 poorly known. To date, only a handful species from the genera Plectostoma, Georis 45 Gyliotrachela, Diplommatina have been studied in terms of their growth (Berry, 1962, 1963; L 46 et al., 2014b), reproduction (Berry, 1965), and demography (Berry, 1966; Schilthuizen et 47 2003). 48 Land snails of the genus Pollicaria, commonly known as elephant pupinid snails, belong to 49 family Pupinidae. All seven Pollicaria species and subspecies from Indochina and Peninsu 50 Malaysia are endemic to this region. P. elephas is the only Pollicaria species found on Peninsu 51 Malaysia (Kongim et al., 2013) (Fig. 1A). This species was described by de Morgan (1885) in 52 state of Perak, Malaysia. P. elephas specimens were recorded in various localities from 53 limestone hills in Perak and from two other locations in Pahang (Chan, 1998; Kongim et al., 20 54 Foon et al., 2017; Minton et al., 2017; GBIF.org, 2018). 55 Some aspects of P. elephas’ morphology, taxonomy, karyotypes, and geographical distribut 56 have been studied (Pain, 1974; Chan, 1997; Kongim et al., 2010; Kongim et al., 2013). Howev 57 the ecology and small-scale distribution of this ground-dwelling species remain unknown. 58 found localities with very high densities of P. elephas during a preliminary survey of a limest 59 hill in Perak, while just tens of meters away, no snails were found. This patchy distribution is 60 unusual. A previous study on another similarly-sized land snail, Limicolaria martensiana, a 61 showed an uneven distribution with more than 100 individuals per m2 at one locality at Ugan 62 (Owen, 1969). 63 Studies of other macro snails from other regions show that a higher land snail abundance can 64 explained by vegetation or habitat characteristics, such as a denser and heterogeneous canopy 65 understory, higher litter humidity and thickness, older and bigger trees, rotten logs, and calci 66 availability (Boag, 1985; Martin and Sommer, 2004; Müller et al., 2005; Horsák et al., 20 67 Dvořáková and Horsák, 2012). The flora composition can be difficult to measure directly but m 68 be a very useful predictor for snail communities (Dvořáková and Horsák, 2012). 69 We examined specific environmental parameters that may be responsible for the unev 70 distribution of P. elephas on a limestone hill. 40 Introduction To date, the limestone hill in Perak is the o 71 location where a sizeable living population of P. elephas is found based on several system 72 samplings of land snails throughout limestone hills in Peninsular Malaysia (Chan, 1997; Cleme 73 et al., 2008; Foon et al., 2017). We first assessed the population size and density of P. elepha 74 different localities on the hill in Perak. We then examined the vegetation and topographic 75 microclimatic variables for each locality to characterise species-specific requirements. 41 There are approximately 1,000 recognised land snail species in Malaysia 42 (http://malaypeninsularsnail.myspecies.info/, http://opisthostoma.myspecies.info/, 43 http://borneanlandsnails.myspecies.info/). However, the ecology of the land snail species is often 44 poorly known. To date, only a handful species from the genera Plectostoma, Georissa, 45 Gyliotrachela, Diplommatina have been studied in terms of their growth (Berry, 1962, 1963; Liew 46 et al., 2014b), reproduction (Berry, 1965), and demography (Berry, 1966; Schilthuizen et al., 47 2003). 48 Land snails of the genus Pollicaria, commonly known as elephant pupinid snails, belong to the 49 family Pupinidae. All seven Pollicaria species and subspecies from Indochina and Peninsular 50 Malaysia are endemic to this region. P. elephas is the only Pollicaria species found on Peninsular 51 Malaysia (Kongim et al., 2013) (Fig. 1A). This species was described by de Morgan (1885) in the 52 state of Perak, Malaysia. P. elephas specimens were recorded in various localities from the 53 limestone hills in Perak and from two other locations in Pahang (Chan, 1998; Kongim et al., 2013; 54 Foon et al., 2017; Minton et al., 2017; GBIF.org, 2018). 48 Land snails of the genus Pollicaria, commonly known as elephant pupinid snails, belong to the 49 family Pupinidae. All seven Pollicaria species and subspecies from Indochina and Peninsular 50 Malaysia are endemic to this region. P. elephas is the only Pollicaria species found on Peninsular 51 Malaysia (Kongim et al., 2013) (Fig. 1A). This species was described by de Morgan (1885) in the 52 state of Perak, Malaysia. P. elephas specimens were recorded in various localities from the 53 limestone hills in Perak and from two other locations in Pahang (Chan, 1998; Kongim et al., 2013; 54 Foon et al., 2017; Minton et al., 2017; GBIF.org, 2018). 48 Land snails of the genus Pollicaria, commonly known as elephant pupinid snails, belong to the 49 family Pupinidae. 40 Introduction All seven Pollicaria species and subspecies from Indochina and Peninsular 50 Malaysia are endemic to this region. P. elephas is the only Pollicaria species found on Peninsular 51 Malaysia (Kongim et al., 2013) (Fig. 1A). This species was described by de Morgan (1885) in the 52 state of Perak, Malaysia. P. elephas specimens were recorded in various localities from the 53 limestone hills in Perak and from two other locations in Pahang (Chan, 1998; Kongim et al., 2013; 54 Foon et al., 2017; Minton et al., 2017; GBIF.org, 2018). 55 Some aspects of P. elephas’ morphology, taxonomy, karyotypes, and geographical distribution 56 have been studied (Pain, 1974; Chan, 1997; Kongim et al., 2010; Kongim et al., 2013). However, 57 the ecology and small-scale distribution of this ground-dwelling species remain unknown. We 58 found localities with very high densities of P. elephas during a preliminary survey of a limestone 59 hill in Perak, while just tens of meters away, no snails were found. This patchy distribution is not 60 unusual. A previous study on another similarly-sized land snail, Limicolaria martensiana, also 61 showed an uneven distribution with more than 100 individuals per m2 at one locality at Uganda 62 (Owen, 1969). 55 Some aspects of P. elephas’ morphology, taxonomy, karyotypes, and geographical distribution 56 have been studied (Pain, 1974; Chan, 1997; Kongim et al., 2010; Kongim et al., 2013). However, 57 the ecology and small-scale distribution of this ground-dwelling species remain unknown. We 58 found localities with very high densities of P. elephas during a preliminary survey of a limestone 59 hill in Perak, while just tens of meters away, no snails were found. This patchy distribution is not 60 unusual. A previous study on another similarly-sized land snail, Limicolaria martensiana, also 61 showed an uneven distribution with more than 100 individuals per m2 at one locality at Uganda 62 (Owen, 1969). 55 Some aspects of P. elephas’ morphology, taxonomy, karyotypes, and geographical distribution 56 have been studied (Pain, 1974; Chan, 1997; Kongim et al., 2010; Kongim et al., 2013). However, 57 the ecology and small-scale distribution of this ground-dwelling species remain unknown. We 58 found localities with very high densities of P. elephas during a preliminary survey of a limestone 59 hill in Perak, while just tens of meters away, no snails were found. This patchy distribution is not 60 unusual. 40 Introduction A previous study on another similarly-sized land snail, Limicolaria martensiana, also 61 showed an uneven distribution with more than 100 individuals per m2 at one locality at Uganda 62 (Owen, 1969). 62 (Owen, 1969). 63 Studies of other macro snails from other regions show that a higher land snail abundance can be 64 explained by vegetation or habitat characteristics, such as a denser and heterogeneous canopy and 65 understory, higher litter humidity and thickness, older and bigger trees, rotten logs, and calcium 66 availability (Boag, 1985; Martin and Sommer, 2004; Müller et al., 2005; Horsák et al., 2007; 67 Dvořáková and Horsák, 2012). The flora composition can be difficult to measure directly but may 68 be a very useful predictor for snail communities (Dvořáková and Horsák, 2012). 69 We examined specific environmental parameters that may be responsible for the unevent 70 distribution of P. elephas on a limestone hill. To date, the limestone hill in Perak is the only 71 location where a sizeable living population of P. elephas is found based on several systematic 72 samplings of land snails throughout limestone hills in Peninsular Malaysia (Chan, 1997; Clements 73 et al., 2008; Foon et al., 2017). We first assessed the population size and density of P. elephas at 74 different localities on the hill in Perak. We then examined the vegetation and topographic and 75 microclimatic variables for each locality to characterise species-specific requirements. 76 63 Studies of other macro snails from other regions show that a higher land snail abundance can be 64 explained by vegetation or habitat characteristics, such as a denser and heterogeneous canopy and 65 understory, higher litter humidity and thickness, older and bigger trees, rotten logs, and calcium 66 availability (Boag, 1985; Martin and Sommer, 2004; Müller et al., 2005; Horsák et al., 2007; 67 Dvořáková and Horsák, 2012). The flora composition can be difficult to measure directly but may 68 be a very useful predictor for snail communities (Dvořáková and Horsák, 2012). 63 Studies of other macro snails from other regions show that a higher land snail abundance can be 64 explained by vegetation or habitat characteristics, such as a denser and heterogeneous canopy and 65 understory, higher litter humidity and thickness, older and bigger trees, rotten logs, and calcium 66 availability (Boag, 1985; Martin and Sommer, 2004; Müller et al., 2005; Horsák et al., 2007; 67 Dvořáková and Horsák, 2012). 15 Abstract elephas were absent, were similar in 27 habitat, topography, microclimate, and vegetation to the plots in the northern part of the limestone 28 hill, where specimens were mostly present. The absence of this species in suitable habitats may be 29 due to their low dispersal ability rather than adverse environmental conditions. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) 78 Study Site 79 The study site was located on a limestone hill in Perak, Malaysia. We established a total of 17 80 plots, each measuring 2 m x 4 m. Seven plots were located in the northern part, nine at the southern 81 part, and one at the central part of the hill (Fig. 2). Each plot was located next to limestone rock 82 outcrops. A pilot survey was conducted to ensure that these plots covered habitats with different 83 environmental variables and to identify plots with living P. elephas suitable for the population 84 density study. In each plot, leaf litter was searched manually by two people for over 20 minutes to 85 find living snails and empty shells. Environmental variables for each of the 17 plots were measured 86 during the pilot survey on 11 May 2018. Manuscript to be reviewed Manuscript to be reviewed 87 Spatial distribution and population density 88 We did not find any living P. elephas or empty shells in the 11 of the 17 examined plots during 89 the pilot survey (Table 1 in Supplemental File 1). We used the capture-mark-recapture method 90 (CMR) to study the population of P. elephas in five of the six plots with living snails. One of the 91 plots (AP-0) was inaccessible after the pilot survey so the CMR study could not be conducted in 92 that plot. The captured P. elephas were marked with different colours of nail polish (Fig. 1B and 93 1C) during different sampling sessions. The snails were then released back to their respective plots, 94 and plots were resampled after 10 to 15 days. The CMR was conducted four times for all plots 95 with the exception of one plot (AP-2), where only three CMR sessions took place. The first 96 sampling was conducted on 9 July 2018 and the first recapture was on 19 July 2018, the second 97 recapture occurred on 1 August 2018, and the third recapture took place on 16 August 2018. The 98 collected snails from the following demographics were examined: juvenile (< 3 whorls), subadult 99 (3 – 5 whorls), and adult (with aperture lip). All living snails caught were released back to their 100 respective plots while the empty shells were collected and deposited in the BORNEENSIS 101 collection of Universiti Malaysia Sabah. 102 We calculated the P. elephas population density by counting the living snails in individual plots 103 during the different CMR sessions. The population size of P. elephas was estimated based on the 104 data collected using the Schnabel index: 105 ● N = total number of snails (unknown) 106 ● C = number of snails captured on the first sampling 107 ● M = number of snails captured on subsequent sampling 108 ● R = number of snails captured on both samplings 109 Multiple marks and recaptures ensured a more accurate estimation of the population size. The 110 Schnabel method (Alcoy, 2013), which allows multiple capture-recapture encounters, was used: 88 We did not find any living P. elephas or empty shells in the 11 of the 17 examined plots during 89 the pilot survey (Table 1 in Supplemental File 1). We used the capture-mark-recapture method 90 (CMR) to study the population of P. elephas in five of the six plots with living snails. 40 Introduction The flora composition can be difficult to measure directly but may 68 be a very useful predictor for snail communities (Dvořáková and Horsák, 2012). 69 We examined specific environmental parameters that may be responsible for the unevent 70 distribution of P. elephas on a limestone hill. To date, the limestone hill in Perak is the only 71 location where a sizeable living population of P. elephas is found based on several systematic 72 samplings of land snails throughout limestone hills in Peninsular Malaysia (Chan, 1997; Clements 73 et al., 2008; Foon et al., 2017). We first assessed the population size and density of P. elephas at 74 different localities on the hill in Perak. We then examined the vegetation and topographic and 75 microclimatic variables for each locality to characterise species-specific requirements. 76 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) 110 Schnabel method (Alcoy, 2013), which allows multiple capture-recapture e 109 Multiple marks and recaptures ensured a more accurate estimation of the 𝑅𝑖= 𝑇ℎ𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑎𝑟𝑘𝑒𝑑 𝑠𝑛𝑎𝑖𝑙𝑠 𝑐𝑎𝑢𝑔ℎ𝑡 𝑎𝑡 𝑡𝑖𝑚𝑒 𝑖 𝑅𝑖= 𝑇ℎ𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑎𝑟𝑘𝑒𝑑 𝑠𝑛𝑎𝑖𝑙𝑠 𝑐𝑎𝑢𝑔ℎ𝑡 𝑎𝑡 𝑡𝑖𝑚𝑒 𝑖 116 Environmental variables 117 We studied four main environmental variables, namely habitat, topography, microclimate, and 118 vegetation. All of the variables were measured for all 17 plots, with the exception of the 119 microclimate. We measured leaf litter thickness by averaging litter thickness at eight points 120 within each plot for habitat variables and estimated the percentage of canopy cover. To obtain 121 topographic variables, we created a digital elevation model (DEM) of 4 m2 cell-size based on a 122 5-meter interval contour map for each of the northern and the southern study site using 123 Triangular interpolation (TIN) in QGIS (ver. 2.18.24; QGIS Development Team, 2018). We then 124 used terrain analysis tools to derive topographic features including slope, aspect, and the terrain 125 ruggedness index (a quantitative measurement of terrain heterogeneity) (Riley et al., 1999). 126 Topographic parameters were extracted for each sampling plot using the ‘Add raster values to 127 point’ setting in SAGA (Conrad et al., 2015). 𝑀𝑖= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑟𝑒𝑣𝑖𝑜𝑢𝑠𝑙𝑦 𝑚𝑎𝑟𝑘𝑒𝑑 𝑠𝑛𝑎𝑖𝑙𝑠 𝑎𝑡 𝑡𝑖𝑚𝑒 𝑖 𝑀𝑖= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑟𝑒𝑣𝑖𝑜𝑢𝑠𝑙𝑦 𝑚𝑎𝑟𝑘𝑒𝑑 𝑠𝑛𝑎𝑖𝑙𝑠 𝑎𝑡 𝑡𝑖𝑚𝑒 𝑖 𝐶𝑖= 𝑇ℎ𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑐𝑎𝑢𝑔ℎ𝑡 𝑎𝑡 𝑡𝑖𝑚𝑒 𝑖 87 Spatial distribution and population density One of the 91 plots (AP-0) was inaccessible after the pilot survey so the CMR study could not be conducted in 92 that plot. The captured P. elephas were marked with different colours of nail polish (Fig. 1B and 93 1C) during different sampling sessions. The snails were then released back to their respective plots, 94 and plots were resampled after 10 to 15 days. The CMR was conducted four times for all plots 95 with the exception of one plot (AP-2), where only three CMR sessions took place. The first 96 sampling was conducted on 9 July 2018 and the first recapture was on 19 July 2018, the second 97 recapture occurred on 1 August 2018, and the third recapture took place on 16 August 2018. The 98 collected snails from the following demographics were examined: juvenile (< 3 whorls), subadult 99 (3 – 5 whorls), and adult (with aperture lip). All living snails caught were released back to their 100 respective plots while the empty shells were collected and deposited in the BORNEENSIS 101 collection of Universiti Malaysia Sabah. 102 We calculated the P. elephas population density by counting the living snails in individual plots 103 during the different CMR sessions. The population size of P. elephas was estimated based on the 104 data collected using the Schnabel index: 102 We calculated the P. elephas population density by counting the living snails in individual plots 103 during the different CMR sessions. The population size of P. elephas was estimated based on the 104 data collected using the Schnabel index: 109 Multiple marks and recaptures ensured a more accurate estimation of the population size. The 109 Multiple marks and recaptures ensured a more accurate estimation of the population size. The 110 Schnabel method (Alcoy, 2013), which allows multiple capture-recapture encounters, was used: Schnabel method (Alcoy, 2013), which allows multiple capture-recapture encoun 110 Schnabel method (Alcoy, 2013), which allows multiple capture-recapture encounters, was used: PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed 111 𝑁= 𝑚 ∑ 𝑖= 1 𝑀𝑖𝐶𝑖 𝑚 ∑ 𝑖= 1 𝑅𝑖 111 Manuscript to be reviewed Manuscript to be reviewed 129 Microclimatic variables 130 We installed climatic HOBO data loggers to record air temperature and humidity (HOBO MX2301 131 Temperature/RH) approximately one meter above the ground for eight of the 17 plots (A-Polli3, 132 A-Polli9, D-P1, D-P3, D-P8, D-P9, D-P11, D-P12). We recorded the soil temperature using HOBO 133 MX2303 temperature sensors for the same eight plots. External sensors were fully covered by leaf 134 litter. These eight plots have been chosen, as they represent localities with both absence and 135 presence data and different population densities of these snails. Living specimens were found in 136 two northern plots, namely, plot A-Polli9, which had a higher number of snails (15–16 individuals) 137 and plot A-Polli3, which had a lower number of snails (1–2 individuals). There were no living 138 specimens found in the five southern plots. The climatic parameters were logged every 10 minutes 139 in July 2018. Data from the two soil temperature loggers could not be retrieved due to damage by 140 rain and wildlife. 131 Temperature/RH) approximately one meter above the ground for eight of the 17 plots (A-Polli3, 132 A-Polli9, D-P1, D-P3, D-P8, D-P9, D-P11, D-P12). We recorded the soil temperature using HOBO 133 MX2303 temperature sensors for the same eight plots. External sensors were fully covered by leaf 134 litter. These eight plots have been chosen, as they represent localities with both absence and 135 presence data and different population densities of these snails. Living specimens were found in 136 two northern plots, namely, plot A-Polli9, which had a higher number of snails (15–16 individuals) 137 and plot A-Polli3, which had a lower number of snails (1–2 individuals). There were no living 138 specimens found in the five southern plots. The climatic parameters were logged every 10 minutes 139 in July 2018. Data from the two soil temperature loggers could not be retrieved due to damage by 140 rain and wildlife. 141 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) 142 Vegetation data 143 We counted and identified all the vascular plants with diameter at breast height (DBH) above 1 144 cm within a 5-meter radius from the centre of each of the 17 plots to obtain the number of vascular 145 plant individuals and the number of vascular plant species of each plot. Voucher specimens were 146 collected for each species and were subsequently identified by P.K. Hoo based on the reference 147 materials at the Herbarium of Forest Research Institute Malaysia (FRIM). 149 Data analysis We calculated the daily mean air humidity, minimum 150 Principal component analysis (PCA) was conducted to assess the degree of habitat heterogeneity 151 among the 17 plots based on the two habitat variables (leaf litter thickness, and canopy cover), 152 four topographic variables (elevation, slope, aspect, and ruggedness index) and two vegetation 153 variables (number of vascular plant individuals and number of vascular plant species). The 154 abundance of vascular plant species was not included in the PCA analysis due to the number of 155 missing values in the dataset and the absence of certain plant species in plots. We visually explored 156 the PCA plot for habitat heterogeneity according to the plots’ locations on the limestone hill 157 (northern part, southern part, and central part). The analysis was done using R (Supplemental File 158 2). 159 Correction tests were performed to examine any significant relationships between the abundance 160 of snails and each of the habitat, vegetation, and topographic variables. We excluded 43 vascular 161 plant species that were recorded only in one plot before statistical analysis to obtain vegetation 162 data. The final dataset consisted of the abundance data for 20 vascular plant species from 17 plots. 168 There was either complete or partial missing microclimatic data from July for some of the plots, 169 so we did not calculate the mean values for each of the microclimatic variables per month. Hence, 170 we could not perform rigorous statistical analysis to test the relationship between the microclimatic 171 variables and the abundance of snails in the plot. Nevertheless, we explored the relationships 172 between the abundance of snails and microclimatic variables by plotting the mean of each sampled 173 plot’s daily microclimatic variables patterns. We calculated the daily mean air humidity, minimum 174 air humidity, mean air temperature, maximum air temperature, mean soil temperature, and 175 maximum soil temperature. 149 Data analysis 176 177 Results 178 Spatial distribution and population density of the snails PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) 150 Principal component analysis (PCA) was conducted to assess the degree of habitat heterogeneity 151 among the 17 plots based on the two habitat variables (leaf litter thickness, and canopy cover), 152 four topographic variables (elevation, slope, aspect, and ruggedness index) and two vegetation 153 variables (number of vascular plant individuals and number of vascular plant species). The 154 abundance of vascular plant species was not included in the PCA analysis due to the number of 155 missing values in the dataset and the absence of certain plant species in plots. We visually explored 156 the PCA plot for habitat heterogeneity according to the plots’ locations on the limestone hill 157 (northern part, southern part, and central part). The analysis was done using R (Supplemental File 158 2). 159 Correction tests were performed to examine any significant relationships between the abundance 160 of snails and each of the habitat, vegetation, and topographic variables. We excluded 43 vascular 161 plant species that were recorded only in one plot before statistical analysis to obtain vegetation 162 data. The final dataset consisted of the abundance data for 20 vascular plant species from 17 plots. 163 As the data were not normally distributed, we used Spearman correlation testing based on both 164 null-hypothesis significance testing and corroborated our analysis by using the Bayes factor 165 (BF10) (Kass & Raftery, 1995). Our conclusion is based on the inference of both frequentist (p 166 values) and Bayesian (Bayes factor) analyses. All analyses were performed using JASP software 167 version 0.12.2 (JASP Team, 2020; Supplemental File 3). 168 There was either complete or partial missing microclimatic data from July for some of the plots, 169 so we did not calculate the mean values for each of the microclimatic variables per month. Hence, 170 we could not perform rigorous statistical analysis to test the relationship between the microclimatic 171 variables and the abundance of snails in the plot. Nevertheless, we explored the relationships 172 between the abundance of snails and microclimatic variables by plotting the mean of each sampled 173 plot’s daily microclimatic variables patterns. 149 Data analysis 149 Data analysis 150 Principal component analysis (PCA) was conducted to assess the degree of habitat heterogeneity 151 among the 17 plots based on the two habitat variables (leaf litter thickness, and canopy cover), 152 four topographic variables (elevation, slope, aspect, and ruggedness index) and two vegetation 153 variables (number of vascular plant individuals and number of vascular plant species). The 154 abundance of vascular plant species was not included in the PCA analysis due to the number of 155 missing values in the dataset and the absence of certain plant species in plots. We visually explored 156 the PCA plot for habitat heterogeneity according to the plots’ locations on the limestone hill 157 (northern part, southern part, and central part). The analysis was done using R (Supplemental File 158 2). 159 Correction tests were performed to examine any significant relationships between the abundance 160 of snails and each of the habitat, vegetation, and topographic variables. We excluded 43 vascular 161 plant species that were recorded only in one plot before statistical analysis to obtain vegetation 162 data. The final dataset consisted of the abundance data for 20 vascular plant species from 17 plots. 163 As the data were not normally distributed, we used Spearman correlation testing based on both 164 null-hypothesis significance testing and corroborated our analysis by using the Bayes factor 165 (BF10) (Kass & Raftery, 1995). Our conclusion is based on the inference of both frequentist (p 166 values) and Bayesian (Bayes factor) analyses. All analyses were performed using JASP software 167 version 0.12.2 (JASP Team, 2020; Supplemental File 3). 168 There was either complete or partial missing microclimatic data from July for some of the plots, 169 so we did not calculate the mean values for each of the microclimatic variables per month. Hence, 170 we could not perform rigorous statistical analysis to test the relationship between the microclimatic 171 variables and the abundance of snails in the plot. Nevertheless, we explored the relationships 172 between the abundance of snails and microclimatic variables by plotting the mean of each sampled 173 plot’s daily microclimatic variables patterns. We calculated the daily mean air humidity, minimum 174 air humidity, mean air temperature, maximum air temperature, mean soil temperature, and 175 maximum soil temperature. fect of environmental variables on snail occurrence and abundance 196 The first three PCA axes explained 78.7% of the habitat, topography, and vegetation variations 197 between plots (Fig. 3, Supplemental File 4, Supplemental File 5). As shown in Fig. 3, the PCA 198 plot did not show apparent differences between the plots in the northern part (most of the plots 199 with living P. elephas) and the southern part (all plots without living P. elephas) on the limestone 200 hill. The abundance of P. elephas per plot was not correlated with canopy cover, leaf litter 201 thickness, elevation, aspect, slope, and the ruggedness of the habitat (Table 2). 196 The first three PCA axes explained 78.7% of the habitat, topography, and vegetation variations 197 between plots (Fig. 3, Supplemental File 4, Supplemental File 5). As shown in Fig. 3, the PCA 198 plot did not show apparent differences between the plots in the northern part (most of the plots 199 with living P. elephas) and the southern part (all plots without living P. elephas) on the limestone 200 hill. The abundance of P. elephas per plot was not correlated with canopy cover, leaf litter 201 thickness, elevation, aspect, slope, and the ruggedness of the habitat (Table 2). 202 The plot A-Polli9 with the higher number of living snails (15 - 16 individuals) had a soil mean 203 temperature lower than 25 °C, and maximum temperature lower than 26 °C (Fig. 4). However, the 204 two plots, namely D-P1 and D-P11, at the southern part of the hill with no snails recorded had a 205 similar mean and maximum soil temperature (~25 °C) to the plot A-Polli9. 202 The plot A-Polli9 with the higher number of living snails (15 - 16 individuals) had a soil mean 203 temperature lower than 25 °C, and maximum temperature lower than 26 °C (Fig. 4). However, the 204 two plots, namely D-P1 and D-P11, at the southern part of the hill with no snails recorded had a 205 similar mean and maximum soil temperature (~25 °C) to the plot A-Polli9. 206 All the plots at the southern and northern parts of the hill had a similar mean temperature with 207 differences smaller than 1 °C during most of the days, with the exception of plots D-P1 and D-P3 208 (Fig. 5). fect of environmental variables on snail occurrence and abundance There were no significant differences in the mean temperature among plot A-Polli9 with 209 a high number of living snails (15–16 individuals), plot A-Polli3 with few living snails (one to two 210 individuals), and other plots without living snails. The plots with a higher mean humidity (85% – 211 100%) at the southern part of the hill did not harbour P. elephas as compared to plots with lower 212 humidity (75%–93%) at the northern part of the hill (Fig. 6). 206 All the plots at the southern and northern parts of the hill had a similar mean temperature with 207 differences smaller than 1 °C during most of the days, with the exception of plots D-P1 and D-P3 208 (Fig. 5). There were no significant differences in the mean temperature among plot A-Polli9 with 209 a high number of living snails (15–16 individuals), plot A-Polli3 with few living snails (one to two 210 individuals), and other plots without living snails. The plots with a higher mean humidity (85% – 211 100%) at the southern part of the hill did not harbour P. elephas as compared to plots with lower 212 humidity (75%–93%) at the northern part of the hill (Fig. 6). 178 Spatial distribution and population density of the snails PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed 179 The numbers of living specimens collected over the four CMR sessions are shown in Supplemental 180 File 1. Living specimens were found in six out of seven plots on the northern part of the hill; none 181 were found in plots on the central and southern parts (Supplemental File 1). The smallest marked 182 specimen was 9 mm (shell width), and the majority of the marked snails were subadult and adult 183 (Supplemental File 1). The recapture rates were greater than 80% for the three plots with more 184 than ten snails recorded during the pilot survey and the first capture session of CMR (A-Polli2, A- 185 Polli7, and A-Polli9, see Table 1 - 4 in Supplemental File 1), except for two recapture sessions in 186 plot A-Polli2 (23% and 67%). The recapture rates were between 50% and 100% for the two plots 187 with less than ten snails. 179 The numbers of living specimens collected over the four CMR sessions are shown in Supplemental 180 File 1. Living specimens were found in six out of seven plots on the northern part of the hill; none 181 were found in plots on the central and southern parts (Supplemental File 1). The smallest marked 182 specimen was 9 mm (shell width), and the majority of the marked snails were subadult and adult 183 (Supplemental File 1). The recapture rates were greater than 80% for the three plots with more 184 than ten snails recorded during the pilot survey and the first capture session of CMR (A-Polli2, A- 185 Polli7, and A-Polli9, see Table 1 - 4 in Supplemental File 1), except for two recapture sessions in 186 plot A-Polli2 (23% and 67%). The recapture rates were between 50% and 100% for the two plots 187 with less than ten snails. 188 Of the five plots examined in the CMR study, plot A-Polli2 had the highest population size of 189 Pollicaria elephas (Table 1), and the calculated population density was estimated to be 190 approximately 57 individuals for that plot and its surrounding area. The highest number of snails 191 recorded per sampling event per plot was 26 specimens in plot A-Polli2 (Table 2 in Supplemental 192 File 1). The snails’ population density in the sampling plots varied only slightly during the different 193 sampling sessions for each plot (Supplemental File 1). Manuscript to be reviewed 188 Of the five plots examined in the CMR study, plot A-Polli2 had the highest population size of 189 Pollicaria elephas (Table 1), and the calculated population density was estimated to be 190 approximately 57 individuals for that plot and its surrounding area. The highest number of snails 191 recorded per sampling event per plot was 26 specimens in plot A-Polli2 (Table 2 in Supplemental 192 File 1). The snails’ population density in the sampling plots varied only slightly during the different 193 sampling sessions for each plot (Supplemental File 1). 231 Discussion 232 Synecology studies have focused on the association of habitat features and the composition of 233 communities of land snails (Müller et al., 2005). However, autecology studies on single species in 234 their natural habitat are scarce. The two different approaches of ecology have developed 235 independently, although the knowledge of both is necessary to understand the ecology of an 236 individual population within a species or the whole ecosystem. A broader understanding of a 237 species’ biogeography starts with the knowledge of the species’ autecology on a local scale 238 (Hugall, et al., 2002). Unfortunately, studies on the responses to environmental variables by 239 individual species of large land snails in the tropical ecosystem are lacking (Horsák et al., 2007). Manuscript to be reviewed The total number 229 of vascular plants and plant species was not correlated with the abundance of snails in the plots 230 (Table 2). 222 The abundance of four vascular plant species were positively correlated with the abundance of the 223 land snails based on the null-hypothesis significance testing (p < 0.05) and Bayes factor (BF10) 224 (Table 4, Supplemental File 7). Of these four species, Diospyros toposia var. toposoides 225 (Ebenaceae) was the only plant species found in plots with and without living snails. Two plant 226 species, namely, Croton cascarilloides (Euphorbiaceae) and Kibatalia laurifolia (Apocynaceae), 227 were recorded only in two plots (A-P0 and A-Polli9). Another plant species, Mallotus peltatus 228 (Euphorbiaceae), was recorded in only three plots (A-P0, A-P2 and A-Polli2). The total number 229 of vascular plants and plant species was not correlated with the abundance of snails in the plots 230 (Table 2). 222 The abundance of four vascular plant species were positively correlated with the abundance of the 223 land snails based on the null-hypothesis significance testing (p < 0.05) and Bayes factor (BF10) 224 (Table 4, Supplemental File 7). Of these four species, Diospyros toposia var. toposoides 225 (Ebenaceae) was the only plant species found in plots with and without living snails. Two plant 226 species, namely, Croton cascarilloides (Euphorbiaceae) and Kibatalia laurifolia (Apocynaceae), 227 were recorded only in two plots (A-P0 and A-Polli9). Another plant species, Mallotus peltatus 228 (Euphorbiaceae), was recorded in only three plots (A-P0, A-P2 and A-Polli2). The total number 229 of vascular plants and plant species was not correlated with the abundance of snails in the plots 230 (Table 2). 240 Spatial distribution and population density of P. elephas 241 The capture-mark-recapture technique has been used for estimation of population size and density 242 for land snails (Blinn, 1963; Hänsel et al., 1999; Standish et al., 2002; Parkyn et al., 2014). Previous 243 studies on other land snails showed that the capture rate using the CMR technique can be very 244 high, with up to an 85% recapture rate after one year (Kleewein, 1999). In our study, the recapture 245 rates varied by plot. Using this technique, we found that P. elephas can achieve high population 246 densities, between three and four individuals per square meter, in suitable habitats at the northern 247 part of the examined limestone hill. We could not find other population density studies on 248 similarly-sized caenogastropod land snails for comparison. However, studies on similarly-sized 249 pulmonate land snails showed that snails can occur in very low densities: less than one individual 250 per m2 to very high densities of up to 100 individuals per m2 for Limicolaria martensiana (Owen, 251 1969). However, Owen (1969) did not investigate environmental factors that could determine the 252 variation of densities in different population. 241 The capture-mark-recapture technique has been used for estimation of population size and density 242 for land snails (Blinn, 1963; Hänsel et al., 1999; Standish et al., 2002; Parkyn et al., 2014). Previous 243 studies on other land snails showed that the capture rate using the CMR technique can be very 244 high, with up to an 85% recapture rate after one year (Kleewein, 1999). In our study, the recapture 245 rates varied by plot. Using this technique, we found that P. elephas can achieve high population 246 densities, between three and four individuals per square meter, in suitable habitats at the northern 247 part of the examined limestone hill. We could not find other population density studies on 248 similarly-sized caenogastropod land snails for comparison. However, studies on similarly-sized 249 pulmonate land snails showed that snails can occur in very low densities: less than one individual 250 per m2 to very high densities of up to 100 individuals per m2 for Limicolaria martensiana (Owen, 251 1969). However, Owen (1969) did not investigate environmental factors that could determine the 252 variation of densities in different population. 253 We could not find P. elephas in the southern part of the limestone hill, while large populations 254 occurred on the northern part of the limestone hill. 214 Association between the abundance of P. elephas and vegetation 215 215 216 Sixty-three taxa of vascular plants were recorded in the 17 plots. Species identifications were 217 obtained for 46 species. The identity of 14 species could only be confirmed at the genus level, and PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed 218 the remaining three species could not be identified. Altogether 43 vascular plant species were 219 recorded in only one plot, of which 27 species were singletons (Table 3). The number of species 220 per plot ranged between three and 11 species, and the number of individuals ranged between four 221 and 42 (Supplemental File 4). 222 The abundance of four vascular plant species were positively correlated with the abundance of the 223 land snails based on the null-hypothesis significance testing (p < 0.05) and Bayes factor (BF10) 224 (Table 4, Supplemental File 7). Of these four species, Diospyros toposia var. toposoides 225 (Ebenaceae) was the only plant species found in plots with and without living snails. Two plant 226 species, namely, Croton cascarilloides (Euphorbiaceae) and Kibatalia laurifolia (Apocynaceae), 227 were recorded only in two plots (A-P0 and A-Polli9). Another plant species, Mallotus peltatus 228 (Euphorbiaceae), was recorded in only three plots (A-P0, A-P2 and A-Polli2). The total number 229 of vascular plants and plant species was not correlated with the abundance of snails in the plots 230 (Table 2). 218 the remaining three species could not be identified. Altogether 43 vascular plant species were 219 recorded in only one plot, of which 27 species were singletons (Table 3). The number of species 220 per plot ranged between three and 11 species, and the number of individuals ranged between four 221 and 42 (Supplemental File 4). 218 the remaining three species could not be identified. Altogether 43 vascular plant species were 219 recorded in only one plot, of which 27 species were singletons (Table 3). The number of species 220 per plot ranged between three and 11 species, and the number of individuals ranged between four 221 and 42 (Supplemental File 4). 222 The abundance of four vascular plant species were positively correlated with the abundance of the 223 land snails based on the null-hypothesis significance testing (p < 0.05) and Bayes factor (BF10) 224 (Table 4, Supplemental File 7). Of these four species, Diospyros toposia var. toposoides 225 (Ebenaceae) was the only plant species found in plots with and without living snails. Two plant 226 species, namely, Croton cascarilloides (Euphorbiaceae) and Kibatalia laurifolia (Apocynaceae), 227 were recorded only in two plots (A-P0 and A-Polli9). Another plant species, Mallotus peltatus 228 (Euphorbiaceae), was recorded in only three plots (A-P0, A-P2 and A-Polli2). Manuscript to be reviewed Manuscript to be reviewed 257 the microclimate, topography, habitat, and vegetation between the northern and southern part of 258 the limestone hill. 259 However, it is unclear why the snails from high-density spots did not migrate to the other spots at 260 the same hill with similar habitat. The dispersal ability of this species was not determined in this 261 study. Nevertheless, the dispersal distances for other similar-sized land snails are very short, 262 ranging from meters to tens of meters per year (Baur, 1986; Schilthuizen et al., 2005; Edmorthy et 263 al., 2012; Ozgo and Bogucki, 2011; Kramarenko, 2014). One possible explanation could be the 264 homing behaviour found in certain snail species (Rollo & Wellington, 1981, Tomiyama, 1992; 265 Stringer et al., 2018) with highly specialised habitat requirements. These species are not migrating 266 far from their favoured spot and can have narrow-ranged endemics occurring unevenly across a 267 large landscape. Another possible explanation may be that an unfavourable habitat prevents 268 dispersal of this species to isolated spots with suitable habitat. 269 Although the environmental variables included in this study were unlikely to determine the 270 absence and presence of this species in different parts of the hill, the heterogeneity of population 271 densities in the plots at the northern part of the hill showed that higher abundance of P. elephas 272 could be associated with lower soil temperatures. This is expected as P. elephas is a ground- 273 dwelling land snail. From our observation on the snails’ behaviour in the field and in captive 274 populations, snails were active during the night where they were seen feeding on leaf litter. In the 275 day, the snails could be found burying themselves underneath leaf litter. Living snails in the field 276 were never found attached to vegetation or rocks above ground. Hence, we can assume that a 277 constant and relatively low soil ground temperature and a lower air temperature and higher 278 humidity at night is important for the population to thrive (Supplemental File 8). 240 Spatial distribution and population density of P. elephas All examined plots were on the same limestone 255 outcrop, with generally similar climatic condition, soil conditions, and land snail communities 256 (Foon et al., 2017). Furthermore, there were no apparent differences in environmental variables in PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed 80 Association between snail abundance and abundance of plant species 280 Association between snail abundance and abundance of plant species 281 Previous studies on the association between plants and specific land snail species have been 282 conducted outside of the tropical regions (Blinn, 1963; Pollard, 1975; Cowie, 1985; Hänsel et al., 283 1999; Standish et al., 2002; Burrell et al., 2007; Horsák et al., 2011; Parkyn et al., 2013, 2014). 284 Most of these studies suggested that plants act as shelters for land snails (Blinn, 1963; Pollard, 285 1975; Cowie, 1985; Standish et al., 2002; Burrell et al., 2007; Parkyn et al., 2014). 286 The abundance of P. elephas is positively associated with a relatively common vascular plant, 287 Diospyros toposia var. toposoides, on the limestone hill. The other three comparatively uncommon 288 species, namely, Croton cascarilloides, Kibatalia laurifolia, and Mallotus peltatus were also 289 positively correlated with the snails’ abundance. All of the three species are only found in plots 290 with living snails (Table 3). However, it is important to note that due to the nature of high 291 heterogeneity of vascular plants in the forest, as well as the 43 vascular plant species that were 292 recorded in only one plot, there were only two plots for Kibatalia laurifolia and Croton 293 cascarilloides, respectively, and three plots for Mallotus peltatus. Also, this indicates that the 294 presence of the three plant species is not necessary for the presence of the snails as these plants 295 were not found in other plots where snails were present. Hence, future studies of carefully-selected 296 sites with different abundances of living snails and our identified species should be conducted to 297 verify the possible causal relationship. 286 The abundance of P. elephas is positively associated with a relatively common vascular plant, 287 Diospyros toposia var. toposoides, on the limestone hill. The other three comparatively uncommon 288 species, namely, Croton cascarilloides, Kibatalia laurifolia, and Mallotus peltatus were also 289 positively correlated with the snails’ abundance. All of the three species are only found in plots 290 with living snails (Table 3). Manuscript to be reviewed However, a study of other land snails in another part of the world 335 suggested that snail density correlated with calcium content and, to a lesser extent, with the pH of 336 the litter layer (Graveland & Van der Wal, 1996). 337 Lastly, predation pressure could also explain snails’ density distribution patterns (Abramsky et al., 338 1992; Meyer & Cowie, 2010; Gerlach et al., 2020). Unfortunately, data on population densities 339 and life histories for predators were not available from this study. 322 Other factors that may affect the distribution and density of land snails 323 The distribution and density of P. elephas may be influenced by factors that were not investigated 324 in this study, such as calcium availability, pH of the substrates, and predators. In the non-limestone 325 forests, calcium availability plays a major role in determining the population density of snails 326 (Gardenfors, 1992; Graveland & Van der Wal, 1996; Skeldon et al., 2007). However, we assumed 327 that calcium availability may not vary significantly because all the plots were located next to the 328 limestone outcrops (Crowther, 1987a); therefore, calcium availability may not be a factor that 329 requires further attention among the plots. 331 Typically, the pH of the substrates (soil and leaf litter) will be affected by the bedrock, and the 332 variation of pH among the plots on the limestone hills were very small (e.g. Crowther, 1982). 333 However, leaf pH and leaf litter can vary significantly among different plant species (Cornelissen 334 et al., 2011; Tao et al., 2019). However, a study of other land snails in another part of the world 335 suggested that snail density correlated with calcium content and, to a lesser extent, with the pH of 336 the litter layer (Graveland & Van der Wal, 1996). 331 Typically, the pH of the substrates (soil and leaf litter) will be affected by the bedrock, and the 332 variation of pH among the plots on the limestone hills were very small (e.g. Crowther, 1982). 333 However, leaf pH and leaf litter can vary significantly among different plant species (Cornelissen 334 et al., 2011; Tao et al., 2019). However, a study of other land snails in another part of the world 335 suggested that snail density correlated with calcium content and, to a lesser extent, with the pH of 336 the litter layer (Graveland & Van der Wal, 1996). Manuscript to be reviewed 298 A plausible explanation for this relationship could be that the leaf litter from these plant species is 299 suitable for the snails’ diet. However, it is also possible that rather than a direct causal relationship, 300 both plants and snails prefer the same parameters of the local environment. A specially designed 301 experiment is needed to test these hypotheses. To our knowledge, there were no in situ experiments 302 on food preferences of land snails conducted in the field. However, there have been laboratory 303 experiments conducted with decaying leaves of selected plant species (Puslednik, 2002; Proćków 304 et al., 2013). In situ experiments on food preferences in a tropical rainforest are challenging 305 because identifying leaf litter from plants is difficult, as plants are very species rich even within a 306 small area, as was shown in this study (Crowther, 1982, 1987a, 1987b). We cannot rule out the 307 hypothesis that plants and leaf litters provide shelter for land snails (e.g. Blinn, 1963; Pollard, 308 1975; Cowie, 1985; Standish et al., 2002; Burrell et al., 2007; Parkyn et al., 2014). 298 A plausible explanation for this relationship could be that the leaf litter from these plant species is 299 suitable for the snails’ diet. However, it is also possible that rather than a direct causal relationship, 300 both plants and snails prefer the same parameters of the local environment. A specially designed 301 experiment is needed to test these hypotheses. To our knowledge, there were no in situ experiments 302 on food preferences of land snails conducted in the field. However, there have been laboratory 303 experiments conducted with decaying leaves of selected plant species (Puslednik, 2002; Proćków 304 et al., 2013). In situ experiments on food preferences in a tropical rainforest are challenging 305 because identifying leaf litter from plants is difficult, as plants are very species rich even within a 306 small area, as was shown in this study (Crowther, 1982, 1987a, 1987b). We cannot rule out the 307 hypothesis that plants and leaf litters provide shelter for land snails (e.g. Blinn, 1963; Pollard, 308 1975; Cowie, 1985; Standish et al., 2002; Burrell et al., 2007; Parkyn et al., 2014). Manuscript to be reviewed However, it is important to note that due to the nature of high 291 heterogeneity of vascular plants in the forest, as well as the 43 vascular plant species that were 292 recorded in only one plot, there were only two plots for Kibatalia laurifolia and Croton 293 cascarilloides, respectively, and three plots for Mallotus peltatus. Also, this indicates that the 294 presence of the three plant species is not necessary for the presence of the snails as these plants 295 were not found in other plots where snails were present. Hence, future studies of carefully-selected 296 sites with different abundances of living snails and our identified species should be conducted to 297 verify the possible causal relationship. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed 309 There were single-species land snail studies to investigate the effects of vegetation on the 310 population density in non-tropical (Hänsel et al., 1999; Horsák et al., 2011; Barrientos, 2000, 2019; 311 Caldwell et al., 2014) and tropical regions (Barrientos, 2000). However, these studies relate the 312 abundance of land snails, either with the general characteristic of vegetation structures (e.g., 313 herbaceous vegetation in Hänsel et al., 1999; sparse herb vegetation in Horsák et al., 2011; 314 thickness of herbaceous vegetation in Barrientos, 2000) or common plant species in the study sites 315 (e.g., Barrientos, 2000; Caldwell et al., 2014). These studies could not establish proof of causation 316 between specific plant species and the abundance of land snails. Although we could not confirm 317 the in-depth association between the vascular plant species and snail feeding ecology, we were 318 able to identify candidate plants to be included in future experiments. It would be worthwhile to 319 investigate the pH and nutrient content of the plant species as the possible food sources and shelters 320 for the land snail since there was an association between snail abundance and particular plant 321 species. 322 Other factors that may affect the distribution and density of land snails 323 The distribution and density of P. elephas may be influenced by factors that were not investigated 324 in this study, such as calcium availability, pH of the substrates, and predators. In the non-limestone 325 forests, calcium availability plays a major role in determining the population density of snails 326 (Gardenfors, 1992; Graveland & Van der Wal, 1996; Skeldon et al., 2007). However, we assumed 327 that calcium availability may not vary significantly because all the plots were located next to the 328 limestone outcrops (Crowther, 1987a); therefore, calcium availability may not be a factor that 329 requires further attention among the plots. 330 331 Typically, the pH of the substrates (soil and leaf litter) will be affected by the bedrock, and the 332 variation of pH among the plots on the limestone hills were very small (e.g. Crowther, 1982). 333 However, leaf pH and leaf litter can vary significantly among different plant species (Cornelissen 334 et al., 2011; Tao et al., 2019). 340 Conclusion 341 We determined the ecological aspects of P. elephas in terms of the habitat, topography, 342 microclimate, and vegetation variables. We also found that ground temperature and a few vascular 343 plant species were positively associated with snail abundance. Although our study was limited by 344 its short duration and the absence of replicate sites on other hills, our findings can be used to 345 formulate testable hypotheses when another population of this snail is found on further sites. After 346 this exploratory study, we suggest a more focused, hypothesis-driven study to determine: (1) how 347 the microclimates variations affect the land snails’ activities during the day and night; (2) the roles 348 of the four vascular plant species that were found associated with living snails as food or shelter. 349 Acknowledgements 350 We thank Jaap Vermeulen and the two anonymous reviewers for their cons 350 We thank Jaap Vermeulen and the two anonymous reviewers for their constructive comments. 351 We want to thank Richard Chung Cheng Kong, the curator of The Herbarium in Forest Research 352 Institute Malaysia (KEP), for allowing us to access the herbarium and its facilities. Postar Miun, 353 a field botanist from Forest Research Center (FRC), has helped tremendously identify plant 354 voucher specimens, for which we are grateful. p y 351 We want to thank Richard Chung Cheng Kong, the curator of The Herbarium in Forest Research 352 Institute Malaysia (KEP), for allowing us to access the herbarium and its facilities. Postar Miun, 353 a field botanist from Forest Research Center (FRC), has helped tremendously identify plant 354 voucher specimens, for which we are grateful. Manuscript to be reviewed 337 Lastly, predation pressure could also explain snails’ density distribution patterns (Abramsky et al., 338 1992; Meyer & Cowie, 2010; Gerlach et al., 2020). Unfortunately, data on population densities 339 and life histories for predators were not available from this study. 337 Lastly, predation pressure could also explain snails’ density distribution patterns (Abramsky et al., 338 1992; Meyer & Cowie, 2010; Gerlach et al., 2020). Unfortunately, data on population densities 339 and life histories for predators were not available from this study. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed 373 Berry, A. J. (1965). reproduction and breeding fluctuations in Hydrocena monterosatiana a 374 Malayan limestone archaeogastropod. In Proceedings of the Zoological Society of London, 144(2), 375 219-228. 373 Berry, A. J. (1965). reproduction and breeding fluctuations in Hydrocena monterosatiana a 374 Malayan limestone archaeogastropod. In Proceedings of the Zoological Society of London, 144(2), 375 219-228. 73 Berry, A. J. (1965). reproduction and breeding fluctuations in Hydrocena 373 Berry, A. J. (1965). reproduction and breeding fluctuations in Hydrocena monterosatiana a 374 Malayan limestone archaeogastropod. In Proceedings of the Zoological Society of London, 144(2), 375 219-228. 374 Malayan limestone archaeogastropod. 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Manuscript to be reviewed Journal of the Asiatic Society of Bengal 55: 466 299-316. 467 Müller, J., Strätz, C., & Hothorn, T. (2005). Habitat factors for land snails in European beech 468 forests with a special focus on coarse woody debris. European Journal of Forest Research, 124(3), 469 233-242. 470 Owen, D. F. (1969). Ecological aspects of polymorphism in an African land snail, Limicolaria 471 martensiana. Journal of Zoology, 159(1), 79-96. 472 Ozgo, M., Bogucki, Z. (2011). Colonisation, stability, and adaptation in a transplant experiment 473 of the polymorphic land snail Cepaea nemoralis (Gastropoda: Pulmonata) at the edge of its 474 geographical range. Biological Journal of the Linnean Society, 104(2), 462-470. 475 Pain, T. (1974) The land operculate genus Pollicaria Gould (Gastropoda), a systematic revision. 476 Journal of Conchology 28: 173-178. 477 Parkyn, J., & Newell, D. A. (2013). Australian land snails a review of ecological research and 478 conservation approaches. Molluscan research, 33(2), 116-129. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Table 1(on next page) Manuscript to be reviewed P., & Blum, J. D. (2007). Terrestrial gastropod 502 responses to an ecosystem-level calcium manipulation in a northern hardwood forest. Canadian 503 Journal of Zoology, 85(9), 994-1007. S , , V , , g, S , & , J ( ) g p 502 responses to an ecosystem-level calcium manipulation in a northern hardwood forest. Canadian 503 Journal of Zoology, 85(9), 994-1007. 504 Standish, R. J., Bennett, S. J., & Stringer, I. A. (2002). Habitat use of Tradescantia fluminensis by 505 Powelliphanta traversi. Science for Conservation, 195A, 35. 506 Stringer, I. A. N., Parrish, G. R., & Sherley, G. H. (2018). Homing, dispersal and mortality after 507 translocation of long-lived land snails Placostylus ambagiosus and P. hongii (Gastropoda: 508 Bothriembryontidae) in New Zealand. Molluscan Research, 38(1), 56-76. 509 Tao, J., Zuo, J., He, Z., Wang, Y., Liu, J., Liu, W., & Cornelissen, J. H. (2019). Traits including 510 leaf dry matter content and leaf pH dominate over forest soil pH as drivers of litter decomposition 511 among 60 species. Functional Ecology, 33(9), 1798-1810. 512 Tomiyama, K. (1992). Homing behaviour of the giant African snail, Achatina fulica 513 (Ferussac)(Gastropoda; Pulmonata). Journal of Ethology, 10(2), 139-146. 514 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Table 1(on next page) Table 1(on next page) Population estimation for Pollicaria elephas for each plot based on capture-mark- recapture technique and Schnabel index. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Manuscript to be reviewed 1 Plots Population densitya Population estimation by CMR (number of snails) A-Polli2 23.0±3.4 56.9 A-Polli9 15.8±1.6 20.8 A-Polli7 17.4±2.8 19.5 A-Polli3 1.4±0.5 2.0 A-P2 6.3±2.1 12.1 2 a Average number of snails ± standard deviation for all of the CMR sessions at each 8 m2 plot. 1 3 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Manuscript to be reviewed Table 2(on next page) Table 2(on next page) Manuscript to be reviewed 1 Habitat and topographic variables Bayesian Kendall's Tau Correlations BF₁₀ Kendall's Tau Correlations p-value Number of vascular plant individuals for each plot -0.186 0.512 -0.186 0.353 Number of vascular plant species for each plot 0.128 0.392 0.128 0.525 Canopy cover (%) 0.261 0.84 0.261 0.216 Leaf litter thickness (cm) 0.154 0.437 0.154 0.436 Elevation (meters) 0.183 0.504 0.183 0.355 Aspect (counter-clockwise in degrees from 0 (due north) to 360 (again due north)) 0.086 0.343 0.086 0.662 Ruggedness index 0.092 0.372 0.092 0.669 Slope (degrees) 0.241 0.721 0.241 0.224 Table 2(on next page) Correlation between the abundance of Pollicaria elephas for each plot with the habitat and topographic parameters. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Manuscript to be reviewed Table 3(on next page) Table 3(on next page) Table 3(on next page) Checklist and the number of individuals per vascular plant species in each plot. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Plots Plots with living Pollicaria elephas snails Plots without living Pollicaria elephas snails Family and species A-P0 A-P2 A-Polli2 A-Polli3 A-Polli7 A-Polli9 A-P14 B-P6 B-P7 C-P4 D11 D-P10 D-P12 D-P3 D-P8 D-P9 Intro-P1 The total number of individuals of each species in 17 plots Th num plot this was Anacardiaceae 1 Mangifera sp. 1 1 1 2 Annonaceae 2 Annonaceae sp. 1 1 1 3 Cananga odorata 1 1 4 Orophea cuneiformis 2 2 5 Polyalthia sp. 1 4 2 6 6 Polyalthia sp. 2 1 1 7 Xylopia sp. 1 1 1 2 Apocynaceae 8 Kibatalia laurifolia* 1 1 2 Bignoniaceae 9 Radermachera pinnata acuminata 1 1 Celastraceae 10 Euonymus javanicus 1 1 Clusiaceae 11 Garcinia cowa 1 1 Cycadaceae 12 Cycas clivicola 1 1 Dipterocarpaceae 13 Vatica kanthanensis 1 2 1 4 Ebenaceae 14 Diospyros frutescens 1 1 15 Diospyros toposia var. toposoides* 3 2 7 8 7 2 1 3 33 16 Diospyros transitoria 2 2 Euphorbiaceae 17 Croton cascarilloides* 2 1 3 18 Macaranga gigantea 1 1 19 Macaranga tanarius 1 3 4 20 Mallotus barbatus 3 3 21 Mallotus brevipetiolatus 7 1 2 3 5 2 29 22 Mallotus peltatus* 6 7 2 15 23 Pimelodendron griffithianum 1 1 Lamiaceae 24 Vitex siamica 2 2 Lauraceae 25 Dehaasia cuneata 1 1 2 Leguminosae 26 Archidendron jiringa 1 1 27 Bauhinia sp. 1 1 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Manuscript to be reviewed Malvaceae 28 Hibiscus macrophyllus 8 8 29 Leptonychia caudata 6 5 5 16 30 Pterospermum acerifolium 1 1 31 Sterculia rubiginosa 1 1 Melastomataceae 32 Memecylon lilacinum 8 8 Meliaceae 33 Aglaia grandis 1 1 Moraceae 34 Ficus fistulosa 1 1 35 Ficus hispida 2 2 36 Ficus schwarzii 1 1 37 Ficus sp. 1 1 1 38 Ficus sp. 2 2 2 Palmae 39 Arenga westerhoutii 4 1 5 40 Borassodendron machadonis 1 1 41 Caryota mitis 1 1 Pandanaceae 42 Pandanus piniformis 5 3 2 10 Phyllanthaceae 43 Bridelia tomentosa 1 1 0 2 5 8 44 44 Cleistanthus gracilis 1 1 5 5 3 24 45 Cleistanthus myrianthus 1 6 7 Primulaceae 46 Ardisia sp. 2 2 47 Myrsine perakensis 1 1 Rubiaceae 48 Aidia densifolia 3 2 7 3 6 21 49 Canthium sp. 1 3 3 50 Canthium sp. 2 4 4 51 Saprosma sp. 1 1 Rutaceae 52 Micromelum minutum 1 1 53 Murraya paniculata 1 1 1 1 4 54 Rutaceae sp. 1 2 2 55 Rutaceae sp. 2 1 1 Salicaceae 56 Homalium dasyanthum 2 2 57 Homalium grandifllorum 1 1 Sapindaceae 58 Paranephelium spirei 1 1 Sapotaceae 59 Isonandra perakensis 2 2 Violaceae 60 Rinorea bengalensis 2 2 3 4 3 2 5 2 7 6 36 Indet 61 Indet sp. 1 2 2 62 Indet sp. 2 1 1 63 Indet sp. 3 2 2 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Table 4(on next page) Table 4(on next page) Manuscript to be reviewed 2 * Plant species were a positive correlation between the abundances of plants and snails was 3 found. 2 * Plant species were a positive correlation between the abundances of plants and snails was 3 found. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Table 4(on next page) Correlation between the abundance of Pollicaria elephas for each plot with the abundance of the vascular plant species. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) , , ; p , p , p , caranga tanarius is equal to 0 Manuscript to be reviewed Manuscript to be reviewed Figure 1 Photographs of specimens of Pollicaria elephas and the study site. Manuscript to be reviewed Family Species Occurrence in the 17 plots Bayesian Kendall's Tau Correlations BF₁₀ Kendall's Tau Correlations p-value Anacardiaceae Mangifera sp. 1 2 -0.243 0.735 -0.243 0.295 Annonaceae Polyalthia sp. 1 2 -0.239 0.714 -0.239 0.296 Annonaceae Xylopia sp. 1 2 -0.243 0.735 -0.243 0.295 Apocynaceae Kibatalia laurifolia 2 0.527* 17.738 0.527* 0.023 Dipterocarpaceae Vatica kanthanensis 3 -0.050 0.320 -0.050 0.826 Ebenaceae Diospyros toposia var. toposoides 8 0.519* 15.604 0.519* 0.015 Euphorbiaceae Croton cascarilloides 2 0.499* 11.617 0.499* 0.030 Euphorbiaceae Macaranga tanarius 2 -0.239 0.714 -0.239 0.296 Euphorbiaceae Mallotus brevipetiolatus 5 -0.398 3.139 -0.398 0.070 Euphorbiaceae Mallotus peltatus 3 0.547* 23.867 0.547* 0.016 Lauraceae Dehaasia cuneata 2 0.162 0.454 0.162 0.485 Malvaceae Leptonychia caudata 3 -0.302 1.167 -0.302 0.187 Palmae Arenga westerhoutii 2 0.060 0.325 0.060 0.794 Pandanaceae Pandanus piniformis 3 -0.298 1.133 -0.298 0.187 Phyllanthaceae Bridelia tomentosa 4 -0.350 1.854 -0.350 0.116 Phyllanthaceae Cleistanthus gracilis 4 0.131 0.397 0.131 0.556 Phyllanthaceae Cleistanthus myrianthus 2 0.020 0.310 0.020 0.931 Rubiaceae Aidia densifolia 5 0.000 0.308 0.000 1.000 Rutaceae Murraya paniculata 4 0.370 2.279 0.370 0.112 Violaceae Rinorea bengalensis 10 0.257 0.809 0.257 0.223 1 * BF₁₀ > 10, BF₁₀ > 30, * BF₁₀ > 100; * p < 0.05, p < 0.01, * p < 0.001, ᵃ The variance in 2 Macaranga tanarius is equal to 0 3 PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Photographs of specimens of Pollicaria elephas and the study site. (A) Male individual of Pollicaria elephas. Scale: 10 mm. (B) and (C) Capture-mark-recapture experiment in the ﬁeld. Diﬀerent colours of nail polish were used to mark the shells of living Pollicaria elephas that were collected during diﬀerent sampling sessions. (D) and (E) Habitat of Pollicaria elephas at two localities at northern part of the limestone hill. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewe Manuscript to be reviewed PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Figure 2 Study sites and location of the 17 studied plots at a limestone hill in the state of Perak, Malaysia. Manuscript to be reviewed Study sites and location of the 17 studied plots at a limestone hill in the state of Perak, Malaysia. (A) Topographic map showing the seven plots at the northern part, nine plots at the southern part and one plot (C-P4) at the central part of the hill. (B) Aerial photograph from the north-western ﬂank (blue arrow) of the hill and location of the seven plots on a topographic map. Five of the plots were selected for the capture-mark-recapture study. The blue box indicates an area of 500 m ✕ 150 m. (C) Aerial photograph from the southern ﬂank (blue arrow) of the hill and the location of the nine plots on a topographic map. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Figure 3 Principal components analysis (PCA) plot of the ﬁrst two axes for habitat, topography, and vegetation variables for the 17 plots. The colour of the labels represents the plot location on the limestone hill and red vectors represent the habitat, topography, and vegetation variables. No individuals of Pollicaria elephas were found in plots at the southern and central part of the limestone hill, while at the northern part living snails were found in six of the seven plots (none were found in A-P14). PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Soil temperature for each day in July 2018 in the six plots. A-Polli3 and A-Polli9 are the plots with living Pollicaria elephas land snails, while the other four plots are without living P. elephas land snails. The insets represent the same plots with a diﬀerent colour legend for the plots with living snails. (A) Mean soil temperature for each day. (B) Maximum soil temperature for each day. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Figure 5 Air temperature for each day in July 2018 in the eight plots. Manuscript to be reviewed Air temperature for each day in July 2018 in the eight plots. A-Polli3 and A-Polli9 are the plots with living Pollicaria elephas land snails, while the other six plots are without living P. elephas land snails. The insets represent the same plots with a diﬀerent colour legend for the plots with living snails. (A) Mean air temperature for each day. (B) Maximum air temperature for each day. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed Humidity for each day in July 2018 in the seven plots. Humidity for each day in July 2018 in the seven plots. Plot A-Polli3 is the plot with living Pollicaria elephas land snails, while the other six plots are without living P. elephas land snails. The insets represent the same plots with a diﬀerent colour legend for the plots with living snails. (A) Mean humidity for each day. (B) Maximum humidity for each day. PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) Manuscript to be reviewed PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021) PeerJ reviewing PDF \| (2020:09:53097:3:0:NEW 5 Jul 2021)
https://openalex.org/W4280566856	https://www.researchsquare.com/article/rs-1110581/latest.pdf	English	null	Temporary cable force monitoring techniques during bridge construction-phase: the Tajo River Viaduct experience	Scientific reports	2,022	cc-by	8,328	Temporary Cable Force Monitoring Techniques During Bridge Construction-phase. The Tajo River Viaduct Experience Álvaro Gaute-Alonso (  gautea@unican.es ) University of Cantabria David Garcia-Sanchez Tecnalia Carlos Alonso Cobo University of Cantabria Iñigo Calderon-Uriszar-Aldaca University of Navarra Álvaro Gaute-Alonso (  gautea@unican.es ) University of Cantabria Iñigo Calderon-Uriszar-Aldaca University of Navarra Research Article Keywords: Structural monitoring systems, Load cells, Vibrating wire technique, Unidirectional strain gauges, Unidirectional accelerometers, Cable force Posted Date: November 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1110581/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Structural monitoring systems, Load cells, Vibrating wire technique, Unidirectional strain gauges, Unidirectional accelerometers, Cable force Posted Date: November 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1110581/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. R d F ll Li Temporary cable force monitoring techniques during the bridge construction phase. The Tajo River Viaduct Experience Alvaro Gaute Alonso, PhD. Eng. (Corresponding author; alvaro.gaute@unican.es; ORCID: https://orcid.org/0000-0002-6195-7397; Grupo de Instrumentación y Análisis Dinámico de Estructuras, University of Cantabria); David Garcia-Sanchez, PhD. Eng. (david.garciasanchez@tecnalia.com; ORCID: https://orcid.org/0000-0002-4763-206X; TECNALIA Basque Research and Technology Alliance (BRTA)); Carlos Alonso Cobo, PhD (carlos.alonso@unican.es; ORCID: https://orcid.org/0000-0002-0845-3616; Structural and Mechanical Engineering Area, University of Cantabria); and Iñigo Calderon-Uriszar-Aldaca, PhD Eng (inigo.calderon@tecnalia.com; ORCID: https://orcid.org/0000-0002-6911-161X; University of Navarra; TECNALIA Basque Research and Technology Alliance (BRTA)) Keywords Structural monitoring systems; Load cells; Vibrating wire technique; Unidirectional strain gauges; Unidirectional accelerometers; Cable force. Abstract This article addresses the comparative analysis of current techniques for monitoring cable forces. Additionally, the experience of three cable force different monitoring techniques for strain control in bridge cables during the construction phase is included: a) installing load cells on the active anchorages of the cables, b) installing unidirectional strain gauges, and c) assessing the stress on cables by applying the vibrating wire technique through the installation of accelerometers. The main advantages and disadvantages of each technique analyzed are highlighted in the construction process context of the Tajo Viaduct, one of the most singular viaducts recently built in Spain. 1 1 2. Assessing safety conditions of construction cables Several nondestructive tests are used to diagnose the safety conditions of stay cables in bridges6, such as ultrasonic testing, the magnetic flux leakage detection technique7, or X-rays. Although effective, these techniques are more suitable for the evaluation of out- of-service stay cables. On the one hand, various devices have been developed for the direct measurement of stress in bridge cables, such as load cells8-12, fiber Bragg grating optical sensors13, or elastomagnetic stress sensors14,15. These sensors are capable, thanks to their specific technologies, of accurately determining the stress experienced by the cable, and when connected to a structural monitoring system (SMS), it is possible to create a long-term historical record of cable stresses and access these data in real time from any remote location. On the other hand, a common indirect methodology for quick stress assessment in bridge cables is the vibrating wire technique. This method is based on the relationship between cable stress and its vibration frequency, which can be correctly identified from the recording of accelerations during free vibration of suspension cables16-21. For the application of this method, it is necessary to use spectral decomposition techniques that allow for a real-time determination of the stress on the suspension cables in bridges via the identification of vibration frequencies associated with their main vibration modes during a free vibration regime22-28. 1. Introduction One of the solutions proposed by civil engineers to overcome large spans is to employ cable-stayed or hanging structures. The critical component of these structures lies in the vulnerability of the cables to problems or damage associated with fatigue and/or corrosion 1 caused by dynamic loads such as cyclic traffic loads, wind loads, and other operational loads as well as environmental effects. Temporary suspension cables for the constructive phase present the same problems, and it is at this phase where the paper aims to impact. The most important parameter for the assessment of stress and fatigue and corrosion damage to cables in service is the historical record of the axial effort over time of these structural elements. This parameter has been recognized as a useful feature damage indicator of the safety condition of stay and suspension cables in bridges2, and the real- time monitoring of this indicator has become essential for the assessment of potential fatigue damage in these structural elements. Nevertheless, the monitoring and evaluation of structural behavior has become standard practice to ensure the safety and durability of cable-stayed or suspended structures3-6. 2 2 3. Direct and indirect monitoring technologies Many types of sensors and suppliers exist for each of the measuring techniques29-32. 3 3 3.2. Vibrating wire Vibrating wire (VW) sensors34 transform the measuring into a chance in the vibration frequency of a string. In the case of the VW strain sensor, a strain change results in the variation of the tension of a steel wire, while in the case of VW piezometers, the deformation of a membrane due to a water pressure change also induces a change of tension of the sensing wire. Once the wire is excited with an electromagnet, it becomes possible to accurately measure its vibration frequency. Frequency measurements are very accurate and stable, so those sensors have become a standard for long-term accurate measurement in geotechnics and structural monitoring. 3.1. Electronic and microelectromechanical system sensors (MEMSs) Electronic sensors transform the measurement (parameter to be measured) into a change in voltage, current, resistance, capacity or inductance. Electrical sensors come in many varieties and are usually very simple to interface to any datalogger system. Examples include potentiometer displacement sensors, resistive strain gauges, load cells, MEMS33 tiltmeters and piezo pressure sensors. Electrical sensors tend to be relatively inexpensive but can suffer from drift and can be affected or damaged by electromagnetic disturbances such as power lines of lightning. 3.4. Distributed sensing Distributed fiber sensors37,38 represent a change of paradigm in terms of monitoring and sensing. Distributed sensors are able to sense at any point along a single standard telecommunication optical fiber line, allowing the discrimination of different positions of the measured parameter along the fiber transferring strain and temperature39 from the structure to the fiber. Distributed sensors are particularly recommended40 to detect and discriminate events at any location within or among a structure. 3.3. Optical fiber From many points of view, optical fiber sensors35 are ideal transducers for structural health monitoring. These sensors transform the quantities to be measured into a change in the propagation characteristic of the light traveling through the optical fiber. Being durable, stable and insensitive to external perturbations, they are especially useful for long-term health assessments of civil structures and geostructures. Many different 4 optical fiber sensor technologies exist, including fiber Bragg gratings, SOFO interferometers, Fabry Perot interferometers and distributed Brillouin and Raman sensors, and offer a wide range of performances and suitability for different applications. In the earliest 90 s, optical fiber sensors made a significant entrance into the sensor industry and currently have a consolidated presence in the structural sensing industry36. 3.6. Image based techniques Olaszek42 developed a method that incorporated the photogrammetric principle with the computer vision technique to investigate the slow dynamic characteristics of bridges. Olaszek42 developed a method that incorporated the photogrammetric principle with the computer vision technique to investigate the slow dynamic characteristics of bridges. Patsias and Staszewski42 and Yoshida et al.44 started the use of videogrammetric techniques to measure the mode shapes of a beam and to capture the 3D dynamic behavior of different structures. Chung et al.45 used digital image techniques to identify nonlinear characteristics in more complex structural systems. Chang and Ji46 developed a two-camera videogrammetric technique for measuring a 3D structural vibration response at the laboratory level. In47, Ji and Chang proposed a novel nontarget technique based on image analysis using one digital camera for cable vibration measurement, but again, it is a proof-of-concept. These techniques, although promising and most of them verified at the laboratory level or under controlled conditions, do not seem to provide complete dynamic information, only partial to free vibration. That is why application during construction needs to wait for a more advanced development level. 3.5. Optical/radar/laser These techniques are especially useful to monitor global movements of structures41. They include traditional geodesy methods, total stations with or without target prisms installed on the structure, laser distance meters and laser profilometers as well as terrestrial radars (including synthetic aperture radar). These techniques can be used, for example, to measure the deformation of a bridge, the movement of a tower, the progression of a landslide or the deflection of a dam. 5 5 4.1. General Tajo River Viaduct description box section, except for their heads, where the section is solid to receive the loads transmitted by the deck. 4.1. General Tajo River Viaduct description The Tajo River Viaduct belongs to the Madrid–Extremadura high-speed rail. It is located in the province of Caceres and has a total length of 1,488 m. Its span distribution is determined by the width of the Tajo River, over which the arched viaduct spans 324 m. The above deck is divided into six spans of 54 m each. The access spans are 60 m long, with two 57 m transition spans between these and the deck spans above 6 6 the arch, one on each springer. This results in a suitable and harmonious distribution of the 26 spans that compose the deck of the Tajo River Viaduct: 45 m + 9x60 m + 57 m + 6x54 m + 57 m + 7x60 m + 45 m48,49. Figure 1. Tajo River Viaduct during its construction. Figure 1. Tajo River Viaduct during its construction. The layout of the viaduct first presents a very wide curve and then a straight line throughout the rest of the bridge. The most emblematic element of this viaduct is the arch, with a span of 324 m between its supports, rising above its foundations to a height of 70 m and above the maximum level of the Alcántara Reservoir to a height of over 80 m. The arch is formed by a box section that varies in edge and width, with an edge height of 4 m and a width of 12 m at the springers and an edge of 3.50 m and a width of 6 m at the keystone section. The deck has a pretensioned, concrete box section with a constant edge of 3.60 m and a total width of 14 m, thereby accommodating the double- track railway of 10.10 m width and two walkways of 1.75 m width on each side. The lower width of the box is 5 m, the top width is 6.50 m, and it has side overhangs that fill the total width of the cross-section up to 14 m. The piers have a variable height to adapt to the profile of the layout, reaching heights of over 70 m in the area nearest to the springers of the arch-type main span. All the piers consist of a single shaft with a hollow 7 box section, except for their heads, where the section is solid to receive the loads transmitted by the deck. 4.3. Analysis of cable monitoring methodologies during bridge construction processes During the structural monitoring of the Tajo River Viaduct, three methodologies were used to monitor the stress in the temporary suspension cables of the arch-type main span: (a) strain gauge load cells arranged at the active anchorage of the temporary cables, (b) unidirectional strain gauges installed on one of the seven wires belonging to one of the strands in the cable, and (c) instrumentation of the cable by means of a unidirectional accelerometer. During the structural monitoring of the Tajo River Viaduct, three methodologies were used to monitor the stress in the temporary suspension cables of the arch-type main span: (a) strain gauge load cells arranged at the active anchorage of the temporary cables, (b) unidirectional strain gauges installed on one of the seven wires belonging to one of the strands in the cable, and (c) instrumentation of the cable by means of a unidirectional accelerometer. 4.2. Construction Process The construction of the arch was carried out using the technique of successive, cable- stayed overhangs by means of two temporary towers located on the piers at the springers of the arch on both banks of the river (see Fig. 1). Each tower was cable- stayed to the foundations of the neighboring piers, which is why it was necessary to provide these foundations with ground-anchoring prestressed units. For the construction phase of the arch, fifteen pairs of cables held each complete half arch, and another fifteen pairs held the tower. The length of these cables ranged from 80 to 180 m. Each half arch is composed of a total of 46 voussoirs, each of 4 m length. It was necessary to place a keystone to complete and close the arch. The concrete trolley is a metal element that supports the formwork of each voussoir and its concrete. This trolley was located in the area of the arch that had been most recently concreted to prepare the concrete of the next voussoir. Once the arch was finished, the temporary cable system consisting of the temporary towers, cables and ground anchors was dismantled. At this point, two adjacent piers on each side of the arch were built; these, together with the keystone, support the deck above the arch. The construction of the deck was carried out span by span by means of a self-supporting formwork arranged from both abutments. To avoid the creation of excessive stresses on the arch, the deck was symmetrically concreted, and a maximum offset of a single span was admitted. 8 8 4.3. Analysis of cable monitoring methodologies during bridge construction processes 4.3.1. Direct monitoring of cables with strain gauge load cells The strain gauge load cells designed by the authors for the instrumentation of the temporary cables of the Tajo River Viaduct were composed of a metal ring arranged between the cable anchorage plate and the distribution plate in the pile or temporary tower. Due to the dimensions of the suspension cables, the average diameter of the load cells of the Tajo River Viaduct varied from 200 mm for the lowest loaded cables (2,000 kN) to 500 mm for the highest loaded cables (5,500 kN) (see fig 2). Figure 2. Tajo river viaduct load cells: a) pile load cell; b) load cell in temporary stay tower. (a) (b) (b) (a) (a) (b) Figure 2. Tajo river viaduct load cells: a) pile load cell; b) load cell in temporary sta Figure 2. Tajo river viaduct load cells: a) pile load cell; b) load cell in temporary stay tower. Figure 2. Tajo river viaduct load cells: a) pile load cell; b) load cell in temporary stay tower. 9 9 This technology allows for the determination of the stress in the stay cable from the empirical characterization of the average normal strain in the central ring of the load cell; see eq. (1). To do this, the outer perimeter of the central ring is instrumented by the uniform distribution of bidirectional strain gauges connected in series by a full Wheatstone bridge configuration50,51. This technology allows for the determination of the stress in the stay cable from the empirical characterization of the average normal strain in the central ring of the load cell; see eq. (1). To do this, the outer perimeter of the central ring is instrumented by the uniform distribution of bidirectional strain gauges connected in series by a full Wheatstone bridge configuration50,51. 𝐹= ∯𝜎· 𝑑Ω = ∑𝜀𝑖 𝑛1 𝑛 · 𝐸𝑎· Ω𝑐 (1) (1) where F = stress in the suspension cable; σ = normal stress; dΩ = differential area at the central ring; ɛi = normal strain at the ith strain gauge; n = number of strain gauges; Ea = modulus of elasticity of steel; Ωc = area of the central ring. where F = stress in the suspension cable; σ = normal stress; dΩ = differential area at the central ring; ɛi = normal strain at the ith strain gauge; n = number of strain gauges; Ea = modulus of elasticity of steel; Ωc = area of the central ring. 4.3.1. Direct monitoring of cables with strain gauge load cells The load cells installed on the cables allowed us to certify the correct tensioning of the cable families and to characterize, in real time, the variations in the stress experienced by the suspension cables during the different phases of the construction process (see Fig. 3). These devices allowed for the detection of any of the following structural phenomena occurring during the construction process: (1) the variation of stress in the cable due to the daily thermal increase, with stress increments of approximately 150 kN for daily temperature variations of 30 K (30°C); (2) the variation of stress due to the concreting of successive voussoirs, with stress variations that could range from 200/300 kN in the cables closest to the concreted voussoir, to values below the daily variation in the furthest cables; (3) the stress increments due to the tensioning of the successive families of cables (see Fig. 4), with stress variations above 500 kN in the families closest to the cables placed under load, and below the daily load variation in the furthest families; or (4) the stress variations due to the load readjustment operations in cables. 10 Figure 3. Evolution of stress on the cables of the rear load cells of the northern half arch: (a) 2nd family of cables; (b) 4th family of cables; (c) 6th family of cables; (d) 8th family of cables; (e) 12th family of cables; (f) 14th family of cables (a) (b) (c) (d) (e) (f) ( ) (c) (d) (c) (d) (d) (f) (e) (f) (e) Figure 3. Evolution of stress on the cables of the rear load cells of the northern half arch: (a) 2nd family of cables; (b) 4th family of cables; (c) 6th family of cables; (d) 8th family of Figure 3. Evolution of stress on the cables of the rear load cells of the northern half arch: (a) 2nd family of cables; (b) 4th family of cables; (c) 6th family of cables; (d) 8th family of cables; (e) 12th family of cables; (f) 14th family of cables gure 3. Evolution of stress on the cables of the rear load cells of the northern half arch: (a) 2nd family of cables; (b) 4th family of cables; (c) 6th family of cables; (d) 8th family o cables; (e) 12th family of cables; (f) 14th family of cables Fig. 4.3.1. Direct monitoring of cables with strain gauge load cells 4 shows the evolution of the stress experienced by the tie rods of the eighth family. This graph shows the evolution of the effort characterized by the load cell installed on one of the rear tie rods, as well as the evolution of the theoretical effort predicted by the theoretical calculation model. The evolution of the experimented stress in the provisional tie-rods of the eighth family can be clearly appreciated, caused by the concreting of successive segments of the arch and by the tensioning of the successive tie-rod families. Fig. 4 shows the evolution of the stress experienced by the tie rods of the eighth family. Fig. 4 shows the evolution of the stress experienced by the tie rods of the eighth family. Fig. 4 shows the evolution of the stress experienced by the tie rods of the eighth family. This graph shows the evolution of the effort characterized by the load cell installed on one of the rear tie rods, as well as the evolution of the theoretical effort predicted by the theoretical calculation model. The evolution of the experimented stress in the provisional tie-rods of the eighth family can be clearly appreciated, caused by the concreting of successive segments of the arch and by the tensioning of the successive tie-rod families. 11 Figure 4. Evolution of the effort experienced by the tie rods of the eighth family. (a) (b) (c) (d) ( ) Figure 4. Evolution of the effort experienced by the tie rods of the eighth family. Figure 4. Evolution of the effort experienced by the tie rods of the eighth family. 12 (a) (b) (c) (d) (e) (f) (d) (f) (e) 12 Figure 5. Evolution of stress on the cables of the front load cells of the northern half arch during tensioning of the thirteenth family of cables: (a) 3rd family of cables; (b) 5th family of cables; (c) 7th family of cables; (d) 9th family of cables; (e) 11th family of cables; (f) 13th family of cables. Figure 5. Evolution of stress on the cables of the front load cells of the northern half arch during tensioning of the thirteenth family of cables: (a) 3rd family of cables; (b) 5th family of cables; (c) 7th family of cables; (d) 9th family of cables; (e) 11th family of cables; (f) 13th family of cables. Fig. 4.3.1. Direct monitoring of cables with strain gauge load cells 3 shows the evolution of the stress experienced by the different families of retaining cables of the northern half arch and its comparison with the theoretical values of the project during all the construction phases of the arch-type main span, while Fig. 5 shows the evolution of the stress in the suspension cables of the northern half arch during the loading of the cables corresponding to the 13th family of temporary cables. 4.3.2. Direct monitoring of cables by installing unidirectional strain gauges on a wire from one of the strands in the temporary cable During the instrumentation of the cables corresponding to the first two families of temporary cables, the authors detected that the edge initially given to the strain gauge load cells was not sufficient to minimize the error induced by the irregularities in the boundary conditions. These sensors had to be complemented with other types of sensors that would allow the characterization of stress variations on the cables. Two unidirectional strain gauges were installed on one of the seven wires that compose one of the strands in the cable, connected to each other by a full Wheatstone bridge configuration50,51. This technology allows for the instrumentation of cables once they are loaded and makes it possible to empirically characterize the stress increments experienced by the cable. 13 The steel wires that compose the strand are twisted together so that the directrix of these wires has some angular deviation from the strand directrix (see Fig. 6). This fact makes the retrieval of the strand strain from the wire strain indirect and implies the need to perform the calibration of the measurement system in a tensile tester where the kN/µɛ correlation between cable stress and wire strain can be obtained (see Fig. 7). The steel wires that compose the strand are twisted together so that the directrix of these wires has some angular deviation from the strand directrix (see Fig. 6). This fact makes the retrieval of the strand strain from the wire strain indirect and implies the need to perform the calibration of the measurement system in a tensile tester where the kN/µɛ correlation between cable stress and wire strain can be obtained (see Fig. 7). Figure 6. Installation of unidirectional strain gauges on one of the wires of a strand in a temporary cable Figure 6. Installation of unidirectional strain gauges on one of the wires of a strand in a temporary cable 14 Figure 7. Calibration of the cable instrumentation method by the installation o unidirectional strain gauges: a) prestressing strand in load-bearing gantry; b load/deflection curve during calibration test. (a) (b (a) (b (b Figure 7. Calibration of the cable instrumentation method by the installation of unidirectional strain gauges: a) prestressing strand in load-bearing gantry; b) load/deflection curve during calibration test. Figure 7. Calibration of the cable instrumentation method by the installation of unidirectional strain gauges: a) prestressing strand in load-bearing gantry; b) load/deflection curve during calibration test. 4.3.2. Direct monitoring of cables by installing unidirectional strain gauges on a wire from one of the strands in the temporary cable The simultaneous instrumentation of the first two families of tie rods by means of load cells and unidirectional extensometers installed in one of the strands that make up the 15 tie facilitated a double contrast of the value of the stress experienced by these structural elements. Fig. 8 shows the evolution of the stress value in the intrumented tie rod of the fourth family of tie rods provided by the load cell (CNT-4), by the extensometers (CNT- 4_ext) and by the theoretical calculation model (CNT-4_theoretical). The graph shows the evolution of the stress in the instrumented tie rod due to the concrete of the successive segments of the arch and the tensioning of the successive families of temporary tie rods. tie facilitated a double contrast of the value of the stress experienced by these structural elements. Fig. 8 shows the evolution of the stress value in the intrumented tie rod of the fourth family of tie rods provided by the load cell (CNT-4), by the extensometers (CNT- 4_ext) and by the theoretical calculation model (CNT-4_theoretical). The graph shows the evolution of the stress in the instrumented tie rod due to the concrete of the successive segments of the arch and the tensioning of the successive families of temporary tie rods. Figure 8. Evolution of the effort experienced by the tie rods of the fourth family. Figure 8. Evolution of the effort experienced by the tie rods of the fourth family. 4.3.3. Indirect monitoring of cables with unidirectional accelerometers Due to the problems encountered in the measurements of the direct methodologies explained before arranged in the first two families of cables of the Tajo River Viaduct, the authors proposed carrying out a weighing campaign of the cables belonging to these families using the vibrating wire technique. This technique enables the measurement of the stress in bridge cables from the characterization of their vibration frequency and 16 their mass16,52,53. The differential equation that relates the axial stress of a cable to its mass, its bending stiffness and its vibration frequency is as follows: their mass16,52,53. The differential equation that relates the axial stress of a cable to its mass, its bending stiffness and its vibration frequency is as follows: 𝐸· 𝐼· 𝜕4𝜗(𝑥,𝑡) 𝜕𝑥4 −𝑇· 𝜕2𝜗(𝑥,𝑡) 𝜕𝑥2 + 𝑚· 𝜕2𝜗(𝑥,𝑡) 𝜕𝑡2 = 0 (2) (2) The solution eq. (3) to the differential eq. (2) allows for the axial stress of the cable to be obtained. The solution eq. (3) to the differential eq. (2) allows for the axial stress of the cable to be obtained. 𝑓= 𝑢 2·𝐿√ 𝑇 𝑚· [1 + 2 · √ 𝐸·𝐼 𝐹·𝐿2 + (4 + 𝑢2·𝜋2 2 ) · 𝐸·𝐼 𝐹·𝐿2] (3) (3) (3) The bending stiffness (E·I) in the bridge cables is negligible with respect to their axial stiffness. This fact makes the second and third sums of eq. (3) negligible with respect to the first, resulting in eq. (4), which relates the axial stress of the cable to its mass and oscillation frequency16. Fig. 9 shows the evolution of the error made, assuming the simplification set forth in eq. (4) for each of the families of temporary tie rods of the Tajo River Viaduct as a function of the stress experienced by these structural elements. The bending stiffness (E·I) in the bridge cables is negligible with respect to their axial stiffness. This fact makes the second and third sums of eq. (3) negligible with respect to the first, resulting in eq. (4), which relates the axial stress of the cable to its mass and oscillation frequency16. Fig. 9 shows the evolution of the error made, assuming the simplification set forth in eq. (4) for each of the families of temporary tie rods of the Tajo River Viaduct as a function of the stress experienced by these structural elements. 4.3.3. Indirect monitoring of cables with unidirectional accelerometers 𝑓= 𝑢 2·𝐿· √ 𝑇 𝑚 (4) 𝑓= 𝑢 2·𝐿· √ 𝑇 𝑚 (4) where T = Axial stress on the cable; u = Considered vibration mode; f = Vibration frequency corresponding to u-mode; E = Modulus of elasticity of the material composing the cable; I = Moment of inertia of the cable; L = Vibration length of the cable; m = Mass per linear meter of the cable. 17 (a) (b) (a) (b) (c) (d) (a) (b) (d) (d) 18 Figure 9. Evolution of the error made by the simplified formulation of the vibrating wire: a) north stay cables; b) north retaining cables; c) south stay cables; d) south retaining cables. To carry out the weighing of the cables by means of the application of the vibrating wire technique, a piezoelectric accelerometer was installed on the cable under testing through a tool that picks up the sheath–strand assembly, the cable was excited, and the accelerations experienced during its movement in free vibration were measured. To obtain the axial stress of the cable by applying eq. (4), it is necessary to take into account the total mass mobilized. In the case of the temporary cables of the Tajo River Viaduct, it was necessary to take into account the mass of the strands that composed the cable together with the mass of the protection sheath (see Fig. 10). The recordings of acceleration experienced by the cables were analyzed by applying the fast Fourier transform54-56, obtaining the eigenfrequencies of the cables and consequently their axial stress (see Fig. 11). Because of prior experience with the construction of similar bridges5,55,57-59 where there were episodes of confluence between the frequency of generation of whirlwinds by the action of the wind on the structure and its frequency of vibration, the vibrating wire testing campaign was used to obtain the damping factor of the cables of the Tajo River Viaduct. Established to obtain the damping factor (see Fig. 12), the test consisted of subjecting the cable to an initial strain by applying a displacement in its main span and then releasing it abruptly, letting it oscillate in a free vibration regime. The damping factor of the cable is obtained from the logarithmic decrease between the maximum amplitudes of its oscillation during free vibrations; see eq. 4.3.3. Indirect monitoring of cables with unidirectional accelerometers (5)60: ƞ = 𝑐 𝑐𝑐= 1 2·𝜋·𝑛· 𝑙𝑛( 𝐴𝑜 𝐴𝑛) (5) 19 where ƞ = damping factor with respect to the critical; n = number of cycles considered for the analysis; A0 = maximum amplitude in the initial oscillation considered for the analysis; and An = maximum amplitude in the nth oscillation considered for the analysis. where ƞ = damping factor with respect to the critical; n = number of cycles considered for the analysis; A0 = maximum amplitude in the initial oscillation considered for the analysis; and An = maximum amplitude in the nth oscillation considered for the analysis. Figure 10. Piezoelectric accelerometer on the temporary tie of the Tajo Viaduct. Figure 10. Piezoelectric accelerometer on the temporary tie of the Tajo Viaduct. Looking for a twofold purpose, the authors proposed a solution that enabled the monitoring of the evolution of the stress in the cable and the characterization of the possible appearance of aeroelastic phenomena caused by resonance or similar phenomena5,55,57-59. This solution consisted of the permanent instrumentation of the right-hand retaining cable belonging to the fifth family of temporary cables of the northern half arch of the Tajo River Viaduct. To accomplish this, a piezoelectric accelerometer was installed on the selected cable using a tool identical to the one used 20 in the vibrating wire spot testing campaign, and this sensor was connected to the SMS of the Tajo River Viaduct. Fig. 13 shows the monitoring of the evolution of stress in the cable obtained from the analysis of the data provided by the piezoelectric accelerometer. (a) (b) (a) (b) 21 Figure 11. Obtaining the stress in the fifth family of ties rod from their vibration frequencies: a) accelerogram; b) frequency spectrum. a) accelerogram; b) frequency spectrum. (b) (a) (c) encies: a) accelerogram; b) frequency spectrum. (b) (a) g ; ) q y p (a) (a) (b) ( ) (c) (c) 22 Figure 12. Retrieval of the damping factor of the right-hand retaining cable of the fifth family of temporary cables of the northern half arch of the Tajo River Viaduct: a) recording of accelerations associated with the first mode of vibration; b) speed record; c) displacement record. Figure 12. 4.3.3. Indirect monitoring of cables with unidirectional accelerometers Retrieval of the damping factor of the right-hand retaining cable of the fifth family of temporary cables of the northern half arch of the Tajo River Viaduct: a) recording of accelerations associated with the first mode of vibration; b) speed record; c) displacement record. Figure 1. Monitoring of the evolution of the stress in the right-hand retaining cable belonging to the fifth family of temporary cables of the northern half arch of the Tajo River Viaduct using a piezoelectric accelerometer. Figure 1. Monitoring of the evolution of the stress in the right-hand retaining cable belonging to the fifth family of temporary cables of the northern half arch of the Tajo River Viaduct using a piezoelectric accelerometer. Figure 1. Monitoring of the evolution of the stress in the right-hand retaining cable belonging to the fifth family of temporary cables of the northern half arch of the Tajo River Viaduct using a piezoelectric accelerometer. The simultaneous instrumentation of the first two families of tie rods using load cells and unidirectional strain gauges installed in one of the strands that make up the tie, together with the tie weighing campaign using the vibrating chord technique, allowed a comparative analysis of the effort facilitated for each of the structural monitoring techniques. Fig. 14 and Table 1 show the effort value provided by each of the structural monitoring techniques used by the authors. 23 Figure 2. Effort facilitated by each of the structural monitoring techniques used. .00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 3500.00 4000.00 C-N-D-1 C-N-T-2 C-S-D-1 C-S-T-2 Force (KN) Load Cells - Extensometers - Vibrating Chord Load Cells Extensometers Vibrating Chord Load Cells - Extensometers - Vibrating Chord Figure 2. Effort facilitated by each of the structural monitoring techniques used. Figure 2. Effort facilitated by each of the structural monitoring techniques used. Time Stamp Load Cell (KN) Extensometer (KN) Vibration Chord (KN) C-N-D-1 16/05/2014 9:06 3.501,4 3.591,1 3.525,0 C-N-T-2 16/05/2014 10:36 1.632,7 1.586,4 1.624,7 C-S-D-1 17/05/2014 9:30 3.493,0 3.465,6 3.511,2 C-S-T-2 17/05/2014 10:10 1.299,9 1.319,6 1.295,2 Table 1. Effort facilitated by each of the structural monitoring techniques used. Table 1. Effort facilitated by each of the structural monitoring techniques used. Table 1. Effort facilitated by each of the structural monitoring techniques used. 5. Lessons learned The lessons learned during Tajo’s Viaduct monitoring thanks to the installation of the different sensor technologies are synthesized in Table 2 and can be summarized as follows: Strain gauge load cells Strain gauge load cells  Advantages:  Advantages: o Allows measurement of the stress on the cables with an error lower than 1%. o Robust, shock-resistant, and weather-resistant solutions.  Disadvantages:  Disadvantages: o Entails the greatest economic investment. 24 24 o The instrumentation of heavy-duty cables involves the design of large, very heavy load cells that are difficult to handle and install on-site. o Installation is only possible prior to the cable installation operation. Unidirectional strain gauges Unidirectional strain gauges  Advantages:  Advantages: o Technique that entails the lowest economic investment. o Installation is possible at any stage of the construction process.  Disadvantages: o Least robust solution, very sensitive to shocks and weather. o In the case of its installation, once in service, the cable provides the stress increments on the structural element, but it is not possible to obtain the absolute value of the stress on the cable. o The accuracy of the measurement of stress on the cables is greatly influenced by the correct on-site installation of the device. Unidirectional accelerometers Unidirectional accelerometers Unidirectional accelerometers  Advantages:  Advantages: o Installation is possible at any stage of the construction process. o Allows the recording of the absolute value of the stress on the cable regardless of the phase of the construction process in which the sensor has been installed. o Allows measurement of the stress on the cables with an error lower than 1%. o Allows measurement of the stress on the cables with an error lower than 1%. Disadvantages:  Disadvantages: o The technique generates a high volume of data, and it is necessary to resort to spectral decomposition techniques to obtain the stress on the cable. Cost sensor Cost readout Long-term accuracy Durability Density of info. Cost sensor 25 Load cells (D) $$$ $ Very Good Very Good Low Electrical/MEM S (D) $ $$ Poor Poor Low Vibrating Wire (I) $$ $$ Very Good Medium High Table 23. Summary table for the validation of different tested technologies based on Table 23. Summary table for the validation of different tested technologies based on Tajo viaduct experience. Table 23. Summary table for the validation of different tested technologies based on Tajo viaduct experience. Table 23. Summary table for the validation of different tested technologies based on Tajo viaduct experience. 6. Conclusions Thanks to this work, the authors intend to give an overview of the different monitoring systems currently used for temporary cable force monitoring techniques during the bridge construction phase. Therefore, a review of the state of the art has been conducted. An overview of load cells, unidirectional strain gauge systems and accelerometers is also provided. All these methods are well established and very accurate. Nonetheless, each of them has its own advantages and disadvantages regarding installation and implementation. The most promising technique currently under development is image-based techniques and can be considered complementary to these currently available methods. Today, this technique cannot provide the same level of accuracy as the current methods, but it is inexpensive and simple to use when compared to the accelerometer, i.e.. Further development is still needed before it can be implemented on long-span cable-stayed bridges because their behavior becomes nontrivial. In fact, a long-span bridge is susceptible to environmental and traffic- induced vibration. Taking into account all these points, this article aims to serve as a base for all work related to the world of cable monitoring during bridge construction. 26 Data Availability Statement The data that support the findings of this study are available from the corresponding author, Gaute A., upon reasonable request. Acknowledgments The authors would like to thank “Administración de Infraestructuras Ferroviarias ADIF” (Administrator of Railway Infrastructures) for trusting the University of Cantabria’s Structures Laboratory to carry out the structural monitoring of the Tajo River Viaduct. This work has received funding from the European Union Horizon 2020 research and innovation program under grant agreement No 769373 (FORESEE project). This paper reflects only the author’s views. The European Commission and INEA are not responsible for any use that may be made of the information contained therein. References 1. H. Tabatai, Transportation Research Board, Inspection and Maintenance of Bridge Stay Cable Systems, National Cooperative Highway Research Program, 2005, Washington, D.C. 2. Y. Yang, S. Li, S. Nagarajaiah, H. Li and P. 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https://openalex.org/W2268483841	http://eprints.whiterose.ac.uk/96126/6/Simvastatin%20Sodium%20Salt%20and%20Fluvastatin%20Interact%20with%20Human%20Gap%20Junction%20Gamma-3%20Protein.pdf	English	null	Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein	PloS one	2,016	cc-by	16,514	Abstract Citation: Marsh A, Casey-Green K, Probert F, Withall D, Mitchell DA, Dilly SJ, et al. (2016) Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein. PLoS ONE 11(2): e0148266. doi:10.1371/journal.pone.0148266 Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover addi- tional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied inter- actions of clones of the bacteriophage, each expressing a different cardiovascular polypep- tide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analy- sis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/ 31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide repre- senting the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states. Editor: Eliana Scemes, Albert Einstein College of Medicine, UNITED STATES Editor: Eliana Scemes, Albert Einstein College of Medicine, UNITED STATES Received: February 21, 2013 Accepted: January 15, 2016 Published: February 10, 2016 Editor: Eliana Scemes, Albert Einstein College of Medicine, UNITED STATES Received: February 21, 2013 Accepted: January 15, 2016 Published: February 10, 2016 Copyright: © 2016 Marsh et al. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein Andrew Marsh3, Katherine Casey-Green3, Fay Probert3, David Withall3, Daniel A. Mitchell5, Suzanne J. Dilly1, Sean James2, Wade Dimitri2, Sweta R. Ladwa1, Paul C. Taylor3, Donald R. J. Singer2,4,5 1 Tangent Reprofiling Ltd, c/o SEEK, Central Point, 45 Beech Street, London, EC2Y 8AD, United Kingdom, 2 University Hospital Coventry and Warwickshire, Clifford Bridge Road, Coventry CV2 2DX, United Kingdom, 3 Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom, 4 Fellowship of Postgraduate Medicine, 11 Chandos St, London W1G 9EB, United Kingdom, 5 Division of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, Coventry, CV2 2DX, United Kingdom * a.marsh@warwick.ac.uk (AM); fpm.chandos@gmail.com (DRJS) * a.marsh@warwick.ac.uk (AM); fpm.chandos@gmail.com (DRJS) RESEARCH ARTICLE Introduction arising from this research to declare. The SPR equipment used in this research was obtained through Birmingham Science City: Innovative Uses for Advanced Materials in the Modern World (West Midlands Centre for Advanced Materials Project 2), with Support from Advantage West Midlands (AWM) and partly funded by the European Regional Development Fund (ERDF). This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Treatment with statins has had a major impact on cardiovascular disease and mortality. The primary mechanism by which statins reduce ischaemic cardiovascular disease is accepted as a reduction in circulating cholesterol achieved both by inhibiting HMG CoA reductase [1], and increased cholesterol scavenging by upregulation of LDL receptors [2]. However, statins have important pleiotropic actions [3,4], independent of their lipid-lowering properties. These pleio- tropic actions may contribute both to the cardiovascular benefits of statins and to their adverse effects. Simvastatin is a lactone prodrug, hydrolysed by non-specific carboxyesterases or non- enzymatic processes to its active hydroxyacid (Fig 1); lactonisation of the hydroxyacid form of all statins can occur in vivo by the action of UDP-glucuronosyl transferases [5,6]. Therapeutic or adverse effects of simvastatin and other statins may occur due to actions of these lactones or due to active hydroxyacid statins and their metabolites. For example β2 integrin leukocyte function antigen-1, has been identified as a significant, beneficial off-target effect of the lactone form of several statins [7], although the lactone form has also been implicated in statin-associ- ated myotoxicity [8]. New approaches to understanding pleiotropic effects of drugs and their metabolites on molecular, cellular and other systemic networks are important both for early stage drug discovery and safety pharmacology [9,10]. Competing Interests: Suzanne J. Dilly, Sweta R. Ladwa, Andrew Marsh and Paul C. Taylor are all shareholders in Tangent Reprofiling Limited, part of SEEK, a trading style of PepTCell Limited (Reg. No. 04983543). Suzanne J. Dilly and Paul C. Taylor are Directors of Tangent Reprofiling Limited (Reg. No. 06704572), the company that provided part-funding for this study. Katherine Casey-Green received an Engineering and Physical Sciences Research Council (EPSRC) Small and Medium-sized Enterprise Collaborative Award in Science and Engineering (CASE) Studentship with Tangent Reprofiling Limited. Andrew Marsh and Paul Taylor received EPSRC Impact Funding from the University of Warwick, which employed Fay Probert and David Withall. Introduction EPSRC played no part in the design of the research or preparation of the manuscript. Employees of Tangent Reprofiling Limited contributed to the research and writing of the manuscript as described in the Contributorship Statement. Wade Dimitri, Andrew Marsh and Donald R. J. Singer are members of the Fellowship of Postgraduate Medicine (a non-profit organization, Company Limited By Guarantee Reg. No. 00721213 and Registered Charity No. 313355) and Donald R. J. Singer is the president of the same organisation. Donald R. J. Singer is a member of the Healthcare Professionals Working Party of the European Medicines Agency. There are no patents, products in development or marketed products arising from this research to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. We have developed a novel approach to understanding the impact of drugs on such a net- work pharmacology approach. This combines chemical genomics phage display [11,12] with rapid immobilisation of a bioactive molecule in multiple orientations (Magic Tag1) [13] to explore beneficial and adverse targets and actions of therapeutic drugs [14], metabolites and other ligands [15]. We here apply a modification of this approach to identifying new molecular targets for the pharmacological effects of simvastatin, one of which is of particular interest and is confirmed as present in the tissue under investigation. Classic biochemical approaches to identifying cellular and molecular targets [16] often rely on displaying a ligand on a support which is screened against cell lysate, which has several drawbacks. Immobilising the ligand in multiple orientations [13,14,17–22] offers improved exploration of chemical space (Fig 2) and the use of genomic polypeptide display libraries [23] offers the chance to better explore the breadth of biological sequence space [24,25]. The chemistry, morphology of the support [26] and screening conditions can also be tuned to improve selection of meaningful interactors [27] and reduce non-specific bind- ing [28]. Rather than selecting bound phage by elution with the small molecule active, which can result in inhibition of phage replication, we innovate herein the direct use of the host bacte- rium, Escherichia coli. BLT5615 in a concomitant elution and amplification step. We provide biophysical evidence, using surface plasmon resonance measurements [29], that the discovered N-terminal sequence codes for a peptide that interacts in a meaningful way with statins. Statin Acids Interact with the N-Terminus of Human GJC3 Protein Introduction In addition, a selection of known, structurally diverse and commercially available gap junction inhibitors [30,31] were investigated providing new structure-function insight for the connexin proteins. Abstract This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank the Engineering and Physical Sciences Research Council, United Kingdom (EPSRC), and Tangent Reprofiling Ltd, for funding this study. Katherine Casey-Green received an EPSRC Small and Medium-sized Enterprise Collaborative Award in Science and Engineering (CASE) Studentship with Tangent Reprofiling Limited. Andrew Marsh and Paul Taylor received EPSRC Impact Funding from the University of Warwick, which employed Fay Probert and David Withall. EPSRC played no part in the design of the research or preparation of the manuscript. There are no patents, products in development or marketed products 1 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Preparing the viral library Standard procedures to extract and purify mRNA from total RNA obtained from internal mammary artery samples (Fig 3) were used see S1 Protocol and K. Casey-Green Ph. D. Thesis, University of Warwick [32] for full detail. A cDNA library was then created using an oligo(dT) priming approach which was ligated into phage vector arms using the mid-copy number dis- play vector T7Select1 10–3, leading to 5–15 copies of the insert on the head group of the phage PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 2 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 1. Simvastatin pro-drug compared with its hydrolysis product and pravastatin. doi:10.1371/journal.pone.0148266.g001 Fig 1. Simvastatin pro-drug compared with its hydrolysis product and pravastatin. Fig 1. Simvastatin pro-drug compared with its hydrolysis product and pravastatin. doi:10.1371/journal.pone.0148266.g001 doi:10.1371/journal.pone.0148266.g001 [33] at a molar ratio of 2:1. This was in order to minimise the number of vectors which self- ligate, leading to 'empty' vectors [34,35]. The diversity of the library (number of unique clones) was determined prior to amplifica- tion by selecting two samples, for which titres were assessed by a plaque counting assay on E. coli lawns, which were seen to be entirely lysed after 3 hours. This suggested a population of greater than 5,000 plaques for 100 μl at a dilution of 10−2, inferring a diversity of greater than 1.5 x 105 unique clones. The remaining library was amplified in two halves (to avoid possible disproportionation, detrimental to library diversity) in E. coli culture, strain BLT5615, at its exponential replication stage (i.e. ‘log phase’). Sixteen plaques were picked from the surface of agar titre plates and their genetic material amplified by touchdown PCR with agarose gel elec- trophoresis confirming inserts of length between 200–1500 base pairs (Fig 4). Fig 2. Immobilisation of a small molecule active using multiple photochemistries. The use of five different photochemistries in a multiwall format allows the simultaneous, rapid exploration of several regio- and other isomeric derivatives. doi:10 1371/journal pone 0148266 g002 Fig 2. Immobilisation of a small molecule active using multiple photochemistries. The use of five different pho the simultaneous, rapid exploration of several regio- and other isomeric derivatives. Fig 2. Immobilisation of a small molecule active using multiple photochemistries. The use of five different photochemistries in a multiwall format allows the simultaneous, rapid exploration of several regio- and other isomeric derivatives. Fig 2. Immobilisation of a small molecule active using multiple photochemistries. The use of five different photochemistries in a multiwall format allows the simultaneous, rapid exploration of several regio- and other isomeric derivatives. doi:10.1371/journal.pone.0148266.g002 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 3 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 3. Preparation of a T7 phage display library from vascular tissue. doi:10.1371/journal.pone.0148266.g003 Fig 3. Preparation of a T7 phage display library from vascular tissue. doi:10.1371/journal.pone.0148266.g003 Fig 3. Preparation of a T7 phage display library from vascular tissue. doi:10.1371/journal.pone.0148266.g003 doi:10.1371/journal.pone.0148266.g003 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Screening against simvastatin pro-drug Simvastatin-associated phage clones bearing polypeptide sequences were corrected for the 60 non-specific binding clones in control wells (S2 Dataset) leaving 23 consensus sequences from these contiguous regions, or ‘contigs’. These were first searched against the NCBI Genome (all assemblies scaffolds) using BLASTN optimized for highly similar sequences [36] and the human genome results further assessed by searching against the human RefSeq database [37] using the BLASTX algorithm (S4 Table). This led to identification of 11 sequences (‘contigs’) sufficiently annotated with biological function in the literature to be considered of significance to human health (S1 Summary) [32]. We note that one of these contigs has excellent homology with myosin light chain which has previously been identified as a strong interactor in a T7 phage screen [38]. In this communica- tion, we focus on the sequence of our contig29, which we recognised to have an identical amino acid sequence to the N-terminal residues 1–25 of the human gap junction gamma 3 pro- tein GJC3 [39,40][41], (human connexin 30.2/31.3, hCx30.2/31.3, NP_853516, UniProt Q8NFK1.1) synonymous with mouse connexin mCx29 [39,42–46]. Fig 5. Biopanning against the genomic phage library displaying 5–15 polypeptide copies per headgroup. Elution is carried out herein using the host bacterium, E. coli BLT5615 avoiding potential inhibitory or other effects of an active small molecule. doi:10.1371/journal.pone.0148266.g005 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 5 / 27 doi:10.1371/journal.pone.0148266.g005 doi:10.1371/journal.pone.0148266.g005 Following this biopanning process, phages selected from the final E. coli agarose lawn were submitted to Sanger sequencing (S1 Dataset) to give 120 sequences (S1 Table) which were aligned using the MegaBLAST tool against the coding region for T7Select 10–3 vector (18 clones vector only, S2 Table) from which coding regions for the T7Select1 10–3 vector were removed. Highly similar sections of clone sequence were noted and all low quality ends trimmed, resulting in 31 contiguous sequences (S3 Table). Simvastatin-associated phage clones bearing polypeptide sequences were corrected for the 60 non-specific binding clones in control wells (S2 Dataset) leaving 23 consensus sequences from these contiguous regions, or ‘contigs’. These were first searched against the NCBI Genome (all assemblies scaffolds) using BLASTN optimized for highly similar sequences [36] and the human genome results further assessed by searching against the human RefSeq database [37] using the BLASTX algorithm (S4 Table). Screening against simvastatin pro-drug Screening of the phage library was carried out using simvastatin immobilized on 5 different photosensitive moieties following published procedures (Fig 5) [13,14]. In brief, by using these different chemical linkers in separate wells on a test plate, we sought to display simvastatin in many orientations to potential binding polypeptides of interest. Following a usual wash proce- dure to remove non-specifically bound phage, phage selected against the immobilized simva- statin were eluted and amplified by directly adding host E. coli BLT5615 to the plate. After three rounds of binding, elution and amplification, phage clones binding to simvastatin in given wells were detected from areas of lysis in an E. coli agarose lawn grown over fresh agar plates. Fig 4. Ethidium bromide stained agarose gel showing a sample of clones from the phage display library after PCR to amplify the insert DNA. Hyperladder in lanes 1 and 25 allows estimation of insert length. PCR controls in lanes 22, 23 and 24 (positive, positive and negative). doi:10 1371/journal pone 0148266 g004 Fig 4. Ethidium bromide stained agarose gel showing a sample of clones from the phage display library after PCR to amplify the insert DNA. Hyperladder in lanes 1 and 25 allows estimation of insert length. PCR controls in lanes 22, 23 and 24 (positive, positive and negative). doi:10.1371/journal.pone.0148266.g004 4 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 5. Biopanning against the genomic phage library displaying 5–15 polypeptide copies per headgroup. Elution is carried out herein using the host bacterium, E. coli BLT5615 avoiding potential inhibitory or other effects of an active small molecule. d i 10 13 1/j l 0148266 00 Fig 5. Biopanning against the genomic phage library displaying 5–15 polypeptide copies per headgroup. Elution is carried out herein using the host bacterium, E. coli BLT5615 avoiding potential inhibitory or other effects of an active small molecule. Following this biopanning process, phages selected from the final E. coli agarose lawn were submitted to Sanger sequencing (S1 Dataset) to give 120 sequences (S1 Table) which were aligned using the MegaBLAST tool against the coding region for T7Select 10–3 vector (18 clones vector only, S2 Table) from which coding regions for the T7Select1 10–3 vector were removed. Highly similar sections of clone sequence were noted and all low quality ends trimmed, resulting in 31 contiguous sequences (S3 Table). PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Screening against simvastatin pro-drug This led to identification of 11 sequences (‘contigs’) sufficiently annotated with biological function in the literature to be considered of significance to human health (S1 Summary) [32]. We note that one of these contigs has excellent homology with myosin light chain which has previously been identified as a strong interactor in a T7 phage screen [38]. In this communica- tion, we focus on the sequence of our contig29, which we recognised to have an identical amino acid sequence to the N-terminal residues 1–25 of the human gap junction gamma 3 pro- tein GJC3 [39,40][41], (human connexin 30.2/31.3, hCx30.2/31.3, NP_853516, UniProt Q8NFK1.1) synonymous with mouse connexin mCx29 [39,42–46]. Following this biopanning process, phages selected from the final E. coli agarose lawn were submitted to Sanger sequencing (S1 Dataset) to give 120 sequences (S1 Table) which were aligned using the MegaBLAST tool against the coding region for T7Select 10–3 vector (18 clones vector only, S2 Table) from which coding regions for the T7Select1 10–3 vector were removed. Highly similar sections of clone sequence were noted and all low quality ends trimmed, resulting in 31 contiguous sequences (S3 Table). Simvastatin-associated phage clones bearing polypeptide sequences were corrected for the 60 non-specific binding clones in control wells (S2 Dataset) leaving 23 consensus sequences from these contiguous regions, or ‘contigs’. These were first searched against the NCBI Genome (all assemblies scaffolds) using BLASTN optimized for highly similar sequences [36] and the human genome results further assessed by searching against the human RefSeq database [37] using the BLASTX algorithm (S4 Table). This led to identification of 11 sequences (‘contigs’) sufficiently annotated with biological function in the literature to be considered of significance to human health (S1 Summary) [32]. We note that one of these contigs has excellent homology with myosin light chain which has previously been identified as a strong interactor in a T7 phage screen [38]. In this communica- tion, we focus on the sequence of our contig29, which we recognised to have an identical amino acid sequence to the N-terminal residues 1–25 of the human gap junction gamma 3 pro- tein GJC3 [39,40][41], (human connexin 30.2/31.3, hCx30.2/31.3, NP_853516, UniProt Q8NFK1.1) synonymous with mouse connexin mCx29 [39,42–46]. 5 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Immunohistochemistry There was selective GJC3 expression in vascular tissue from patients with ischaemic cardiovas- cular disease (Fig 10). Expression was localized to diffuse staining in media and intima but not in adventitia (Fig 10). Immunohistochemistry also showed expression of GJC3 in capillary endothelium [data not shown]. Structure of discovered sequence A molecular structure has not been solved for GJC3 (hCx30.2) or its C-terminal splice variant hCx31.3, but the X-ray crystal structure of human connexin 26 (hCx26) gap junction GJB2 (2zw3.pdb) [47] allowed us to perform simple homology modelling [48]. Alignment of our contig29 sequence, and NP_853516.1 against that for GJB2 (Fig 6) highlights the sequence identity between the first two, but difference between the latter two, as might be expected. S1 Fig shows the full sequence alignment for GJC3 and GJB2 from S1 Alignment performed using T-Coffee [49] (www.ebi.ac.uk) and rendered using UCSF Chimera.[50] Hydropathy plots (Figs 7 and 8) were nonetheless well matched for the two full-length pro- teins, indicating that a similar transmembrane structure is retained. Both the Kyte-Doolittle and Wimley-White scales show good agreement between predicted transmembrane regions for GJC3 and GJB2. The discovered sequence, corresponding to residues 1–25 of GJC3 is highlighted in orange on the secondary structure for the known coordinates of GJB2 (2zw3. pdb) in Fig 9. Its position corresponds to the non-transmembrane portion residing in the cyto- plasmic channel opening expected to be formed by homology [48,51] with a number of gap junction proteins in hemichannels and connexons. We continue to develop a more thorough homology model of GJC3 in a phospholipid bilayer and to explore its structure and function in detail. Additional Bioinformatics Wimley-White octanol-to-water scale smoothed values for GJC3 and GJB2 are shown, where positive hydropathy values indicate expected membrane associated residues. The predicted transmembrane (TM) segments indicated are at least 19 residues in length and were calculated using a validated algorithm [126]. Wimley-White smoothed hydropathy calculated using MPEx [127] http://blanco.biomol.uci.edu/mpex/ and replotted using ggplot2 in R [125]. doi:10.1371/journal.pone.0148266.g008 Fig 7. Comparison of Kyte-Doolittle index hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive values. Kyte-Doolittle analysis using ProtScale [124] http://web.expasy.org/protscale/. Parameters used were: window size = 9, relative weight 100%, linear model, not normalized. Data replotted using ggplot2 in R [125]. Fig 7. Comparison of Kyte-Doolittle index hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive values. Kyte-Doolittle analysis using ProtScale [124] http://web.expasy.org/protscale/. Parameters used were: window size = 9, relative weight 100%, linear model, not normalized. Data replotted using ggplot2 in R [125]. 100%, linear model, not normalized. Data replotted using ggplot2 in R [125]. doi:10.1371/journal.pone.0148266.g007 Fig 8. Wimley-White hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive negative values. Wimley-White octanol-to-water scale smoothed values for GJC3 and GJB2 are shown, where positive hydropathy values indicate expected membrane associated residues. The predicted transmembrane (TM) segments indicated are at least 19 residues in length and were calculated using a validated algorithm [126]. Wimley-White smoothed hydropathy calculated using MPEx [127] http://blanco.biomol.uci.edu/mpex/ and replotted using ggplot2 in R [125]. d i 10 1371/j l 0148266 008 doi:10.1371/journal.pone.0148266.g007 j p g Fig 8. Wimley-White hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive negative values. Wimley-White octanol-to-water scale smoothed values for GJC3 and GJB2 are shown, where positive hydropathy values indicate expected membrane associated residues. The predicted transmembrane (TM) segments indicated are at least 19 residues in length and were calculated using a validated algorithm [126]. Wimley-White smoothed hydropathy calculated using MPEx [127] http://blanco.biomol.uci.edu/mpex/ and replotted using ggplot2 in R [125]. Fig 8. Wimley-White hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive negative values. Wimley-White octanol-to-water scale smoothed values for GJC3 and GJB2 are shown, where positive hydropathy values indicate expected membrane associated residues. The predicted transmembrane (TM) segments indicated are at least 19 residues in length and were calculated using a validated algorithm [126]. Additional Bioinformatics A search of the Human Protein Atlas [52] for GJC3 reveals wide tissue (S5 Table) and cellular (S3 Dataset) expression of this protein. Data on vascular tissue is absent, although the presence of GJC3 at medium levels of expression is noted in heart muscle. S4 Dataset summarises tissue expression for the wider set of connexins from the same database. The ICR CanSAR resources [53] and EBI Catalogue of Somatic Mutations in Cancer (COSMIC) [54], accessed Dec 2015, reveal over-expression and copy number variant gain (42/278 samples) in a variety of cancer cell lines and tumour samples [623/28982 tested with 46 unique samples with mutations, e.g. adrenal gland (10/79 tested), breast (68/1092 tested), oesophagus (24/125 tested) stomach (31/ 285 tested) pancreas (17/168 tested)]. Single nucleotide polymorphism (SNP) missense muta- tions for GJC3 present in the 1000 Genomes Project [55] from NCBI search 19 Dec 2015 are listed in S5 Dataset and insertion/deletion mutations in S6 Dataset. A search of Fig 6. Alignment of discovered contig29 versus GJC3 (residues 1–25) and the sequence for GJB2 (residues 1–25). Carried out using T-Coffee (www.ebi.ac.uk) with GJC3 NCBI sequence NP_853516.1 and the protein databank file corresponding to GJB2 code 2zw3.pdb. Consensus residues are shown in the upper row; figure prepared using UCSF Chimera [50]. Fig 6. Alignment of discovered contig29 versus GJC3 (residues 1–25) and the sequence for GJB2 (residues 1–25). Carried out using T-Coffee (www.ebi.ac.uk) with GJC3 NCBI sequence NP_853516.1 and the protein databank file corresponding to GJB2 code 2zw3.pdb. Consensus residues are shown in the upper row; figure prepared using UCSF Chimera [50]. Fig 6. Alignment of discovered contig29 versus GJC3 (residues 1–25) and the sequence for GJB2 (residues 1–25). Carried out using T-Coffee (www.ebi.ac.uk) with GJC3 NCBI sequence NP_853516.1 and the protein databank file corresponding to GJB2 code 2zw3.pdb. Consensus residues are shown in the upper row; figure prepared using UCSF Chimera [50]. doi:10.1371/journal.pone.0148266.g006 6 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 7. Comparison of Kyte-Doolittle index hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive values. Kyte-Doolittle analysis using ProtScale [124] http://web.expasy.org/protscale/. Parameters used were: window size = 9, relative weight 100%, linear model, not normalized. Data replotted using ggplot2 in R [125]. doi:10.1371/journal.pone.0148266.g007 Fig 8. Wimley-White hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive negative values. PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 11. Alternative mRNA splicing and selected single nucleotide polymorphisms for GJC3 products. CCDS entry CCDS34697.1 (accessed 24 May 2012) for transcribed mRNA from GJC3 (NM_181538.2 and NP_853516.1) and its translated 279 amino acid polypeptide showing the alternate exon region, designated with a blue rectangle. Residues highlighted in purple (M1V, R4V, P19H, R22H, E268D) indicate single nucleotide polymorphisms (SNPs: see main text) leading to variation in coded amino acids within predicted geometrically neighbouring and functionally significant N- and C-terminal regions. V261 highlighted in orange indicates the C-terminal splice variant start point. Discovered residues correspond to amino acids 1–25. Underlined residues S227S, T234N highlighted in green indicate phosphorylation sites identified from PhosphositePlus and genomic SNPs. d i 10 1371/j l 0148266 011 Fig 11. Alternative mRNA splicing and selected single nucleotide polymorphisms for GJC3 products. CCDS entry CCDS34697.1 (accessed 24 May 2012) for transcribed mRNA from GJC3 (NM_181538.2 and NP_853516.1) and its translated 279 amino acid polypeptide showing the alternate exon region, designated with a blue rectangle. Residues highlighted in purple (M1V, R4V, P19H, R22H, E268D) indicate single nucleotide polymorphisms (SNPs: see main text) leading to variation in coded amino acids within predicted geometrically neighbouring and functionally significant N- and C-terminal regions. V261 highlighted in orange indicates the C-terminal splice variant start point. Discovered residues correspond to amino acids 1–25. Underlined residues S227S, T234N highlighted in green indicate phosphorylation sites identified from PhosphositePlus and genomic SNPs. Fig 11. Alternative mRNA splicing and selected single nucleotide polymorphisms for GJC3 products. CCDS entry CCDS34697.1 (accessed 24 May 2012) for transcribed mRNA from GJC3 (NM_181538.2 and NP_853516.1) and its translated 279 amino acid polypeptide showing the alternate exon region, designated with a blue rectangle. Residues highlighted in purple (M1V, R4V, P19H, R22H, E268D) indicate single nucleotide polymorphisms (SNPs: see main text) leading to variation in coded amino acids within predicted geometrically neighbouring and functionally significant N- and C-terminal regions. V261 highlighted in orange indicates the C-terminal splice variant start point. Discovered residues correspond to amino acids 1–25. Underlined residues S227S, T234N highlighted in green indicate phosphorylation sites identified from PhosphositePlus and genomic SNPs. doi:10.1371/journal.pone.0148266.g011 PhosphositePlus [56] for human GJC3 shows sites for post-translational phosphorylation found by proteomic mass spectrometry to be confined to the C-terminal portion: S227 and T234 (Fig 11). In the orthologous mouse Gjc3 gene product mCx29 (Uniprot Q921C1; see S7 Dataset and NCBI HomoloGene 15399 used to generate a Multiple Sequence Alignment such as S2 Fig), different phosphorylation sites have been identified: mCx29_S247, mCx29_S249, mCx29_S250, mCx29_S253, mCx29_S261, together with sites of ubiquitinylation mCx29_K254, mCx29_K266 and a site of N-acetylation mCx29_K141. Additional Bioinformatics Wimley-White smoothed hydropathy calculated using MPEx [127] http://blanco.biomol.uci.edu/mpex/ and replotted using ggplot2 in R [125]. doi:10.1371/journal.pone.0148266.g008 doi:10.1371/journal.pone.0148266.g008 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 7 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 9. Discovered N-terminal residues 1–25 (orange) aligned on a simple model based on GJB2.pdb X-ray structure. Hexameric structure viewed from cytoplasmic side of the transmembrane hemichannel (prepared using UCSF Chimera [50]). doi:10.1371/journal.pone.0148266.g009 Fig 9. Discovered N-terminal residues 1–25 (orange) aligned on a simple model based on GJB2.pdb X-ray structure. Hexameric structure viewed from cytoplasmic side of the transmembrane hemichannel (prepared using UCSF Chimera [50]). Fig 9. Discovered N-terminal residues 1–25 (orange) aligned on a simple model based on GJB2.pdb X-ray structure. Hexameric structure viewed from cytoplasmic side of the transmembrane hemichannel (prepared using UCSF Chimera [50]). doi:10 1371/journal pone 0148266 g009 Fig 9. Discovered N-terminal residues 1–25 (orange) aligned on a simple model based on GJB2.pdb X-ray structure. Hexameric structure viewed from cytoplasmic side of the transmembrane hemichannel (prepared using UCSF Chimera [50]). doi:10.1371/journal.pone.0148266.g009 doi:10.1371/journal.pone.0148266.g009 doi:10.1371/journal.pone.0148266.g009 Fig 10. Immunohistochemistry for human artery with antibody against GJC3, hCx30.2/31.3. (A) H-86— sc-68376, Santa Cruz Biotechnology, Inc., vs. (B) control section, both micrographs at 20 x magnification. Brown staining in (A) indicates selective localisation of GJC3 in media and endothelium of the artery. doi:10 1371/journal pone 0148266 g010 Fig 10. Immunohistochemistry for human artery with antibody against GJC3, hCx30.2/31.3. (A) H-86— sc-68376, Santa Cruz Biotechnology, Inc., vs. (B) control section, both micrographs at 20 x magnification. Brown staining in (A) indicates selective localisation of GJC3 in media and endothelium of the artery. doi:10.1371/journal.pone.0148266.g010 8 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Surface Plasmon Resonance (SPR) Each chemically synthesized peptide corresponding to the expected reading frame for contig29 (peptide A), frame +1 nucleotide (peptide B), frame +2 nucleotides (peptide C) (S3 Fig) was successfully immobilised in separate lanes of a 6-channel ProteOn NLC sensor chip (S4 Fig) and then exposed to increasing concentrations of a range of statins (simvastatin, fluvastatin and pravastatin hydroxyacids, as well as simvastatin lactone). Fig 12 shows the SPR chromato- grams of the statins interacting with the in-frame peptide A while the corresponding chro- matograms for the out-of-frame peptides B and C are shown in S5 Fig. Simvastatin (i.e. the lactone) showed no interaction with the immobilised peptides, whereas the sodium salt of its hydroxyacid demonstrated a weak concentration dependent profile with the in-frame peptide A only (Fig 12A). When each peptide was exposed to fluvastatin sodium salt, more pronounced binding is seen to peptide A (Fig 12C), with little interaction seen with peptide B and no interaction with peptide C (S5 Fig, Panel C). Interestingly, pravastatin sodium salt, which is more hydrophilic, showed no interaction with any of the immobilised peptides (Fig 12D and S5 Fig, Panel D) suggesting the hydroxyacid pharmacophore alone is not suffi- cient for binding of statins to peptide A. 9 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 12. Surface plasmon resonance sensorgrams of peptide A interacting with statins. (A) Simvastatin hydroxyacid, (B) simvastatin lactone, (C) fluvastatin hydroxyacid, and (D) pravastatin hydroxycid, each at concentrations of 400 μM (light blue), 200 μM (red), 100 μM (grey), 50 μM (yellow), 25 μM (dark blue) 0 μM (green) for each ligand in PBS. Concentration dependent interactions are observed for both simvastatin and fluvastatin hydroxyacid sodium salts with the most pronounced interaction observed for fluvastatin. doi:10.1371/journal.pone.0148266.g012 Fig 12. Surface plasmon resonance sensorgrams of peptide A interacting with statins. (A) Simvastatin hydroxyacid, (B) simvastatin lactone, (C) fluvastatin hydroxyacid, and (D) pravastatin hydroxycid, each at concentrations of 400 μM (light blue), 200 μM (red), 100 μM (grey), 50 μM (yellow), 25 μM (dark blue) 0 μM (green) for each ligand in PBS. Concentration dependent interactions are observed for both simvastatin and fluvastatin hydroxyacid sodium salts with the most pronounced interaction observed for fluvastatin. Discussion We report here a previously unrecognized molecular interaction between simvastatin in its HMG CoA reductase inhibitory hydroxyacid form and the N-terminal domain of gap junction gamma-3 protein. The position of the N-terminal domain in what is expected to be the cyto- plasmic channel opening formed by the gap junction proteins indicates that this putative inter- action site for simvastatin hydroxyacid may influence function as a component either of hemichannels or cell-cell gap junctions. Interactions of additional small molecules explored in this work including niflumic acid, flufenamic acid and carbenoxolone with the N-terminus may explain the known inhibitory effect on other connexins of these compounds. Diffuse stain- ing of immunohistochemically labelled samples herein is consistent with expression of GJC3 in human arteries. Tissue-specific data [59] from The Human Protein Atlas [60] suggest much wider expression of GJC3 than previously recognized, although the vasculature does not appear as a separate entry in that database [61]. Connexins in cardiovascular tissue The connexins are a large structurally-related family of membrane proteins capable of building gap junction channels, which permit direct exchange of small molecules ( 1200 Da) and elec- trical current among neighbouring cells; and hemichannels, capable of allowing cells to com- municate between the intra-cellular and extra-cellular space [62]. Both amino and carboxy termini of connexins are found in the cytoplasm [40]. A large number of genes are involved in direct cell-cell communication in cardiovascular and other tissues [63]. Genetic and acquired variability in connexin structure and function may contribute to disease risk, natural history and variable response to treatment including adverse events [64,65]; and provide biomarkers of disease for diagnosis, prognosis and prediction of and monitoring of treatment response. Connexins have in particular been suggested to play a role in the pathogenesis of atherosclero- sis [66]. Each of the three key cell types involved in atherogenesis, monocytes/macrophages, endothelial cells, and smooth muscle cells, express a distinct pattern of connexins, with recog- nized vascular connexins, Cx37, Cx40, and Cx43, differentially modified as atherosclerosis develops [67,68]. Of particular interest to this current work is the observation that lovastatin has been found to inhibit gap junction intercellular communication in cultured aortic smooth muscle cells [69]. Surface Plasmon Resonance (SPR) doi:10.1371/journal.pone.0148266.g012 doi:10.1371/journal.pone.0148266.g012 In order to further investigate the binding characteristics of peptide A, a number of known gap junction inhibitors [30,57] (for a list of inhibitors abstracted from these references and their known and shared targets from ChEMBL_20, [58] see S8 Dataset) were chosen for SPR analysis. Fig 13 illustrates increasing concentrations of niflumic acid (A), flufenamic acid (B), carbenoxelone (C), and its parent 18β-glycyrrhetinic acid (D) binding to peptide A while the corresponding chromatograms of peptides B and C are shown in S6 Fig. As expected, no inter- action was observed for any of the compounds tested with the out-of-frame peptides B and C. A concentration dependent binding profile of niflumic acid was observed with peptide A and a stronger interaction observed for flufenamic acid. The glycyrrhetinic acid derivative, carbenox- olone, exhibited a pronounced binding interaction, while there was no evidence for the parent Fig 13. Surface plasmon resonance sensorgrams of peptide A interacting with known hemichannel blockers. (A) Niflumic acid, (B) flufenamic acid, (C) carbenoxolone, and (D) 18β-glycyrrhetinic acid each at concentrations of 400 μM (light blue), 200 μM (red), 100 μM (grey), 50 μM (yellow), 25 μM (dark blue) 0 μM (green) for each ligand in PBS. Concentration dependent binding profiles are observed for niflumic acid with more pronounced effects observed for flufenamic acid and carbenoxolone. doi:10.1371/journal.pone.0148266.g013 Fig 13. Surface plasmon resonance sensorgrams of peptide A interacting with known hemichannel blockers. (A) Niflumic acid, (B) flufenamic acid, (C) carbenoxolone, and (D) 18β-glycyrrhetinic acid each at concentrations of 400 μM (light blue), 200 μM (red), 100 μM (grey), 50 μM (yellow), 25 μM (dark blue) 0 μM (green) for each ligand in PBS. Concentration dependent binding profiles are observed for niflumic acid with more pronounced effects observed for flufenamic acid and carbenoxolone. doi:10.1371/journal.pone.0148266.g013 doi:10.1371/journal.pone.0148266.g013 10 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein 18β-glycyrrhetinic acid or 18α-glycyrrhetinic acid binding to peptide A. Additional com- pounds which were investigated using SPR included 3-hydroxymethylglutaric acid, arachido- nylethanolamide, heptanol, steviol, stevioside and 18α-glycyrrhetinic acid. None exhibited any binding to the peptides (S7 Fig). Data for SPR experiments is included in S9 Dataset. Physiological significance of human gap junction gamma-3 protein Mouse Cx29 expression by contrast is considered to be restricted to the central and peripheral nervous system, including brain, spinal cord and sci- atic nerve [42], where it is expressed in Schwann cells in small myelinating fibres and is postu- lated to exist as hemichannels [43,79,80]. In humans, hCx30.2/Cx31.3 R15G and L23H mutants expressed in the inner ear have, along with several other connexins [81], been impli- cated in non-syndromic hearing impairment [82,83]. Its over-expression in a variety of cancer tumour and cell line samples together with copy number variant gain seen in data from COS- MIC [54], confirm unexplored tissue profiles and linkage with disease. Hence the observed function of GJC3 in mediating ATP release in conjunction with acknowledged experimental difficulty in differentiating connexin from pannexin 1 purinergic signalling [84,85] (which has been positively linked with P2 receptors and sympathetic vasoconstriction [86]), make this and its related channels of particular interest [87]. N-Terminal sequences are recognized to be crucial for function in many connexins [88,89] and there are 4 single nucleotide polymorphisms (SNPs) observed in this discovered region of GJC3 (Fig 11: M1V (C/T rs192406037), F5V (A/T rs187126645), P19H (G/T rs201016616), and R22H (C/T rs200074250)) and three somatic mutations p.R8Q (COSM3267470, c.23G/A; carcinoma, upper aerodigestive tract), p.L25I (COSM1093913, c.73C/I; carcinoma, endome- trium) and p.V27M (COSM5184309, c.79G/A; carcinoma, large intestine). Other SNPs are seen in the putative structurally neighbouring cytoplasmic C-terminus with at least 35 docu- mented in the RefSeq genome NM_181538.2 associated with GJC3 gene products, not includ- ing two in the 5’-untranslated region, one of which is associated clinically with peripheral neuropathy induced by bortezomib [90,91]. The C-terminus of hCx30.2/31.3 also contains two sites for post-translational phosphorylation, S227 and T234 (Fig 11), identified by proteomic mass spectrometry, according to PhosphositePlus. Those sites of phosphorylation, the only ones reported in GJC3, are subject to genomic variation (Fig 11: the synonymous variant S227S [rs769486892 C/T] and T234N [rs140894833 C/A] and in the C-terminus from amino acids 219–279 there are 14 known somatic mutations including the neighbouring R220S (COSM219466, c.660G/T; breast carcinoma) and S228T (COSM4149446, c.682.T/A; carci- noma, ovary). They introduce potentially complementary phosphate groups to the N-terminal arginine functions seen at R4, R7, R8, R15, R16, offering a testable hypothesis by which the gat- ing of the channel might be controlled (see ‘Functional significance of GJC3 N-terminus‘ below). Physiological significance of human gap junction gamma-3 protein Human connexin 30.2 [61] (NP_853516, hCx30.2) is an integral membrane protein expressed from GJC3 (NCBI Gene ID: 349149) and is known by its official name GJC3, “human gap junc- tion gamma-3 protein, 30.2 kDa”, which also has a splice isoform of molecular mass 31.3 kDa, hence it is also called hCx30.2/31.3. It is now recognised as a member of the γ-subfamily of connexins [70], although “connexin 31.3” [71], has previously been referred to as gap junction ε-1 protein GJE1 [71,72] (‘gje1’ is the approved symbol for an orthologous protein in frogs [70,73] and GJE1 in primates is now considered a pseudogene for Cx23 whose expression is prevented by a nonsense mutation [74]) and the ortholog mouse connexin 29 (mCx29) [42,43,45,46] has been used synonymously. Hence in this publication we use names for all three gene products “hCx30.2/31.3 (mCx29)” where possible, noting for clarity that mouse PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 11 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein connexin 30.2 (mCx30.2) is not the same as our protein, but is orthologous with human con- nexin hCx31.9 [75]. The human genetic locus for GJC3 maps to 7q22.1 (CCDS 34697.1 GRCh37.p5) [71,72] and has been reported to group with [76,77] Gjc3 in Mus musculus, gjc3 in Canis familiaris [78] and other mammals (Homologene 15399 alignment, S7 Dataset and S7 Fig). As with other mouse/human connexin orthologs, expression profiles across the species differ. Our study is the first we are aware of to suggest that this connexin plays a vascular role, although hCx30.2/ 31.3 (mCx29) is reported in humans to be expressed strongly in heart, liver and skeletal muscle (1.9 kb transcript), with a 1.6 kb transcript in pancreas [61]. Immunohistochemistry data recently made available through the Human Protein Atlas [60] shows much wider expression of GJC3 than previously recognized, although that antibody data is marked as “uncertain reli- ability”. In that work, strong expression (assayed through polyclonal antibody HPA015024 raised against a hapten representing GJC3 amino acids 220–279) was observed in many tissues, although only low expression was detected in skeletal muscle, or the liver (S5 Table, and expression of GJC3 in primary cell types S3 Dataset comparing the wider set of gap junction proteins’ tissue expression in S4 Dataset). PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Physiological significance of human gap junction gamma-3 protein Importantly the clinically observed R15G mutant has already been shown to lead to decreased ATP release, but not affect trafficking to the membrane [83]. 12 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein We note that an alternative translation initiation site in the mouse gene Gjc3 has been shown to contain a 450 bp exon, overlapped with the main exon when spliced, which produces an 11 amino acid residue extended N-terminal sequence in mCx29 [46] containing what may be, for that species, a potentially important glutamic acid and a lysine residue (see multiple sequence alignments, S7 Dataset and S2 Fig). The mRNA splice variant of GJC3, hCx31.3 is known to be expressed in human oligoden- drocytes [41] and in line with earlier observations [43] this protein, as with mCx29 [92] has been found not to form full connexon or gap junction channels, but “shares functional proper- ties with pannexin (hemi) channels” [93]. Nonetheless, connexins such as these are both dis- ease-mediating and drug-modifiable targets of cardiovascular relevance [64] and their observation in internal mammary artery, and from The Human Protein Atlas, many other tis- sues with the exception of liver and skeletal muscle, is thus significant. Statins and human gap junction gamma-3 protein Statins have previously been linked indirectly to other connexins, for example exhibiting potential vascular anti-proliferative effects by reducing Cx43 expression in primary human vascular cells, and in pravastatin-treated LDLR-/- mice [67]. In contrast both mCx37 and mCx40 have been reported to be down-regulated in aortic endothelial cells from cholesterol- enriched diet mice, with selective recovery of mCx37 but not mCx40 endothelial cell expression by simvastatin treatment [68]. Expression of Cx40 and Cx43 mRNA and protein is reduced both by lovastatin and fluvastatin [66]. Statins have not previously been linked with hCx30.2/ 31.3 (mCx29), but the possibility that GJC3 is expressed within internal mammary artery and that simvastatin hydroxyacid may interact with its N-terminal sequence offers potential new insight into additional mechanisms for the clinically observed actions of statins. In order to be functionally active as an inhibitor of HMG CoA reductase, simvastatin used herein is interconverted enzymatically in vivo between the lactone and HMG CoA reductase- inhibitory hydroxyacid form [6] and this varies according to genetic subpopulations [5,94]. The pharmacokinetics and clinical effects of statins are also known to be associated with tissue- specific membrane protein expression of anion transporters and neutral ATP binding cassette (ABC)-type transporters [95] and thus this new linkage to what can be classified as a mem- brane transporter is of particular relevance. An example of how recognition events at N-termi- nal sequences affect the channel behavior of connexins is the formation of a carbamate within human connexin 26, GJB2 by carbon dioxide [96]. PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Biophysical investigation of the new interaction Further investigation of the uncovered interaction is therefore important and we chose to use surface plasmon resonance [29,97–99] to study the interaction between simvastatin, simva- statin hydroxyacid as its sodium salt, along with fluvastatin, and pravastatin which are both administered as hydroxyacids and the N-terminal polypeptide discovered in the phage display screen. It is recognised that viruses are susceptible to the expression of frame-shifted peptides [100], something we have observed for the T7 phage system [101]. In order to explore this pos- sibility and provide biophysical validation that the uncovered sequence in contig29 interacts in a meaningful way with simvastatin we purchased chemically synthesized peptides A, B, C in the expected reading frame, frame + 1 nucleotide and frame + 2 nucleotides respectively. We presented each peptide on a neutravidin-coated gold SPR chip by adding a C-terminal lysine bearing an ω-NH2-coupled biotin moiety (S3 Fig) thereby mimicking its position within the connexin. This approach has previously been used to successfully investigate binding selectivity in many systems, including the discovery of candidate binding partners for the carboxyl PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 13 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein terminus of connexin 43 [99] and the first demonstration of C-terminal peptide tail/intracellu- lar loop interactions [98]. No interactions were observed for any of the compounds tested with the out-of-frame pep- tides B or C (S5 and S6 Figs). While simvastatin i.e. the lactone showed no interaction with the uncovered sequence contig29 (peptide A), simvastatin acid sodium salt exhibited a weak, con- centration dependent binding profile (Fig 12A). This validates the results obtained by the T7 phage system by confirming that the sequence contig29 (and not the out of frame peptides) does interact in a meaningful way with simvastatin. However, this also suggests that hydrolysis to the active, hydroxyacid form has taken place during the immobilisation or biopanning pro- cess. In order to investigate this further, fluvastatin and pravastatin (which are both adminis- tered in the hydroxyacid form) were investigated. A significant binding profile, exhibiting some non-ideality not untypical of small molecule SPR, was observed for fluvastatin (Fig 12C) while pravastatin showed no interaction with peptide A indicating that the hydroxyacid alone is not sufficient for statin binding to the GJC3 N-terminus (Fig 12D). Biophysical investigation of the new interaction In order to further inves- tigate contig29 a number of known gap junction (albeit not validated ‘hemichannel’) inhibitors were investigated (niflumic acid, flufenamic acid, carbenoxolone, 18α-glycyrrhetinic acid (Fig 13), 18β-glycyrrhetinic acid, arachidonylethanolamide, and heptanol (S6 Fig, Panel C)) [30] along with compounds sharing functional significance with statins (3-hydroxy-3-methylgluta- ric acid (S6 Fig, Panel A), or other chemical structural similarities (the sweeteners steviol and stevioside S6 Fig, Panels D and E)). To the best of our knowledge little is known regarding the mechanism of action of the gap junction inhibitors and so it is not clear if these compounds bind directly to particular features within connexin channels, or perhaps act through some other membrane-mediated process [102]. Indeed, differentiating functional activity of con- nexin 43 hemichannels from pannexin 1 [84,103–105] in assays such as ATP release is recog- nised as problematic [84,106,107] and we note that carbenoxolone for example is also an inhibitor of pannexin 1 activity [84,86,108]. Investigation of simvastatin, fluvastatin and prava- statin hydroxyacids as potential inhibitors of pannexin 1 (Panx1) channel function is thus warranted. Fenamates are known reversible inhibitors of gap junctions and so we also chose to investi- gate binding of niflumic acid and flufenamic acid to peptide A using SPR (Fig 13). Dose depen- dent binding was observed for both compounds with the most pronounced effect observed for flufenamic acid. The data presented here suggest that carbenoxolone, a derivative of 18β-glycyrrhetinic acid may function by direct interaction with the N-terminal domain, while 18α-glycyrrhetinic acid, lacking the flexible succinate moiety and 18β-glycyrrhetinic acid (enoxolone, Fig 14) do not. This data should not be over-interpreted, but indicates the latter connexin gap junction inhibi- tors may have a different selectivity or mode of action, if indeed they act against GJC3 or other hemichannels rather than gap junctions, which does not seem to have been fully explored. Functional significance of GJC3 N-terminus As noted above, the N-terminus of many connexins is well-known to be important in deter- mining their function as channels [89,109]. For example, in connexin 32 hemichannel the N- terminus has been shown to act as a voltage sensor in a proposed gating mechanism [110] and is recognized as bearing many loss of function genetic mutations associated with Charcot- Marie-Tooth disease [111]. The fact that acidic residues including glutamate and aspartate are conserved in the N-terminus of many connexins (see for example Fig 1 in Beyer et al. ref [89]) together with the observed gap junction inhibitory properties of polyamines has led to the investigation of “interfering N-terminal peptides” such as Ac-KLLDK-NH2 from Cx43 as PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 14 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 14. Chemical structures of enoxolone (18β-glycyrrhetinic acid), 18α-glycyrrhetinic acid) and carbenoxolone (18β-glycyrrhetinic acid). g 14. Chemical structures of enoxolone (18β-glycyrrhetinic acid), 18α-glycyrrhetinic acid) and carbenoxolone (18β-glycyrrhetinic acid). 10 1371/j l 0148266 014 g 14. Chemical structures of enoxolone (18β-glycyrrhetinic acid), 18α-glycyrrhetinic acid) and carbenoxolone (18β-glycyrrhetinic acid). 14. Chemical structures of enoxolone (18β-glycyrrhetinic acid), 18α-glycyrrhetinic acid) and carbenoxolone (18β-glycyrrhetinic acid). doi:10.1371/journal.pone.0148266.g014 inhibitors of gating functions in other connexins including Cx40 [112]. The presence of basic residues in the form of arginines in our discovered sequence at positions 4, 7, 8 in particular is thus striking (see helical wheel, Fig 15 (A)). The statins and the gap junction inhibitors we find to bind to the hCx30.2/31.3 (mCx29) N-terminus by SPR contain at least one, and in the case of carbenoxolone, two carboxylic acid functionalities that might interact with the guanidinium side-chain(s) of these arginine(s). Furthermore, the same arginines could interact with the C- terminal S227, T234 when phosphorylated (Fig 15 (B)), in order to control movement of N- and C-termini and hence access to the channel [113]. As has been proposed for rat Cx43 [114], such a mechanistic hypothesis would enable a serine/threonine kinase e.g. protein kinase C to control the opening of GJC3 in response to an intracellular signal thereby releasing ATP; see for example Kang et al. rat Cx43 phosphorylation regulated by IP3/IP3 receptor [115]. We have now expressed human GJC3 in order to further study the discovered ligand–target interactions and the proposed mechanistic hypothesis. Additional sequences identified and a network perspective From this screen of the new internal mammary artery genomic library we identified an addi- tional six ‘contig’ sequences with sufficient homology and annotated biomedical literature to PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 15 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Fig 15. Helical wheel projections of: (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 226–227. Serine 227 and threonine 234 subject to post-translational phosphorylation circled in red. Fig 15. Helical wheel projections of: (A) discovered N-terminal residues 1–25 and (B) C-terminal residues subject to post-translational phosphorylation circled in red. Fig 15. Helical wheel projections of: (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 22 subject to post-translational phosphorylation circled in red. (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 226–227. Serine 227 and threonine 234 l ti i l d i d (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 226–227. Serine 227 and threonine 234 ylation circled in red. Fig 15. Helical wheel projections of: (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 226–227. Serine 227 and threonine 234 subject to post-translational phosphorylation circled in red. doi:10.1371/journal.pone.0148266.g015 merit further investigation (S1 Summary). Several are of significance for developing a cardio- vascular network of interacting proteins and the statins in order to better contextualize their polypharmacology, which is work in progress. Gap junction inhibitors also possess additional molecular targets, including other channels and transporters (S8 Dataset) and our data will aid further characterizing their in vitro and in vivo activity. PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Conclusions As test of concept, we applied a new chemical genomics approach, using a viral display library in conjunction with the photo-immobilised simvastatin ligand, to study possible new targets for this widely prescribed lipid-lowering pro-drug. Using these methods, supported by in silico studies, immunohistochemistry and biophysical data, we have provided evidence for the previ- ously unrecognized presence of gap junction gamma-3 protein in arteries from patients with ischaemic heart disease and interaction of simvastatin and fluvastatin hydroxyacids with the N-terminal sequence of this human gap junction gamma-3 protein. We also demonstrate for the first time a selective interaction of the same sequence with the known inhibitors of con- nexin channel function: niflumic acid, flufenamic acid and carbenoxolone, offering a new entry with which to study their mechanism of action. There are few pharmacological tools with which to probe connexin function making statins high priority for further investigation against these channels. Our work raises several key questions relevant to understanding aetiology and improving treatment of cardiovascular disease, in particular relating to treatments using statins. Cardiac PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 16 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein gap junction channels comprising connexins are known to undergo remodeling in disease [116] and hetero-oligomerisation of connexins is a recognized phenomenon [117,118] raising the possibility that gap junction gamma-3 protein may play a role within homo- or hetero-hex- amers in the pathophysiology of human conduit arteries. Interaction of simvastatin hydroxy acid or other statins such as fluvastatin may also thus take place in connexon channels with intercellular conductive functions, as well as hemichannels expected to show extracellular com- munication as part of signalling microdomains [119]. Whether interaction with gap junction gamma-3 protein contributes to the therapeutic or adverse effects of statin hydroxyacids remains to be investigated and these effects may be tissue-specific. We speculate that the low expression of GJC3 in human liver compared to many other tissues is noteworthy given the high retention of clinically used statins by that organ. A more complete understanding of the biological significance of the novel statin-GJC3 interaction and whether or not particular stat- ins might interact with other connexins is thus important. That knowledge may lead to the development of new classes of treatment with cardiovascular connexins as therapeutic targets [120], perhaps by influencing intercellular conductive function of gap junctions, or connexin derived hemichannels associated with extracellular communication. Ethics Statement All patients gave written informed consent and the study was approved by the Local Research Ethics Committee (NHS National Research Ethics Service, Coventry Research Ethics Commit- tee, Coventry UK: ref—07/H1210/126). We studied the interactions of simvastatin with a viral library of clones prepared from mRNA extracted from internal mammary arteries from 7 patients. Their cardiovascular risk factors included smoking, hypertension, hyperlipidaemia and diabetes mellitus. These samples were obtained as segments of surplus internal mammary artery harvested during coronary artery bypass surgery for treatment of ischaemic heart disease. All work involving the phage library was carried out under the UK Health and Safety Execu- tive's Genetically Modified Organisms (HSE GMO) guidelines (contained use). Phage library preparation from mRNA, validation, and subsequent work involving the phage library was per- formed at Tangent Reprofiling Ltd, Warwick HRI, Wellesbourne, Warwickshire, UK. All plasticware was purchased as RNase-free. RNAlater RNase inhibitor solution from Ambion [121] (Catalogue #7020) was used to transport the tissues on ice from theatre to the biological lab with at most 30 minutes between extraction of the tissue and processing, or freez- ing to –80°C. mirVana™miRNA Isolation Kit from Ambion [122,123] (Catalogue #1560, 1561) was used to extract total RNA from the tissues obtained. Straight A’s™mRNA extraction Kit from Novagen (Catalogue #69962–3) was used to extract mRNA from the total RNA samples. OrientExpress™Oligo(dT) Primer cDNA synthesis kit from Novagen (Catalogue #69992–3) and the T7 Select1 Phage Display System from Novagen (Catalogue #70550–3) were used to prepare the T7 Phage Display Library. Water used in total RNA and mRNA extraction was RNase free water, purchased from Ambion. Water used in cDNA and Phage Library prepara- tion was Milli-Q filtered sterile water prepared on site. Tris-buffered saline (TBS: 0.05 M tris(hydroxyethyl)aminomethane (tris), 0.15 M NaCl, pH 7.6 at 25°C) with Tween1 20 surfactant was prepared using 13 TBS tablets (Sigma Aldrich, Cat# T5030) added to Milli-Q1 water (195 mL). Two concentrations of Tween1 20 were used, 0.5% and 0.1%, 975 μL and 195 μL respectively. For preparation of agarose gels, GelRed1 (10,000X in water, Biotium, Cambridge Biosci- ences, Cat# BT41003) was substituted for ethidium bromide, except during library preparation 17 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein (Fig 4). GelRed1 was added to molten agarose solution (5 μL per 100 mL) and not added to the running buffer. Biopanning The combined phage library (50 μL library 1 and 50 μL library 2: see library preparation, S1 Protocol) was amplified in 5 mL log phase E. coli BLT5615 solution at OD600 0.5, with agita- tion for 3 h at 37°C. The lysate was centrifuged at 12k × g and the supernatant decanted into fresh tubes. For round 1 of biopanning, amplified human vascular phage library (200 μL per well) was added to wells containing bound simvastatin and to control wells containing water, and the plate agitated for 45 minutes. Excess phage solution was then removed and wells washed with 0.5% Tween1 in tris-buffered saline (TBS) six times, with 2 minutes agitation for each wash. Log phase E. coli was prepared as follows. Stock E. coli strain BLT5615 (1 mL, OD600 ~2.0 in lysogeny broth (LB) medium with ampicillin was added to LB medium (100 mL) with ampicillin (1 μL/mL) for 2 h at 37°C. At 1.5 h isopropyl β-D-1-thiogalactopyranoside (IPTG, 500 μL, 100 mM) was added. When this mixture reached OD600 ~0.5 it was added to each well (200 μL), and the plate agitated for 10 minutes to elute the remaining bound phage. The first round well contents were again amplified in log phase E. coli BLT5615 (5 mL) in labelled 50 mL tubes. The tubes were incubated and shaken for 3 h at 37°C, during which time the solution cleared as the E. coli were lysed. Aliquots (2 mL) of amplified phage from each tube were separated from any cellular debris by centrifuge and the clarified lysates decanted into fresh, labelled tubes. The contents of each well from the first round of biopanning (200 μL per well) were then introduced to the next row of immobilized drug and control wells and a second biopanning process performed. On the third round of biopanning, the contents of each well from the elu- tion step were sampled and diluted to 10−3, 10−5 and 10−7, then mixed with stock E. coli BLT5615 culture and poured on to agarose/agar assay plates directly to avoid any dispropor- tionation of the phage present. Ethics Statement All other processes for preparation of agarose gel and loading of wells are identical to those described in the supporting information. Sequencing Master Mix (per sample): 2 μL Big Dye1 (Applied Biosystems, Cat# 4337454), 1 μL 5X sequencing buffer (Applied Biosystems, Cat# 4336697), 0.4 μL T7Select Up Primer, water to make up to 9 μL. Cleaned DNA solution (1 μL) was added before submission for Sanger dideoxynucelotide sequencing. The amount of water in the sequencing PCR master mix was adjusted to account for 1 μL sequencing buffer. Simvastatin Immobilisation Simvastatin (5 mg, Sigma-Aldrich, Cat# S6196, 97% (HPLC) solid) was dissolved in DMSO (1 mL) as a working stock solution (5 mg/mL, 0.012 M, stored at 4°C, allowed to return to room temperature before use). A 100-fold dilution of the stock drug solution was prepared (4 mL, 40 μL in 3.96 mL water, 0.12 mM), and this solution, in a dark room, was added to three wells each of 5 Magic Tag1 photochemically active moieties as previ- ously described [13–15] (200 μL, 10 ng, 0.024 nmol per well). Water (200 μL per well) was added to three wells each of the same moieties to form the negative controls. Photochemical activation was achieved by irradiating wells for 10 minutes under a 254 nm UV lamp (220 W). All wells were then washed with water six times and the plate stored at 4°C until use. PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Biopanning From these agarose lawns, plaques of lysed bacteria were selected that were non-confluent and regular in shape and size to give: (a) 24 phage clones from those wells that contained sim- vastatin (positive wells, a total of 120 clones) and (b) 12 phage clones from control wells con- taining water (a total of 60 clones, S2 Dataset). These selected phage clones were submitted to PCR and sequencing (see S1 Dataset). All phage clones and products from PCR were stored at PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 18 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein 4°C initially, then archived as glycerol stocks (1:9 80% glycerol solution to phage solution) at – 80°C. Immunohistochemistry Sections of internal mammary artery were prepared using standard methods. We performed immunohistochemistry using a rabbit polyclonal antibody [sc-68376, described as “connexin 29 Antibody (H-86)”: Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA] raised against an epitope corresponding to amino acids 194–279 of NP_853516.1 mapping at the C-terminus of human connexin 30.2/31.3 (NP_853516.1; 86/86 Identities, 86/86 Positives, 0/86 Gaps), to identify the presence and location of human gap junction gamma-3 protein. Sequencing The PCR products were placed in a 96-well Millipore filter plate (Cat# LSKMPCR10) and fil- tered under vacuum. Water (100 μL) was added and the plate was agitated for 5 minutes. Big Dye™sequencing master mix (9 μL) was added to each well, followed by DNA solution (1 μL), then submitted for Sanger dideoxynucleotide sequencing. Initial alignment, to identify and remove T7Select1 10–3 vector sequence, correct any base mis-reads, and note highly similar sequences, was carried out using Lasergene1 SeqMan Pro (DNAStar Inc.). PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Supporting Information S1 Alignment. T-Coffee aligned sequences in.afasta format for GJC3 NP_853516.1 and GJB2_NP003995.2. (AFASTA) S1 Dataset. The complete Sanger dideoxy sequencing data for a biopanning screen of sim- vastatin, immobilised from solution to a set 5 photochemistries on a 96-well polystyrene plate, versus a genomic T7 phage display created from samples of human vascular tissue total mRNA. (ZIP) S2 Dataset. Negative controls for sequencing of phage which bind to the same 5 photo- chemistries on a 96-well plate in the absence of simvastatin ligand. (ZIP) S3 Dataset. Normal tissue cell expression of gap junction protein, gamma 3, 30.2kDa (GJC3) analyzed using polyclonal antibody HPA015024. Accessed from Human Protein Atlas using keyword GJC3, July 2015 and saved as.xlsx spreadsheet. (XLSX) S4 Dataset. The Human Protein Database annotates 19 gap junction proteins as connexins. http://www.proteinatlas.org/search/connexin with CNST, CAV3, CXADR data removed; exported as TAB data. Saved as.xslx file at http://figshare.com/articles/connexin_annotated_ HTA_2015_xlsx/1422052 (XSLX) S5 Dataset. Human GJC3 missense single nucleotide polymorphisms from NCBI present in 1000 Genomes Project data (19 Dec 2015). (TXT) Surface Plasmon Resonance (SPR) Normal tissue cell expression of gap junction protein, gamma 3, 30.2kDa (GJC3) analyzed using polyclonal antibody HPA015024. Accessed from Human Protein Atlas using keyword GJC3, July 2015 and saved as.xlsx spreadsheet. (XLSX) S4 Dataset. The Human Protein Database annotates 19 gap junction proteins as connexins. http://www.proteinatlas.org/search/connexin with CNST, CAV3, CXADR data removed; exported as TAB data. Saved as.xslx file at http://figshare.com/articles/connexin_annotated_ HTA_2015_xlsx/1422052 (XSLX) S5 Dataset. Human GJC3 missense single nucleotide polymorphisms from NCBI present in 1000 Genomes Project data (19 Dec 2015). (TXT) S6 Dataset. Human GJC3 insertion/deletion polymorphisms from NCBI (19 Dec 2015). (TXT) S7 Dataset. GJC3 multiple sequence alignment for human, mouse and rat proteins showing how an alternative site of translation initiation results in extended N-terminus for mCx29. (RTF) S8 Dataset. Gap junction inhibitors and known targets from ChEMBL_20 database. (XLSX) S9 Dataset. Complete set of SPR graphs as.xlsx spreadsheets. (ZIP) S1 Fig. T-Coffee (www.ebi.ac.uk) sequence alignment for GJC3 NP_853516.1 and GJB2 NP_003995.2. Graphical output in Kyte-Doolittle hydrophobicity colour scheme. (PDF) S2 Fig. Multiple sequence alignment for mammalian GJC3 from HomoloGene 15399. Sequences from H. sapiens, Pan trogolodytes, Macaca mulatta, Canis lupus, Bos taurus, M. mus culus, R. norvegicus. (PDF) Surface Plasmon Resonance (SPR) The following chemically synthesized peptides were purchased at >90% purity (Abingdon Health Laboratory Services, UK; HPLC-MS identity and purity analysis). 1 10 20 Peptide A: MCGRFLRRLL AEESRRSTPV GRLLL K(biotin) CONH2 The following chemically synthesized peptides were purchased at >90% purity (Abingdon Health Laboratory Services, UK; HPLC-MS identity and purity analysis). 1 10 20 Peptide A: MCGRFLRRLL AEESRRSTPV GRLLL—K(biotin)-CONH2 Peptide A: MCGRFLRRLL AEESRRSTPV GRLLL—K(biotin)-CONH2 Peptide C: VWQVPAAAAG GGEPALHPRG APLAF—K(biotin)-CONH2 Surface plasmon resonance sensorgrams were recorded on a BioRad ProteOn XPR36 bio- sensor. Biotin labelled peptides A, B, C were immobilized from 1 mg/mL solutions in PBS onto a ProteOn NLC Sensor Chip (BioRad, #176–5021), composed of NeutrAvidin immobi- lized on a modified alginate polymer layer. Exploratory binding experiments were performed using ca. 1500 μM or 400 μM solutions (in PBS buffer) of simvastatin (Cayman Chemicals, Cat#CAYM10010344-100), simvastatin sodium salt (Cayman Chemicals, Cat#CAY10010345- 10 MG), fluvastatin sodium salt (Cayman Chemicals, Cat#CAYM10010337-100), pravastatin sodium salt (Cayman chemicals, Cat#CAYM10010343), niflumic acid (Sigma-Aldrich Cat#N0630), flufenamic acid (Sigma-Aldrich Cat#F9005), carbenoxolone disodium salt (Sigma-Aldrich Cat#C4790), 18α-glycyrrhetinic acid (Sigma-Aldrich Cat#G8503), 18β-glycyr- rhetinic acid (Sigma-Aldrich Cat#G10105), 3-hydroxy-3-methylglutaric acid (Sigma-Aldrich Cat#H4392), 1-heptanol (Sigma-Aldrich Cat#H2805), arachidonylethanolamide (Sigma- Aldrich Cat#A0580), steviol hydrate (Sigma-Aldrich Cat#H8664), and stevioside hydrate (Sigma-Aldrich Cat#S3572). Subsequent titrations were performed in triplicate (simvastatin and fluvastatin hydroxyacid sodium salts, niflumic acid, flufenamic acid and carbenoxolone) or duplicate (simvastatin lactone, pravastatin sodium salt, 3-hydroxy-3-methylglutaric acid, 18β-glycyrrhetinic acid, 18α-glycyrrhetinic acid, arachidonylethanolamide, 1-heptanol, steviol hydrate, and stevioside hydrate) at concentrations of 400 μM, 200 μM, 100 μM, 50 μM, 25 μM 0 μM for each ligand in PBS. Data were analysed at the time using ProteOn Manager™using both Channel and Interspot referencing (the latter was preferred for final analysis and the data presented herein) then exported as.xlsx files for subsequent analysis using Microsoft Excel1. PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 19 / 27 Statin Acids Interact with the N-Terminus of Human GJC3 Protein Supporting Information S1 Alignment. T-Coffee aligned sequences in.afasta format for GJC3 NP_853516.1 and GJB2_NP003995.2. (AFASTA) S1 Dataset. The complete Sanger dideoxy sequencing data for a biopanning screen of sim- vastatin, immobilised from solution to a set 5 photochemistries on a 96-well polystyrene plate, versus a genomic T7 phage display created from samples of human vascular tissue total mRNA. (ZIP) S2 Dataset. Negative controls for sequencing of phage which bind to the same 5 photo- chemistries on a 96-well plate in the absence of simvastatin ligand. (ZIP) S3 Dataset. S6 Dataset. Human GJC3 insertion/deletion polymorphisms from NCBI (19 Dec 2015). (TXT) S6 Dataset. Human GJC3 insertion/deletion polymorphisms from NCBI (19 Dec 2015). (TXT) S7 Dataset. GJC3 multiple sequence alignment for human, mouse and rat proteins showing how an alternative site of translation initiation results in extended N-terminus for mCx29. (RTF) S8 Dataset. Gap junction inhibitors and known targets from ChEMBL_20 database. (XLSX) S9 Dataset. Complete set of SPR graphs as.xlsx spreadsheets. (ZIP) S1 Fig. T-Coffee (www.ebi.ac.uk) sequence alignment for GJC3 NP_853516.1 and GJB2 NP_003995.2. Graphical output in Kyte-Doolittle hydrophobicity colour scheme. (PDF) S2 Fig. Multiple sequence alignment for mammalian GJC3 from HomoloGene 15399. Sequences from H. sapiens, Pan trogolodytes, Macaca mulatta, Canis lupus, Bos taurus, M. mus- culus, R. norvegicus. (PDF) S3 Fig. Chemically synthesized C-terminal K(biotin) labelled peptides. Peptide A in expected reading frame for contig29; peptide B in frame +1 nucleotide, peptide C in frame 20 / 27 PLOS ONE \| DOI:10.1371/journal.pone.0148266 February 10, 2016 Statin Acids Interact with the N-Terminus of Human GJC3 Protein +2 nucleotides. (PDF) S4 Fig. Surface plasmon resonance sensorgrams showing injection onto a neutravidin coated chip pre-equilibrated with phosphate buffered saline (PBS): lane 1 peptide A, lane 2 peptide B, lane 3 peptide C. Chromatograms are corrected to the average values of two refer- ence channels containing PBS alone. Solid black line represents the mean of all channels in the corresponding lane with grey areas representing the standard deviation across channels. (EPS) S5 Fig. Surface plasmon resonance graphs of frame-shifted peptides B and C with statins: (A) simvastatin hydroxyacid, (B) simvastatin lactone, (C) fluvastatin hydroxyacid, and (D) pravastatin hydroxyacid. (TIF) S6 Fig. Surface plasmon resonance graphs of frame-shifted peptides B and C with known connexin inhibitors: (A) niflumic acid, (B) flufenamic acid, (C) carbenoxelone, and (D) 18β-glycyrrhetinic acid. (PDF) S7 Fig. Surface plasmon resonance graphs of peptides A, B, and C with increasing concen- trations of: (A) 3-hydroxy-3-methylglutaric acid, (B) arachidonylethanolamide, (C) hepta- nol, (D) steviol, and (E) stevioside. (PDF) S1 Protocol. Preparation of T7 phage library from human vascular tissue. (DOCX) S1 Summary. Text summary of 30 discovered sequences from T7 phage genomic library screen versus simvastatin on Magic Tag1 plates and BLASTX alignment of NCBI H. sapi- ens RefSeq protein. From K. Casey-Green PhD thesis, University of Warwick 2011. (DOCX) S1 Table. Sanger dideoxy sequencing output for 120 phage clones from simvastatin biopan. (DOCX) S2 Table. Phage clones which align with T7 Select1 vector sequence. (DOCX) S3 Table. S6 Dataset. Human GJC3 insertion/deletion polymorphisms from NCBI (19 Dec 2015). (TXT) Lasergene SeqMan1 analysis of simvastatin biopan sequences to give library con- tiguous sequences (contigs). (DOCX) S4 Table. Simvastatin biopan contigs BLASTX alignment versus NCBI H. sapiens RefSeq protein. (DOCX) S5 Table. Tissue specific expression for gap junction protein, gamma 3, 30.2kDa (GJC3) detected at High or Medium expression level in 54 of 79 analyzed normal tissue cell types using polyclonal antibody HPA015024. Data from Human Protein Atlas using keyword GJC3, http://www.proteinatlas.org/ENSG00000176402-GJC3/tissue accessed 18 May 2015. §RNA-Seq data expressed as number of Fragments Per Kilobase gene model and Million reads (FPKM): Not detected 0–1; Low 1–10; Medium 10–50; High >50. (DOCX) (PDF) S4 Fig. Surface plasmon resonance sensorgrams showing injection onto a neutravidin coated chip pre-equilibrated with phosphate buffered saline (PBS): lane 1 peptide A, lane 2 peptide B, lane 3 peptide C. Chromatograms are corrected to the average values of two refer- ence channels containing PBS alone. Solid black line represents the mean of all channels in the corresponding lane with grey areas representing the standard deviation across channels. (EPS) S5 Fig. Surface plasmon resonance graphs of frame-shifted peptides B and C with statins: (A) simvastatin hydroxyacid, (B) simvastatin lactone, (C) fluvastatin hydroxyacid, and (D) pravastatin hydroxyacid. (TIF) S6 Fig. Surface plasmon resonance graphs of frame-shifted peptides B and C with known connexin inhibitors: (A) niflumic acid, (B) flufenamic acid, (C) carbenoxelone, and (D) 18β-glycyrrhetinic acid. (PDF) S6 Fig. Surface plasmon resonance graphs of frame-shifted peptides B and C with known connexin inhibitors: (A) niflumic acid, (B) flufenamic acid, (C) carbenoxelone, and (D) 18β-glycyrrhetinic acid. (PDF) S7 Fig. Surface plasmon resonance graphs of peptides A, B, and C with increasing concen- trations of: (A) 3-hydroxy-3-methylglutaric acid, (B) arachidonylethanolamide, (C) hepta- nol, (D) steviol, and (E) stevioside. (PDF) S1 Protocol. Preparation of T7 phage library from human vascular tissue. (DOCX) S1 Summary. 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https://openalex.org/W2160900742	https://europepmc.org/articles/pmc4636810?pdf=render	English	null	The direction of cross affects obesity after puberty in male but not female offspring	BMC genomics	2,015	cc-by	11,688	Abstract Background: We investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N). Results: We found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (p = 0.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (p = 0.00198) and the peroxisome pathway were impaired (p = 0.00094). The circadian rhythm was affected as well (p = 0.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, p = 0.022), Cyp4a10 (1.35 fold, p = 0.026) and Cyp4a14 (1.32 fold, p = 0.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, p = 0.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, p = 0.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements. Conclusions: Parent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair β-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (p = 0.074). Keywords: Allele-specific gene expression, Allelic imbalance, High fat diet, Imprinting, Peg3, Circadian rhythm, Sex differences Sex hormones Keywords: Allele-specific gene expression, Allelic imbalance, High fat diet, Imprinting, Peg3, Circadian rhythm, Sex differences, Sex hormones contribution of all detected loci to the variance in a population was small (0.01 % to 0.34 %) [2–6]. There- fore, additional factors could contribute to obesity in an individual. © 2015 Kärst 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. * Correspondence: gudrun.brockmann@agrar.hu-berlin.de †Equal contributors 1Albrecht Daniel Thaer-Institut für Agrar- und Gartenbauwissenschaften, Humboldt-Universität zu Berlin, Invalidenstraße 42, D-10115 Berlin, Germany Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Kärst et al. BMC Genomics (2015) 16:904 DOI 10.1186/s12864-015-2164-2 Kärst et al. BMC Genomics (2015) 16:904 DOI 10.1186/s12864-015-2164-2 Abstract Missing heritability can be hidden in genetic effects of rare alleles, epistatic interaction and gene-by- environment interaction, but can be also contributed to by parent-of-origin specific effects. Parent-of-origin effects include the Y chromosome, mitochondria, parental-specific genetic imprinting, and maternal envir- onmental effects during pregnancy and the suckling period. The direction of cross obesity after puberty in male but not female offspring Stefan Kärst1†, Danny Arends1†, Sebastian Heise1†, Jan Trost1, Marie-Laure Yaspo2, Vyacheslav Amstislavskiy2, Thomas Risch2, Hans Lehrach2 and Gudrun A. Brockmann1* * Correspondence: gudrun.brockmann@agrar.hu-berlin.de †Equal contributors Background Besides nutrition and sedentary lifestyle, genetic predis- position contributes to obesity. To assess the genetic de- terminants of obesity, many large-scale genome-wide association studies were performed in different human populations. These studies led to the identification of about 350 genomic loci contributing to obesity [1]. However, their effects are small and the overall In a mouse model of juvenile obesity, we tested, if parental obesity influences the transmission of Kärst et al. BMC Genomics (2015) 16:904 Page 2 of 14 the German Animal Welfare Authorities (approval no. G016/11). predisposition for adiposity to F1 offspring. We used the Berlin Fat Mouse Inbred line (BFMI) which car- ries a recessive mutation responsible for high fat de- position in particular during early development between four and ten weeks [7]. At ten weeks BFMI males are 5.5 times as obese as C57BL/6Nrl mice (B6N). Phenotyping Body weight and body composition (total lean mass, total fat mass) were recorded weekly from week three and four. Body composition was measured using EchoMRI whole-body composition analyzer (Echo Med- ical Systems, Houston, TX, USA). At ten weeks blood glucose levels were measured prior to dissection after 2 hours of fasting using a glucometer (Contour, Bayer, Leverkusen, Germany). Liver triglycerides and proteins were measured in homogenized liver samples by commercially available kits (DC Protein Assay, Bio-Rad, München, Germany; TRIGS Triglycerides Assay, Randox, Crumlin, United Kingdom). For test statistics between the matBFMI and patBFMI, we applied a linear model using anova in R. Since litter size had a significant effect on all traits, phenotypic values were adjusted for litter size before statistic tests were performed, except for blood glucose and liver triglycerides. In reciprocal crosses between BFMI and B6N mice we tested the development of obesity in F1 males and females and their response to the obese parent with respect to gene expression in the liver. The results provide evidence for regulation of genes by the influ- ence of the direction-of-cross, which leads to obesity and fatty liver exclusively in male offspring of obese BFMI mothers. RNA preparation and sequencing RNA sequencing was performed on 10 liver samples: 1 male and 1 female B6N mouse, 1 male and 1 female BFMI mouse, 3 F1 (BFMI x B6N) males (patBFMI), and 3 F1 (B6N x BFMI) males (matBFMI). The F1 males were of non-cross-fostered mice. Tissue samples were frozen in liquid nitrogen and stored at –80 °C until RNA preparation. RNA was prepared using the RiboZero RNA library protocol. Paired end RNA sequencing was run on an Illumina HiSeq2000 machine. Methods Animals Reciprocal crosses were generated from the Berlin Fat Mouse Inbred line BFMI860-12/Hber (BFMI) and C57BL/6NCrl (B6N). Seven BFMI males were mated with seven B6N females and six B6N males with six BFMI860 females to generate 48 F1 animals (deemed patBFMI and matBFMI, respectively). Each reciprocal F1 offspring groups consisted of 12 males and 12 fe- males. From these animals three F1 males (matBFMI) from three BFMI females and three F1 males (patBFMI) from three B6N females were used for RNA-seq transcriptome measurements. One high fat diet raised male and female from each inbred parental strain (BFMI and B6N) were used as parental strain control animals and measured via RNA-seq as well. For the cross-fostering experiment, six BFMIxB6N and six B6NxBFMI crosses generated two additional groups of reciprocal F1 animals with a total of 88 an- imals, 46 with a BFMI father (23 males, 23 females) and 42 with a BFMI mother (24 males, 18 females). For cross-fostering, litters were exchanged between B6N and BFMI mothers within 12 hours after birth. The litter size ranged between four and eleven off- spring. Two animals per cross-fostering group were excluded due to outlier testing (R-package “outliers”). Parents were fed a standard breeding diet (SBD). F1-animals were fed a high fat diet (HFD) from wean- ing at 21 days until 70 days when offspring was euthanized and dissected. The SBD contained 16.7 MJ/kg of metabolisable energy, 11 % from fat, 36 % from proteins and 53 % from carbohydrates (V1534- 000, ssniff EF R/M, Ssniff GmbH, Soest/Germany). The HFD contained 19.5 MJ/kg of metabolisable energy, 45 % from fat, 24 % from proteins and 31 % from carbohydrates (E15103–34, ssniff EF R/M, Ssniff GmbH, Soest/Germany). All experimental treatments of animals were approved by Analysis of RNA-Seq data After sequencing, bcl2fastq v1.8.4 [8] was used for base calling and demultiplexing. Paired end reads were trimmed using Trimmomatic [9] using standard settings. TruSeq3-PE adapter sequences provided by Illumina were used for adapter trimming. Reads were aligned against Mus musculus GRCm38 reference genome (Ensembl) using BWA [10], after which GATK [11] was applied to do base quality score recalibration, indel realignment, du- plicate removal, SNP/INDEL discovery and genotyping across all 10 samples using standard hard filtering param- eters or variant quality score recalibration according to GATK Best Practices recommendations [12]. Raw reads from RNA-Seq data were extracted from BAM files using the GenomicFeatures package using the Mus_musculus.GRCm38.81.gtf file obtained from ensembl, overlap between genes and raw reads was calculated in ‘union’ mode using the Rsamtools package [13]. Reads were quantile normalized between samples using the pre- processCore package. After sample quantile normalization, RPKM values for each gene were calculated, which were Kärst et al. BMC Genomics (2015) 16:904 Page 3 of 14 SNP was removed due to the fact that the data was not consistent across the individuals. then log transformed ( ln(n + 1) ) to reduce the effect of ex- treme RPKM values in subsequent statistical tests. The bio- maRt R package was used to provide additional gene annotation such as MGI descriptions (using the M. muscu- lus ensembl database GRCm38.p4). After filtering for high quality SNPs which are homozy- gous (but different) between the parental strains, χ 2 scores were calculated for each individual. χ2 scores were adjusted to account for direction of the effect (< 50 % vs. > 50 %) and scores were square root transformed to minimize the impact of a single F1 individual (in the F1 groups) showing a highly significant deviation from the expected 50:50 ratio. Summed scores across all individuals in a F1 cross direction (matBFMI and patBFMI) were calculated to obtain a group based χ2 score, which were then subtracted from each other to get a group difference score per SNP. To account for over dispersion in the read counts obtained from the DP4 values, we permuted our group based χ2 scores to obtain a 5 % FDR threshold for SNPs that show significant ASE in either the patBFMI or the matBFMI group. We analyzed the gene expression of the reciprocal F1 populations with respect to differentially expressed genes, pathways, and allele specific expression. Analysis of transcription factor binding sites Differentially expressed genes (p < 0.05) were submitted to transcription factor binding site (TFBS) analysis. For each gene we extracted the upstream (2000 bp) and down- stream (1000 bp) region around the transcription start using the TxDb.Mmusculus.UCSC.mm10 package from bioconductor [17]. All known mouse TFBS motifs were downloaded from the Jaspar database into R using the MotifDb package [18, 19]. Each gene region of 3000 basepairs was scanned for all estrogen receptor related motifs in the Jasper database using the Biostrings package [20], for each motif we recorded the number of matches within the 3000 bp region (Mmusculus-jolma2013-Esrra-2 -UniPROBE-Esrra. UP00079, -JASPAR_CORE-Esrrb-MA0141.1, -JASPAR_ 2014-Esrrb-MA0141.2). Additionally, position weight matrices for estrogen receptor alpha (ER), estrogen response element (ERE) and the binding site for gluco- corticoid receptor (GR)[21, 22] were applied as well. Analysis of RNA-Seq data Testing for differences between the two groups of reciprocal crosses was done using two sided t-tests. Due to limited sample sizes of three animals per F1 group, adjustment for multiple testing was not applied for differential gene expression analysis. The original transcriptome sequen- cing data for all ten animals tested are available at the GEO database (http://www.ncbi.nlm.nih.gov/geo/). Analysis of allele specific expression (ASE) To assess ASE in the reciprocal F1 crosses, bcftools was used to perform SNPs calling on the entire population (all 10 animals: 2 BFMI, 2 B6N, 3 matBFMI, 3 patBFMI). De- tected SNPs underwent a rigorous data quality checks to make sure detected SNPs showed sufficient coverage in the entire population. The individuals from the BFMI and B6N pure lines were analyzed to detect homozygous SNPs that showed different alleles between B6N and BFMI. We demanded that all SNPs found in the four parental strain animals had non-missing data (neither low confidence nor low coverage). SNPs were called using the samtools mpi- leup tool. Subsequently, the generated bcf file was proc- essed using BCFtools [16] and filtered using the provided vcfutils.pl script for SNPs with a 100 reads minimum for the entire population (over all 10 animals). After using the entire population for SNP calling, we reanalyzed all SNPs per individual using the samtools mpileup tool to obtain DP4 measurements for each F1 individual. SNPs with low confidence (Phred probability threshold < 50) or less than 5 high quality reads in an individual were excluded from fur- ther analyses. Allele ratios alt/(ref + alt) were calculated for all remaining SNPs. Additionally, we filtered for consistency between the different individuals from the same F1 cross direction. If the standard deviation of the alt/(ref + alt) ra- tios across the 3 individuals per F1 group was > 20 %, the Since only one weight matrices (jolma2013-Ar) was available in the Jasper database for androgen response elements (AREs) in mouse, we created position weight matrices for androgen response elements using the motifs from Shaffer et al. for ARE, ARE2, ADR3, IR3 [23]. We used a conservative threshold of 90 % for sensitivity and specificity. A list of the applied pos- ition weight matrices is given in Additional file 1: Table S3. Additionally, we compared the occurrence of TFBS in both differentially and allele specific expressed genes with 1,000 permutations of random sets of genes of the respective same group size. Pathway analyses Genes that were differentially expressed (p < 0.05) between the two groups of F1 males with RPKM values > 0.5 in at least one of the F1 groups and gene expression differences of > = 20 % were subjected to overrepresentation analyses in KEGG pathways using innateDB [14] to identify path- ways associated with these genes [15]. We used the default hypergeometric algorithm and the Benjamini Hochberg algorithm for multiple testing corrections. Heatmaps were created for selected pathways using the mean gene expres- sion per group. Results and discussion Phenotypes Since BFMI animals carry a major recessive mutation lead- ing to juvenile obesity in homozygous carriers [27], we ex- pected lean F1-offspring in either cross direction. However, data provided evidence for the development of obesity in F1 males, if the mother originates from the obese BFMI line (matBFMI), while females of the same cross direction and both sexes of the reciprocal cross had normal body weight and fat mass (Table 1). Body mass did not differ signifi- cantly between offspring of the reciprocal crosses, but F1 males from BFMI mothers (matBFMI) had 1.83 times more adipose tissue mass at 10 weeks (p = 0.0037) than F1 males from BFMI fathers (patBFMI). Since the lean mass was only marginally lower in the obese matBFMI versus the lean patBFMI F1 males (0.98 fold, p = 0.054), the fat to lean mass ratio was also 1.86 times higher in matBFMI males (p = 0.0009). Moreover, matBFMI also showed ectopic fat storage in the liver with fat content being 1.30 times higher than in patBFMI males from the reciprocal cross (p = 0.084). The higher fat mass in matBFMI males began at puberty and was significant from seven weeks on (Fig. 1). A cross-fostering experiment was performed to assess the influence of postnatal maternal effects such as milk energy content and behavior during the suckling period on later development of fat deposition in offspring. This Table 1 Phenotypes of 10 weeks old males and females of reciprocal crosses between BFMI and B6N with and without cross-fostering on high fat diet Phenotypes1 F1(BFMI x B6N) F1(B6N x BFMI) p-Value for direction- of-cross effect BFMI father (patBFMI) BFMI mother (matBFMI) Males Females Males Females Males Females Maternal fostering Body mass (g) 34.23 (3.26) 26.16 (2.02) 36.82 (3.96) 25.27 (2.06) n.s. n.s. Fat mass (g) 3.86 (1.49) 3.34 (1.01) 7.06 (2.56) 3.44 (1.04) 0.0037 n.s. Lean mass (g) 27.54 (2.06) 20.45 (1.00) 26.93 (2.10) 19.68 (1.09) 0.0536 0.0855 Fat / Lean mass ratio 0.14 (0.05) 0.16 (0.04) 0.26 (0.08) 0.17 (0.05) 0.0009 n.s. Liver mass (g) 1.55 (0.23) 1.21 (0.10) 1.68 (0.18) 1.16 (0.12) n.s. n.s. Liver triglycerides/ protein (μg/μg) 0.45 (0.09) 0.47 (0.11) 0.59 (0.22) 0.45 (0.08) 0.0843 n.s. Blood glucose levels (mg/dl) 165.33 (27.20) 135.92 (12.22) 174.25 (23.80) 133.25 (10.71) n.s. n.s. Cross-fostering Body mass (g) 34.27 (3.41) 25.96 (3.75) 35.63 (2.65) 24.36 (1.71) n.s. n.s. Quantitative PCR RNA from ten male matBFMI and ten male patBFMI was extracted from liver tissue. Total RNA was isolated Kärst et al. BMC Genomics (2015) 16:904 Page 4 of 14 Page 4 of 14 Page 4 of 14 Kärst et al. BMC Genomics (2015) 16:904 from liver tissue using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany). One μg of total RNA was reverse-transcribed into cDNA using Accu- Script High Fidelity Transcriptase (Stratagene Europe, Amsterdam, Netherlands) and Oligo (dT) primers (Ark Scientific, Sigma-Aldrich, München, Germany). The cDNA integrity was checked in a polymerase chain reaction (PCR). For semi-quantitative real-time PCR 20 ng cDNA was mixed with the qPCR master- mix of the Plus Kit for SYBR Green (Eurogentec, Co- logne, Germany) and 300 nM of the primers at a final volume of 10 μl per reaction. The amplification protocol included a 10 min DNA denaturation step at 95 °C, followed by 40 cycles consisting of 20 s at 60 °C and 40 s at 72 °C. Quantification of mRNA transcript amounts was performed using the Applied Biosystems® ViiA™ 7 Real-Time PCR System and ViiA™Software v.1.2.1. Primers for target genes Ar, Peg3 and Srebf1 were designed using Primer3web [24]. Sequences for reference gene primers (Actb, Mrpl46, Srp72) were taken from Hruz et al. [25]. The geometric mean of the three reference genes was used to normalize target gene expression. Relative quantifi- cation of gene expression was performed according to ABI's user guide [26]. Further details are provided in Additional file 2: Table S4. dard deviation after correction for litter size, except for blood glucose and liver triglycerides, which were not affected by the litter size. cate significant effects of cross fostering on phenotypes comparing animals of the same direction of cross (patBFMI vs. cross-fostered Results and discussion Phenotypes Fat mass (g) 4.06 (2.06) 3.45 (1.56) 5.54 (1.74) * 2.70 (0.99) 0.0142 n.s. Lean mass (g) 29.54 (1.84) 21.69 (3.06) 29.61 (1.68) 20.64 (1.19) ° n.s. n.s. Fat / Lean mass ratio 0.14 (0.06) 0.16 (0.07) 0.19 (0.06) 0.13 (0.04) ° 0.0067 n.s. Liver mass (g) 1.66 (0.21) 1.22 (0.23) 1.75 (0.15) 1.17 (0.13) n.s. n.s. Liver triglycerides/ protein (μg/μg) 0.15 (0.09) 0.34 (0.17)° 0.21 (0.08) 0.26 (0.13) 0.0307 n.s. Blood glucose levels (mg/dl) 137.82 (19.07) ** 130.36 (13.13) 151.83 (22.7) * 139.29 (20.64) 0.0307 n.s. 1shown are means ± standard deviation after correction for litter size, except for blood glucose and liver triglycerides, which were not affected by the litter size. Asterisks additionally indicate significant effects of cross fostering on phenotypes comparing animals of the same direction of cross (patBFMI vs. cross-fostered patBFMI, matBFMI vs. cross-fostered matBFMI) (*p < 0.01, p < 0.05, °p < 0.1) 0 weeks old males and females of reciprocal crosses between BFMI and B6N with and without cross-fostering Kärst et al. BMC Genomics (2015) 16:904 Page 5 of 14 Fig. 1 Development of phenotypes of males and females on a high fat diet of the two reciprocal F1-crosses until 10 weeks. The figures show means ± standard deviations of the total fat mass and the total fat mass-to-total-lean mass-ratios in F1-animals without (a and b, respectively) and with cross-fostering (c and d, respectively). Paternal and maternal BFMI (pat, mat) indicates direction of cross. Asterisks indicate significant differences between different reciprocal crosses for males (blue) and females (red) and significant differences within the same reciprocal cross between males and females of matBFMI (orange) and patBFMI (grey) (*p < 0.001, p < 0.01, p < 0.05) Fig. 1 Development of phenotypes of males and females on a high fat diet of the two reciprocal F1-crosses until 10 weeks. The figures show means ± standard deviations of the total fat mass and the total fat mass-to-total-lean mass-ratios in F1-animals without (a and b, respectively) and with cross-fostering (c and d, respectively). Paternal and maternal BFMI (pat, mat) indicates direction of cross. Results and discussion Phenotypes Asterisks indicate significant differences between different reciprocal crosses for males (blue) and females (red) and significant differences within the same reciprocal cross between males and females of matBFMI (orange) and patBFMI (grey) (p < 0.001, p < 0.01, p < 0.05) experiment validated the results obtained before, but fat deposition was a little lower (Fig. 1). Cross-fostering led to a reduction of adipose tissue mass in male and female F1 offspring of BFMI mothers by approximately 22 % if they were raised by lean B6N foster mothers. Cross- fostered F1 males of BFMI mothers still developed 1.36 times higher fat mass (p = 0.0142), had a 1.36 times higher fat to lean ratio (p = 0.0067), 1.40 times higher hepatic tri- glyceride levels (p = 0.03070) and higher blood glucose levels (1.10 fold, p = 0.03073) than cross-fostered F1 males of the reciprocal cross (Table 2). These data support an ef- fect of the suckling period on later obesity and diabetes development that has been shown before [28–30]. expressed between the two groups of reciprocal F1 males at p < 0.05 and a fold-change difference of at least 20 % provided evidence for alterations of pathways for lipid me- tabolism, peroxisome, circadian rhythm, cellular transport processes and the endocrine system as a results of the direction of cross (Table 2, Table 3). Most genes with higher expression in the obese F1 males control the cellular utilization of fatty acids for energy production or fat synthesis including the hydrolysis of triglycer- ides into free fatty acids and glycerol, the conversion of free fatty acids into fatty acyl-CoA esters, the bio- synthesis of saturated fatty acids or the degradation and conversion into acetyl-CoA by β-oxidation [31] (Fig. 2). But also genes for the transport between per- oxisome and mitochondria via carnitine [32, 33] and the electron carrier generation by the citrate cycle and the respiratory chain in mitochondria were differ- entially expressed. Differential gene expression between F1 males of BFMI mothers and BFMI fathers Since lipid accumulation of F1 males differed between the reciprocal crosses, we investigated the direction-of-cross effect on hepatic gene expression profiles in males. Over- representation analyses of genes that were differentially The metabolic changes in obese males were associated with a shift of the circadian rhythm, in which the gene Page 6 of 14 Kärst et al. BMC Genomics (2015) 16:904 Table 2 KEGG pathways affected by the direction of cross in F1 males (p < 0.05) KEGG ID Major category/Subcategory Pathway Name Proportion of genes in pathway Identified genes1 fold-change (matBFMI vs. Differential gene expression between F1 males of BFMI mothers and BFMI fathers BMC Genomics (2015) 16:904 Table 2 KEGG pathways affected by the direction of cross in F1 males (p < 0.05) (Continued) Table 2 KEGG pathways affected by the direction of cross in F1 males (p < 0.05) (Continued) Linoleic acid metabolism Esrra, Esrrb Cyp2c39 (1.29) Esrra, Esrrb 04640 Organismal systems/Immune system Hematopoietic cell lineage 2 % Dntt (1.24) - 0.04814 Il1r1 (0.74) - 1P-value for differential gene expression between reciprocal F1 groups of males was p < 0.05, minimum fold-change difference >20 %, The innateDB tool was used for the KEGG pathway analysis. matBFMI – BFMI mother, patBFMI – BFMI father 2Steroid hormone transcription factor binding sites. Esrra/b – estrogen related receptor a/b, ER – estrogen receptor alpha, ERE – estrogen response element, IR3 - androgen inverted repeats of hexameric half-site separated by 3 bp of spacer 1P-value for differential gene expression between reciprocal F1 groups of males was p < 0.05, minimum fold-change difference >20 %, The innateDB tool was used for the KEGG pathway analysis. matBFMI – BFMI mother, patBFMI – BFMI father 2Steroid hormone transcription factor binding sites. Esrra/b – estrogen related receptor a/b, ER – estrogen receptor alpha, ERE – estrogen response element, IR3 - androgen inverted repeats of hexameric half-site separated by 3 bp of spacer Arntl (0.54 fold, p = 0.01), was down- and Per2 (1.32 fold, p =0.02) and Per3 (1.71 fold, p = 0.03) were up- regulated (Table 2). Disruptions of the normal circadian rhythm have been linked to obesity in Clock [34] and Arntl (alias Bmal1) knock-out mice and feeding experi- ments [35]. A shift of the circadian rhythm in offspring as a result of in utero exposure to maternal obesity has been shown in rats [36]. alleles of Srebf1 were found in humans to promote hepatic fibrogenesis [43], which can develop from nonalcoholic fatty liver disease. Consistently with these differences in expression, the hepatic lipase (Lipc) that degrades triglycerides was by trend down- regulated in obese F1 males (0.96 fold, p = 0.01). In a situation, in which fatty acids are preferentially synthesized and accumulated, while their β-oxidation is impaired, the cell has to increase energy generation from alternative fuels. Previous measurements of the respira- tory quotient of the BFMI inbred mice provided evi- dence for a higher use of carbohydrates as the main energy fuel [44]. Differential gene expression between F1 males of BFMI mothers and BFMI fathers Succinyl-CoA, for example, is a citrate cycle intermediate that can be produced from degrad- ation of certain amino acids but also from the propano- ate pathway. The moderate up-regulation of Mgll (1.17 fold, p = 0.01), Aldh3a2 (1.11 fold, p = 0.01) and Dak (1.09 fold, p = 0.02) from the glycerolipid pathway aims at the conversion of triglycerides to precursor substrates for subsequent glycolysis. Increased carbohydrate metab- olism is further supported by up-regulation of the carbo- hydrate transporters Slc16a7 (1.20 fold, p = 0.0007) and Slc16a12 (1.13 fold, p = 0.02) in matBFMI (Table 3, Additional file 3: Table S1). Indications for impaired β-oxidation in the obese males were found from up-regulated expression of cytoplasmic acetyl-CoA carboxylase (Acaca, 1.20 fold, p < 0.06) and cit- rate lyase (Acly, fold 1.11, p < 0.08), which usually inhibits the cytosol-mitochondria transport of the fatty acid de- rived substrates via allosteric inhibition of Crat (1.27 fold, p = 0.01) by malonyl-CoA [37–39]. Furthermore, acyl- CoA thioesterases (Acot) are transporters that regulate the amounts of intermediates from fatty acid oxidation in- side mitochondria. The mitochondrial matrix bound Acot2 (1.62 fold, p = 0.05), for example, enhances hepatic fatty acid oxidation by its transporter activity to prevent substrate overload [40], which otherwise could impair β-oxidation [41]. In obese F1 males also Acot4 (1.82 fold, p < 0.02) and Acot3 (6.49 fold, p < 0.08) were up- regulated, which could indicate high amounts of such intermediates inside the mitochondrial matrix, leading to impaired fatty acid oxidation and increased levels of free fatty acids. In humans, Slc16a7 catalyzes the rapid transport of lactate, pyruvate, branched-chain oxo acids and other substrates across the plasma membrane of mitochondria and is regulated by estradiol [45, 46]. This regulation could contribute to the fact that female F1 offspring of the BFMI mother are protected against elevated hepatic triglyceride storage. Genes encoding peroxisome proliferator-activated re- ceptors (Ppara, Pparg, Ppargc1a, Ppargc1b) that promote β-oxidation and inhibit triglyceride synthesis were not differentially expressed between F1 males of reciprocal crosses. In contrast, the opponent sterol regulatory element–binding factor 1 (Srebf1) which increases fatty acid and triglyceride synthesis [42] was 1.17 fold higher expressed (p = 0.03) in the obese F1 males. Differential gene expression between F1 males of BFMI mothers and BFMI fathers patBFMI) TFBS 2 Corrected P-Value for pathway ORA 04710 Organismal Systems/ Environmental adaptation Circadian rhythm 10 % Arntl (0.54) - 0.00087 Per2 (1.32) - Per3 (1.71) - 04146 Cellular Processes/Transport and catabolism Peroxisome 5 % Abcd1 (1.22) - 0.00094 Abcd2 (1.52) - Acsl3 (1.22) - Crat (1.27) - 00830 Metabolism/Metabolism of cofactors and vitamins Retinol metabolism 5 % Cyp2c38 (1.61) Esrra, Esrrb 0.00113 Cyp2c39 (1.29) Esrra, Esrrb Cyp4a10 (1.35) ERE, IR3 Cyp4a14 (1.32) - 00590 Metabolism/Lipid metabolism Arachidonic acid metabolism 4 % Cyp2c38 (1.61) Esrra, Esrrb 0.00099 Cyp2c39 (1.29) Esrra, Esrrb Cyp4a10 (1.35) ERE, IR3 Cyp4a14 (1.32) - 00071 Metabolism/Lipid metabolism Fatty acid degradation 6 % Acsl3 (1.22) - 0.00198 Cyp4a10 (1.35) ERE, IR3 Cyp4a14 (1.32) - 00061 Metabolism/Lipid metabolism Fatty acid biosynthesis 14 % Acsl3 (1.22) - 0.00282 Fasn (1.22) ERE 03320 Organismal Systems/ Endocrine system PPAR signaling pathway 4 % Acsl3 (1.22) - 0.00617 Cyp4a10 (1.35) ERE, IR3 Cyp4a14 (1.32) - 00062 Metabolism/ Lipid metabolism Fatty acid elongation 8 % Acot4 (1.82) - 0.00627 Elovl6 (1.32) - 00140 Metabolism/Lipid metabolism Steroid hormone biosynthesis 4 % Cyp2c38 (1.61) Esrra, Esrrb 0.00636 Cyp2c39 (1.29) Esrra, Esrrb Cyp7b1 (0.83) - 01040 Metabolism/ Lipid metabolism Biosynthesis of unsaturated fatty acids 8 % Acot4 (1.82) - 0.00635 Elovl6 (1.32) - 04270 Organismal Systems/Circulatory system Vascular smooth muscle contraction 2 % Avpr1a (0.77) - 0.01549 Cyp4a10 (1.35) ERE, IR3 Cyp4a14 (1.32) - Table 2 KEGG pathways affected by the direction of cross in F1 males (p < 0.05) 00071 Metabolism/Lipid metabolism Fatty acid degradation 6 % 00061 Metabolism/Lipid metabolism Fatty acid biosynthesis 14 % 03320 Organismal Systems/ Endocrine system PPAR signaling pathway 4 % 00062 Metabolism/ Lipid metabolism Fatty acid elongation 8 % 00140 Metabolism/Lipid metabolism Steroid hormone biosynthesis 4 % 01040 Metabolism/ Lipid metabolism Biosynthesis of unsaturated fatty acids 8 % 04270 Organismal Systems/Circulatory system Vascular smooth muscle contraction 2 % 02010 Environmental information processing/Membrane transport ABC transporters 4 % 00591 Metabolism/ Lipid metabolism 4 % Page 7 of 14 Kärst et al. Differential gene expression between F1 males of BFMI mothers and BFMI fathers Many Srebf1 target genes were increased as well (Ar: 2.38 fold, p = 0.07; Elovl6: 1.32 fold, p = 0.02; Fads1: 1.07 fold, p = 0.02; Fasn: 1.21 fold, p = 0.01; Gpam: 1.13 fold, p = 0.02; Hmgcr: 1.32 fold, p = 0.003; Hsd17b7: 1.14 fold, p = 0.05; Ldlr: 1.10 fold, p = 0.005; Me1: 1.16 fold, p = 0.01; Pgd: 1.15 fold, p = 0.04). Certain Among the highest up-regulated genes in matBFMI were the cytochrome P450, family 4, subfamily A mem- bers Cyp4a10 (1.35 fold, p = 0.03) and Cyp4a14 (1.32 fold, p = 0.01), which together with an increase of their rate limiting regulator Por (1.16 fold, p = 0.06) is an indi- cator for increased ω-oxidation [47], a prerequisite for subsequent peroxisomal β-oxidation. These genes were found to play a part in the development of liver steatosis [48, 49]. The cytochrome P450 proteins are involved in the synthesis of cholesterol, steroids, and other lipids. Page 8 of 14 Kärst et al. BMC Genomics (2015) 16:904 Table 3 Genes highly differentially expressed between the reciprocal F1 males (p < 0.05, fch > 20 %) Gene Symbol1 Chr Gene description Mean RPKM2 Fch matBFMI/ t Test P- value TFBS3 patBFMI matBFMI patBFMI Slc16a7 10 solute carrier family 16 (monocarboxylic acid transp.), 7 1.91 2.30 1.20 0.0007 Esrrb Nlrp12 7 NLR family, pyrin domain containing 12 2.66 2.19 0.82 0.0009 ERE Il1r1 1 interleukin 1 receptor, type I 2.16 1.60 0.74 0.0019 - St5 7 suppression of tumorigenicity 5 1.97 1.61 0.82 0.0029 - Hmgcr 13 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 3.01 3.98 1.32 0.0034 - Scara5 14 scavenger receptor class A, member 5 (putative) 1.23 0.55 0.44 0.0050 - Gpr110 17 G protein-coupled receptor 110 1.48 0.85 0.57 0.0092 - Grem2 1 gremlin 2 homolog, cysteine knot superfamily 1.50 1.11 0.74 0.0102 - Fasn 11 fatty acid synthase 4.47 5.41 1.21 0.0110 ERE Selenbp2 3 selenium binding protein 2 5.72 4.76 0.83 0.0113 - Cyp4a14 4 cytochrome P450, family 4, subfam. a, polypept. 14 3.58 4.74 1.32 0.0119 - Cyp2c38 19 cytochrome P450, family 2, subfamily c, polypet. 38 1.46 2.33 1.60 0.0122 Esrra, Esrrb Crat 2 carnitine acetyltransferase 1.79 2.27 1.26 0.0128 - Coq10b 1 coenzyme Q10 homolog B (S. Differential gene expression between F1 males of BFMI mothers and BFMI fathers cerevisiae) 1.42 1.98 1.39 0.0138 - Rai14 15 retinoic acid induced 14 1.98 1.64 0.83 0.0144 - Mup-ps20 4 major urinary protein, pseudogene 20 1.46 0.51 0.35 0.0145 - Arntl 7 aryl hydrocarbon receptor nuclear translocator-like 1.90 1.05 0.55 0.0151 - Acnat2 4 acyl-coenzyme A amino acid N-acyltransferase 2 2.37 3.22 1.36 0.0155 Esrrb, IR3 Abcd2 15 ATP-binding cassette, sub-family D (ALD), 2 1.14 1.72 1.51 0.0157 - Cyp2c39 19 cytochrome P450, family 2, subfamily c, polypept.39 2.77 3.57 1.29 0.0157 Esrra, Esrrb Slc45a3 1 solute carrier family 45, member 3 2.15 1.72 0.80 0.0166 - Sntg2 12 syntrophin, gamma 2 1.75 1.45 0.83 0.0187 - Per2 1 period circadian clock 2 1.26 1.67 1.32 0.0208 - Hcn3 3 hyperpolarization-activated, cyclic nucleotide-gated 0.95 1.51 1.60 0.0208 - Gas6 8 growth arrest specific 6 2.26 2.73 1.21 0.0209 - Serpina12 12 ser/cys peptidase inhibitor, member 12 2.40 1.94 0.81 0.0211 - Elovl6 3 ELOVL family member 6, elongation of LCFA 3.20 4.17 1.30 0.0214 - Acsl3 1 acyl-CoA synthetase long-chain family member 3 2.24 2.71 1.21 0.0215 - Acot4 12 acyl-CoA thioesterase 4 0.93 1.66 1.79 0.0219 - Slc20a1 2 solute carrier family 20, member 1 1.54 1.87 1.21 0.0219 - B3galt1 2 UDP beta 1,3-galactosyltransferase, polypept.1 1.52 1.07 0.70 0.0230 - Cyp7b1 3 cytochrome P450, family 7, subfamily b, polypept. 1 5.11 4.23 0.83 0.0257 - Cyp4a10 4 cytochrome P450, family 4, subfamily a, polypept.10 3.78 5.02 1.33 0.0260 ERE, IR3 1Bold gene symbols indicate up regulation in F1 males of a BFMI mother. A list of all differentially expressed genes (p < 0.1) is given in Additional file 1: Table S1. Chr – chromosome, matBFMI – BFMI mother, patBFMI – BFMI father, Fch – fold-changes 2All values were log-transformed (ln) 3TFBS - Transcription factor binding sites for steroid hormones. Esrrb – estrogen related receptor b, ERE – estrogen response element, IR3 – androgen inverted repeats of hexameric half-site separated by 3 bp of spacer Bold gene symbols indicate up regulation in F1 males of a BFMI mother. A list of all differentially expressed genes (p < 0.1) is given in Additional file 1: Table S1. Chr – chromosome, matBFMI – BFMI mother, patBFMI – BFMI father, Fch – fold-changes 2All values were log-transformed (ln) 3TFBS - Transcription factor binding sites for steroid hormones. Differential gene expression between F1 males of BFMI mothers and BFMI fathers Differentially expressed X chromosomal genes that are involved in the lipid metabolism are ATP-binding cassette, sub-family D (ALD), member 1 (Abcd1: 1.22 fold, p = 0.04), HAUS augmin-like complex, subunit 7 (Haus7: 1.21 fold, p = 0.05), NAD(P) dependent steroid dehydrogenase-like (Nsdhl: 1.10 fold, p = 0.03), monocarboxylic acid transporter (Slc16a2) and glycerol kinase (Gyk) (Table 4, Additional file 3: Table S1). Zfx is well studies in cancer as a proliferation factor; it is also involved in animal size and fertility and lower gene expression was found to inhibit cell growth [57, 58]. Dysfunctional Pqbp1 was associated to lipid metabolism in C. elegans. Repression of this gene led to reduction of lipid content in mammalian primary white adipocytes and also triglyceride synthesis from fatty acids was impaired [59]. Since no differences in expression were found for genes on the Y-chromosome, we provide further evidence that the Y chromosome can most likely ruled out as cause for differences in obesity of males. In the class of mitochondrial genes we found mitochondrial NADH de- hydrogenase 1 (mt-Nd1) expressed at a lower level (0.95 fold, p = 0.02) in obese matBFMI F1 males (Table 4). contribute to the sex specific obesity effect. The andro- gen receptor affects the liver metabolism differently in males and females [54]. Besides the liver effects, the androgen receptor protects females against high fat diet-induced obesity and dyslipidemia [55]. Additional studies showed that mice lacking the androgen receptor develop hepatic steatosis and insulin resistance in males but not females under high fat diet [56]. Among the genes that were highly differentially expressed between the two groups of F1 males (p < 0.05, fold-change difference min. 20 %, Table 3), six and two genes contain binding sites for estrogen (Acnat2, Cyp2c38, Cyp2c39, Cyp4a10, Fasn, Nlrp12) and andro- gen (Acnat2, Cyp4a10), respectively. Considering all dif- ferentially expressed genes under p < 0.05 estrogen (ER), androgen (AR) or glucocorticoid response (GR) elements occur in 220 up- (55 ER, 14 AR, 3 GR) and 189 down- regulated (30 ER, 11 AR, 6 GR) genes in the obese ver- sus lean F1 males (Additional file 3: Table S1). We compared the occurrence of these transcription factor binding sites in genes being differentially expressed (p < 0.05, >20 % fold-change difference) be- tween the obese and the non-obese F1 males with ran- domly selected genes. Differential gene expression between F1 males of BFMI mothers and BFMI fathers Esrrb – estrogen related receptor b, ERE – estrogen response element, IR3 – androgen inverted repeats of hexameric half-site separated by 3 bp of spacer 2All values were log-transformed (ln) 3TFBS - Transcription factor binding sites for steroid hormones. Esrrb – estrogen related receptor b, ERE – estrogen response element, IR3 – androgen inverted repeats of hexameric half-site separated by 3 bp of spacer The proteins encoded by Cyp4a10 and Cyp4a14 hydro- xylize fatty acids [50, 51]. Recently, fatty acid-hydroxy fatty acids (FAHFAs) have been discovered as a class of lipids with anti-diabetic and anti-inflammatory effects [52]. Higher concentrations of FAHFAs in the obese F1 males due to increased expression of the two Cyp4A genes would be consistent with similar blood glucose levels in male F1 offspring of both cross directions (Table 1). The hepatic expression of the androgen receptor (Ar) gene was higher in the obese matBFMI F1 males com- pared to the lean counterparts (2.38 fold, p = 0.07). The androgen receptor functions as a steroid-hormone acti- vated transcription factor [53]. Therefore, Ar could Kärst et al. BMC Genomics (2015) 16:904 Page 9 of 14 Fig. 2 Differential gene expression comparing F1 males from reciprocal crosses and parental strains. Heatmap of genes that were differentially expressed between the reciprocal crosses (p < 0.05) and parental strain gene expression for KEGG pathways of a) biosynthesis of unsaturated fatty acids and b) fatty acid degradation Fig. 2 Differential gene expression comparing F1 males from reciprocal crosses and parental strains. Heatmap of genes that were differentially expressed between the reciprocal crosses (p < 0.05) and parental strain gene expression for KEGG pathways of a) biosynthesis of unsaturated fatty acids and b) fatty acid degradation Gene expression of hemizygous genetic elements Differential gene expression on the X and Y chromo- some and mitochondria was examined in particular be- cause these homozygous genomic units can be polymorphic between the reciprocal crosses. Among them are the androgen receptor (Ar: 2.38 fold, p = 0.07), zinc finger protein X-linked (Zfx: 0.84 fold, p = 0.09) and polyglutamine binding protein 1 (Pqbp1: 1.07 fold, p < 0.1), which act as transcription factors. Differential gene expression between F1 males of BFMI mothers and BFMI fathers We found that binding motifs for estrogen seemed to be under-represented in down- regulated genes (p = 0.074). However, when relaxing our restraints and define our down-regulated genes as p < 0.05 and do not filter for only the highest fold-changes (> 20 %), this under-representation of estrogen binding sites becomes highly significant (p = 0.004) while androgen binding sites tended to be over-represented in up-regulated genes (p = 0.056). Therefore, modifications of these genes as a result of epigenetic reprogramming during genetic imprinting or during pregnancy could modify their expression in response to sex-hormone production beginning at puberty. Quantitative PCR validation Quantitative PCR validation We verified our gene expression fold-change results from the RNA-seq analyses performing qPCR for Ar, Peg3 and Srebf1. The qPCR results confirmed the direc- tion of fold-changes in the obese matBFMI animals for Page 10 of 14 Page 10 of 14 Kärst et al. Quantitative PCR validation BMC Genomics (2015) 16:904 Table 4 Differentially expressed genes between patBFMI and matBFMI on X chromosome and mitochondria Gene Symbol1 Chr Gene Description (mgi) Mean RPKM2 Fch matBFMI/ patBFMI tTest F1 TFBS3 patBFMI matBFMI Trmt2b X TRM2 tRNA methyltransferase 2B 2.07 1.83 0.88 0.0008 - Ssr4 X signal sequence receptor, delta 3.01 2.84 0.94 0.0013 - Rps4x X ribosomal protein S4, X-linked 4.3 4.17 0.97 0.0026 - Prdx4 X peroxiredoxin 4 3.63 3.49 0.96 0.0086 - Shroom2 X shroom family member 2 1.66 1.46 0.88 0.0163 - Rps24-ps3 X ribosomal protein S24, pseudogene 3 5.17 5.04 0.97 0.0212 - Gm5763 X predicted pseudogene 5763 0.85 1.52 1.79 0.0255 - Nsdhl X NAD(P) dependent steroid dehydrogenase-like 3.15 3.47 1.10 0.0331 - Abcd1 X ATP-binding cassette, sub-family D (ALD), 1 1.37 1.67 1.22 0.0439 - Mospd2 X motile sperm domain containing 2 1.92 1.88 0.98 0.0456 - Haus7 X HAUS augmin-like complex, subunit 7 1.85 2.23 1.21 0.0486 Esrrb Cd99l2 X CD99 antigen-like 2 1.77 1.87 1.06 0.0489 Esrra,Esrrb Serpina7 X serine (or cysteine) peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 2.01 2.52 1.25 0.0508 ERE Pls3 X plastin 3 (T-isoform) 2.84 2.74 0.96 0.0557 - Tsc22d3 X TSC22 domain family, member 3 3.59 3.20 0.89 0.0667 ADR3 Slc16a2 X solute carrier family 16 (monocarboxylic acid transporters), member 2 2.77 2.62 0.95 0.0668 - Ar X androgen receptor 0.26 0.62 2.38 0.0678 - Hsd17b10 X hydroxysteroid (17-beta) dehydrogenase 10 4.21 4.43 1.05 0.0688 - Tmsb4x X thymosin, beta 4, X chromosome 3.8 3.63 0.96 0.0703 IR3 Gyk X glycerol kinase 2.9 2.97 1.02 0.0790 Esrra,Esrra, ERE Gm14760 X predicted gene 14760 3.02 3.18 1.05 0.0803 Esrra,Esrrb Wdr13 X WD repeat domain 13 1.62 1.7 1.05 0.0831 Esrra, ERE Gm14373 X predicted gene 14373 2.49 2.41 0.97 0.0858 - Cldn2 X claudin 2 3.22 3.45 1.07 0.0870 - Rlim X ring finger protein, LIM domain interacting 2.2 2.12 0.96 0.0910 - Zfx X zinc finger protein X-linked 1.47 1.23 0.84 0.0915 - Pqbp1 X polyglutamine binding protein 1 2.53 2.70 1.07 0.0971 - Lamp2 X lysosomal-associated membrane protein 2 4.14 4.01 0.97 0.0974 Esrra,Esrrb F8 X coagulation factor VIII 1.99 1.83 0.92 0.0979 - Gm6472 X predicted pseudogene 6472 4.99 4.90 0.98 0.0997 - mt-Nd1 MT mitochondrially encoded NADH dehydrogenase 1 6.76 6.42 0.95 0.0165 - 1Bold gene symbols indicate up-regulation in matBFMI. Y chromosomal genes were not differentially expressed. Quantitative PCR validation Chr – chromosome, matBFMI – BFMI mother, patBFMI – BFMI father, Fch – fold-change 2All values were log-transformed (ln) 3TFBS - Transcription factor binding sites for steroid hormones. Esrrb – estrogen related receptor b, ERE – estrogen response element, ADR3 – androgen direct repeats of the hexameric half-site, IR3 – androgen inverted repeats of hexameric half-site separated by 3 bp of spacer known to be caused by obesity, though a study in cell culture found androgens to inhibit Peg3 expression [60]. the most genes with Ar being 3.27 fold (p = 0.004) up- regulated and Peg3 being severely down-regulated (0.36 fold, p = 0.002; Additional file 2: Table S4). Srebf1 was found to be 1.66 fold upregulated, al- though with lower significance (p = 0.065) as com- pared to the RNA-seq results (p = 0.027). It is possible that the upregulation of the androgen recep- tor is a reaction to low androgen levels that are Allele specific expression Since allele-specific expression could result from paren- tal imprinting, we exploited the sequence information of expressed genes. After rigorous quality check, we found Kärst et al. BMC Genomics (2015) 16:904 Page 11 of 14 47,566 SNPs on autosomes (7,470 not in genes, and 40,096 SNPs in 6,260 genes). After selecting SNPs for being different between parental strains BFMI and B6N 26,101 SNPs were left (1,846 not in genes, and 24,255 SNPs in 3,961 genes). After selecting for ASE there were 1,362 SNPs left (139 not in genes, and 1,223 SNPs in 415 genes). Filtering those for concordance yielded 1,315 SNPs (137 not in genes, and 1,178 SNPs in 402 genes, see Additional file 4: Table S2). These SNPs were used to search for parent-of origin-specific expression in the liver of 10 week old males. Deviations from balanced biallelic expression can cause different phenotypes by causing total gene expression differences but also inde- pendently since allele functions can be altered due to se- quence variants, altering the function of gene products. We found evidence for allelic imbalances for protein coding genes Abca8b, Commd1, H13, Harbi1, Mcts2, Peg3, Plg, Prkd3 and Zrsr1 (Table 5). genetically imprinted during gametogenesis and differen- tially expressed in offspring [61–64]. However, even if parent-of-origin specific expression is proven, the ex- pression level of the paternal allele could differ between offspring due to cis or trans acting regulation that can- not be derived from RNA-seq data [64]. This was the case for Peg3 in our study. Interestingly, the obese matBFMI males showed only 0.31 fold expression of Peg3 (p = 0.046), which was one of the genes with the most extreme decrease in expression. Peg3 is a Krüppel C2H2-type zinc finger protein, which plays a role in the olfactory system related sexual learning, cell proliferation and p53-mediated apoptosis [65, 66]. In zebrafish, Peg3 inhibits the Wnt pathway [67]. In our data, lower ex- pression of Peg3 in in obese males was accompanied by lower expressions of the Wnt target genes cadherin 1 (Cdh1: 0.75 fold, p = 0.03), epidermal growth factor re- ceptor (Egfr: 0.85 fold, p = 0.05) and neuropilin 1 (Nrp1: 0.90 fold, p = 0.02). High Peg3 expression was recently suggested to protect from high-fat diet induced obesity in F1 offspring of reciprocal crosses between PWK and B6 [68]. Allele specific expression BMC Genomics (2015) 16:904 Page 12 of 14 puberty only in male F1 offspring of obese BFMI mothers, but not in females of the same cross and males and females of the reciprocal cross. Gene ex- pression analysis of liver RNA provided evidence for higher expression of genes controlling fat deposition and impaired β-oxidation of lipids as well as effects on the circadian rhythm in the obese F1 males. The obese F1 males likely use more carbohydrates and amino acids for energy production than lean males. Among the extremely up-regulated genes were Cyto- chrome A4 genes leading to hydroxylation of fatty acids. Furthermore, typical imprinted genes were dif- ferentially expressed between the two groups of males in reciprocal crosses. obesity under high fat diet, whereas PWK mice do not. Our data suggest that different paternal alleles and ex- pression levels of Peg3 act differently in heterozygous BFMI/B6N mice. Very little is known about the function of the imprinted gene Zrsr1 although it was associated with the pluripotency state in induced pluripotent stem cells [69]. Furthermore, it could be shown that trans-acting DNA binding proteins, such as methyl-CpG binding do- main proteins 2 (Mecp2), methyl-CpG binding domain protein Mbd1 and Mbd3 bind at maternal and paternal Zrsr1 alleles differently [70]. In our F1 male mice, Zrsr1 was 0.80 fold expressed in the obese F1 males (p = 0.08). The predominant expression of the maternal H13 allele was confirmed by our data and the gene was only 0.94 fold expressed (p < 0.05) in the matBFMI males. The observations led us to the conclusion that parent-of-origin effects during gametogenesis and pregnancy play a significant role for the development of obesity in later life. Postnatal maternal effects sig- nificantly influence fat deposition, but could not en- tirely overwrite perinatal parent-of-origin-effects. Reprogramming of gametes and metabolic imprints during pregnancy set epigenetic marks that lead to differential expression of genes in response to sex- hormone activation beginning at puberty. We suggest that the obese F1 males store more fat due to im- paired β-oxidation and cellular compound transport. They compensate the lower energy production from fatty acids by an increased use of alternative fuels for the mitochondrial and non-mitochondrial energy pro- duction. The hydroxylases Cyt4a10 and Cyp4a14 are suggested to contribute to increased fatty liver, but the hydroxylated fatty acids are likely nonhazardous. Allele specific expression The ‘paternal transmission’ of the Peg3 effect was deduced from the observation that B6 develop Paternal allele expression was identified for paternally expressed 3 (Peg3) and zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1 (Zrsr1). Mater- nal allele expression was found for the gene H13. Previ- ously, all three genes have been described to be Table 5 SNPs indicating allelic imbalance in both reciprocal crosses Alternative allele1 proportion Chr SNP- Position Ref/ Alt Gene symbol Gene description patBFMI matBFMI Ratio diff. TFBS2 2 91712576 C/T Harbi1 harbinger transposase derived 1 48.1 79.7 31.5 Esrra, Esrrb 2 152687856 C/T Mcts2 malignant T cell amplified sequence 2 88.9 7.2 81.8 - 2 152704657 A/T H13 histocompatibility 13 18.4 75.9 57.5 - 2 152705549 G/A H13 7.9 100.0 92.1 7 6704437 C/T Peg3 paternally expressed 3 93.6 - - - 7 6706584 A/G Peg3 100.0 - - 11 22956525 G/A Commd1 COMM domain containing 1 37.3 57.7 20.3 - 11 22972794 G/T Commd1 98.8 0.0 98.8 11 22974882 G/A Zrsr1 zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1 95.2 4.6 90.7 - 11 109933190 T/C Abca8b ATP-binding cassette, sub-family A (ABC1), member 8b 57.6 30.9 26.7 - 11 109933214 C/T Abca8b 60.8 38.5 22.4 11 109974591 A/C Abca8b 67.7 45.7 22.0 12 104081368 A/G Serpina4- ps1 serine (or cysteine) peptidase inhibitor, clade A, member 4, pseudogene 1 24.6 60.9 36.3 - 12 104081522 G/A Serpina4- ps1 27.2 58.0 30.8 17 12380069 G/T Plg plasminogen 22.6 71.7 49.1 - 17 78969687 T/C Prkd3 protein kinase D3 77.5 57.3 20.2 - Chr – chromosome, matBFMI – F1 males from BFMI mothers, patBFMI – F1 males from BFMI fathers; * Coverage thresholds for ChiSquare based difference calculations in Peg3 were not met in all matBFMI samples. However, variant calling annotates all matBFMI samples as reference (B6) at these positions, confirming the paternal expression. Further ASE gene details are given in Additional file 2: Table S2 1Alternative allele in these cases is the BFMI allele 2TFBS - Steroid hormone transcription factor binding sites. Esrra/b – estrogen related receptor a/b, ERE – estrogen response element Table 5 SNPs indicating allelic imbalance in both reciprocal crosses Kärst et al. Allele specific expression The imprinted genes Peg3, Zrsr1, und H13 and the androgen receptor might play important sex-specific roles for affected down-stream signaling and meta- bolic pathways. We further suggest that the Y and X chromosomes as well as mitochondria play a minor role in the identified phenotypic differences. Differences in expression of imprinted genes could be causal for regulation of down-stream genes contributing to obesity. Recently, paternal intergenerational metabolic reprogramming was found in drosophila, where paternal fat-diet led to changes in the germ cell chromatin state, transcription patterns and modified offspring metabol- ism. The genetic factors identified in drosophila have been verified in mouse and human studies [71]. We also found a lower proportion of the BFMI Abca8b allele expressed by the obese matBFMI males. While the patBFMI expressed between 58–68 %, the matBFMI expressed only 31–46 % of the BFMI allele. This corre- sponds to a mean difference for the alternative allele pro- portion of 24 % under a fold-change of 0.87 (p = 0.06) in total gene expression. ATP-binding cassette (ABC) trans- porter genes represent the largest family of transmem- brane proteins, which arrange the transport of various molecules across all cell membranes utilizing energy from bound ATP. Most ABC genes move compounds from the cytoplasm to the outside of the cell or into an extracellular compartment (endoplasmic reticulum, mitochondria, per- oxisome). ABC transporters shuttle hydrophobic com- pounds within the cell as part of a metabolic process or outside the cell for transport to other organs or secretion from the body. The Abca subfamily includes the largest Abc genes (several >2000 amino acids) and its members are involved in transport of vitamin A (retinol, etc.) deriv- atives as well as in disorders of cholesterol transport and high-density lipoproteins (HDL) biosynthesis [72]. Our data from pathway analyzes and ASE indicate that altered functions of ABC transporters (mainly subfamily Abca and Abcd) contribute to the accumulation of body fat in the obese matBFMI males. Since the higher adipose tissue mass in F1 males of the BFMI mother was maintained during cross-fostering, parent-of-origin effects of genomic reprogramming during gametogenesis and maternal effects during pregnancy could be causal for the elevated fat accumulation. Although, we found indicators for substrate inhibition circuits in mitochondria, the hypothesis of impaired sub- strate selection or mitochondrial substrate overload by fatty acids in BFMI mitochondria needs further research. Allele specific expression Furthermore, we like to point out that allele-specific- expression analysis with RNA-seq data requires high control standards. Allele-specific expression could only be tested, if sequence variation was identified between the examined mouse strains. Since the analysis per- formed in this study was very conservative, the data we present here are highly reliable. Nonetheless, we miss Conclusions In reciprocal crosses between the obese line BFMI and the lean line B6N, we observed high fat depos- ition with and without cross-fostering beginning at Kärst et al. BMC Genomics (2015) 16:904 Page 13 of 14 information on genes without variation in the coding se- quence and genes with very low transcript levels in liver. Moreover, the molecular mechanisms necessary for allele specific expression in adult tissues need further evalu- ation. A controlling function of sex hormones in metab- olism was shown before and is supported by reduced counts of binding sites for estrogens in down-regulated genes in our data. 3. Willer CJ, Speliotes EK, Loos RJF, Li S, Lindgren CM, Heid IM, et al. Six new loci associated with body mass index highlight a neuronal influence on body weight regulation. Nat Genet. 2009;41:25–34. 4. Zeggini E, Scott LJ, Saxena R, Voight BF, Marchini JL, Hu T, et al. 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All genes being differentially expressed between both reciprocal crosses (p < 0.1). (XLSX 96 kb) Additional file 4: Table S2. Sequence variants measured as monoallelic in one or both reciprocal crosses (XLSX 814 kb) Additional file 1: Table S3. Position weight matrices for scanned TFBS. (XLSX 12 kb) Additional file 1: Table S3. Position weight matrices for scanned TFBS. (XLSX 12 kb) 8. bcl2fastq Conversion. 2013. 9. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. Additional file 2: Table S4. Validation of RNA-seq derived fold-changes via qPCR. (XLSX 10 kb) 10. Li H, Durbin R. Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics. 2009;25:1754–60. Additional file 3: Table S1. All genes being differentially expressed between both reciprocal crosses (p < 0.1). (XLSX 96 kb) Additional file 3: Table S1. 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https://openalex.org/W3093210393	https://repository.ubn.ru.nl/bitstream/handle/2066/229594/1/229594.pdf	English	null	Effects of physical therapy for temporomandibular disorders on headache pain intensity: A systematic review	Musculoskeletal science & practice/Musculoskeletal science and practice	2,020	cc-by	9,351	Effects of physical therapy for temporomandibular disorders on headache pain intensity: A systematic review Meer, H.A. van der; Calixtre, L.B.; Engelbert, R.H.; Visscher, C.M.; Nijhuis-van der Sanden, M.W.G.; Speksnijder, C.M. 2020, Article / Letter to editor (Musculoskeletal Science and Practice, 50, (2020), article 102277) Doi link to publisher: https://doi.org/10.1016/j.msksp.2020.102277 Effects of physical therapy for temporomandibular disorders on headache pain intensity: A systematic review Meer, H.A. van der; Calixtre, L.B.; Engelbert, R.H.; Visscher, C.M.; Nijhuis-van der Sanden, M.W.G.; Speksnijder, C.M. 2020, Article / Letter to editor (Musculoskeletal Science and Practice, 50, (2020), article 102277) Doi link to publisher: https://doi.org/10.1016/j.msksp.2020.102277 Version of the following full text: Publisher’s version Downloaded from: https://hdl.handle.net/2066/229594 Download date: 2024-10-24 A R T I C L E I N F O Keywords: Temporomandibular disorder Orofacial pain Headache Physical therapy Oral rehabilitation Background: Physical therapy is regarded an effective treatment for temporomandibular disorders (TMD). Pa tients with TMD often report concomitant headache. There is, however, no overview of the effect of physical therapy for TMD on concomitant headache complaints. Objectives: The aim of this study is to systematically evaluate the literature on the effectiveness of physical therapy on concomitant headache pain intensity in patients with TMD. erapy on concomitant headache pain intensity in patients wit Data sources: PubMed, Cochrane and PEDro were searched. Data sources: PubMed, Cochrane and PEDro were searched. Study eligibility criteria: Randomized or controlled clinical trials studying physical therapy interventions were included. Participants: Patients with TMD and headache. Participants: Patients with TMD and headache. Appraisal: The Cochrane risk of bias tool was used to assess risk of bias. Synthesis methods: Individual and pooled between-group effect sizes were calculated according to the stan dardized mean difference (SMD) and the quality of the evidence was rated using the GRADE approach. Results: and manual therapy on both orofacial region and cervical spine. There is a very low level of certainty that TMD-treatment is effective on headache pain intensity, downgraded by high risk of bias, inconsistency and imprecision. Limitations: The methodological quality of most included articles was poor, and the interventions included were very different. Conclusions: Physical therapy interventions presented small effect on reducing headache pain intensity on sub jects with TMD, with low level of certainty. More studies of higher methodological quality are needed so better conclusions could be taken. Effects of physical therapy for temporomandibular disorders on headache pain intensity: A systematic review Hedwig A. van der Meer a,b,c,d,e,, Leticia B. Calixtre f, Raoul H.H. Engelbert a,c, Corine M. Visscher e, Maria WG. Nijhuis – van der Sanden b, Caroline M. Speksnijder d a Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Department of Orofacial Pain and Dysfunction, Amsterdam, the Netherlands ntre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Department of Orofacial Pain and Dysfunction, h l d a Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Department of Orofacial Pain an Amsterdam, the Netherlands b Amsterdam, the Netherlands b University Medical Center Utrecht, Utrecht University, Department of Oral-Maxillofacial Surgery and Special Dental Care, Utrecht, the Netherlands c Amsterdam University of Applied Sciences, Education of Physiotherapy, Faculty of Health, Amsterdam, the Netherlands d Radboud University Medical Center, Research Institute for Health Sciences, IQ Healthcare, Nijmegen, the Netherlands e University of Amsterdam, Amsterdam University Medical Centers (AUMC), Department of Rehabilitation, Amsterdam, Meibergdreef 9, Amsterdam Movement Scien the Netherlands f Federal University of S˜ao Carlos (UFSCar), Physiotherapy Department, S˜ao Carlos, Brazil Corresponding author. Hogeschool van Amsterdam, Tafelbergweg 51, 1105, BD, Amsterdam. .. E-mail address: h.a.van.der.meer@hva.nl (H.A. van der Meer). Contents lists available at ScienceDirect Contents lists available at ScienceDirect Available online 16 October 2020 2468-7812/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.msksp.2020.102277 Received 11 March 2020; Received in revised form 2 October 2020; Accepted 13 October 2020 Available online 16 October 2020 2468-7812/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. Hogeschool van Amsterdam, Tafelbergweg 51, 1105, BD, Amsterdam. .. E-mail address: h.a.van.der.meer@hva.nl (H.A. van der Meer). https://doi.org/10.1016/j.msksp.2020.102277 Received 11 March 2020; Received in revised form 2 October 2020; Accepted 13 October 2020 Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Musculoskeletal Science and Practice 50 (2020) 102277 2. Methods Quality assessment of the studies was performed using the Cochrane ‘risk of bias (RoB)’ tool, in contrast with what was registered in PROS PERO, because the Cochrane RoB tool is recommended over the use of the PEDro score (Armijo-Olivo et al., 2015a). The Cochrane RoB tool assesses five domains regarding bias: selection bias, performance bias, detection bias, attrition bias and reporting bias. The tool focuses on the internal validity and does not lead to a quality score (Higgins et al., 2011). This tool shows if there is a high, low, or unclear risk of bias within the study which may influence the internal validity of the study (Higgins et al., 2011). Two independent reviewers performed the quality assessment blinded to each other’s results (HvdM, LBC). Any discrep ancies were discussed and, when needed, a third reviewer (CMS) made the decision regarding the final quality score of the article. A risk of bias graph and risk of bias summary were extracted from the program Re view Manager 5.3 (RevMan. Published online, 2014). 1. Introduction stratified into myalgia (pain in the masticatory muscles), arthralgia (pain in the temporomandibular joint), or functional complaints of the joint, like clicking or locking (Schiffman et al., 2014). Up to 85% of patients with TMD complain about myalgia (List and Jensen, 2017). Physical therapists are equipped to treat patients with these musculo skeletal complaints. In the last five years, six reviews, discuss the effectiveness of several physical therapy modalities on TMD complaints One in five adults in Europe are estimated to have a perceived dysfunction of their masticatory system, which is related to temporo mandibular disorders (TMD) (LeResche, 1997; L¨ovgren et al., 2016). TMDs are defined according to the Diagnostic Criteria for TMD (DC/TMD) as complaints involving the masticatory system and can be Musculoskeletal Science and Practice 50 (2020) 102277 H.A. van der Meer et al. 2.4. Search Key wordsused in the search strategy were, amongst others: “phys ical therapy”, “physiotherapy”, “temporomandibular disorder” and “headache”. In PubMed, we used a combination of MeSH Terms and title/abstract searches and different physical therapy modalities were described. The search strategy for PEDro and Cochrane required adap tation from the PubMed search strategy. The complete search strategies can be found in Appendix 1. There was no hand search. Grey literature was not included. 2.1. Protocol and registration This review has been reported in accordance with PRISMA recom mendations (Moher et al., 2009) and is registered on PROSPERO (registration number CRD42017062487). 2.2. Eligibility criteria Studies had to meet the following inclusion criteria to be eligible: 1) adult participants with TMD based on the diagnostic criteria for TMD (DC/TMD) (Schiffman et al., 2014; Dworkin and LeResche, 1992); 2) headache pain intensity as outcome measure; 3) randomized controlled trial (RCT) or controlled clinical trial (CCT); 4) TMD-treatment within the physical therapy domain (World Confederation for P, 2017); and 5) article is published in English or Dutch. Articles were excluded when an occlusal device (e.g. a stabilization splint) was the only intervention. There were no restrictions on publication dates, or on age and gender of the participants. All headache types were included in the review. 2.6. Data collection process, data items and summary measures Data extraction was performed by one reviewer (HvdM). A second reviewer (LBC) checked the extracted data for accuracy. The following key data were extracted: 1) study characteristics: first author, year of publication, type of study, sample size; 2) participant characteristics: age, gender, and TMD diagnosis (Schiffman et al., 2014); 3) intervention characteristics: type of intervention(s) (World Confederation for P, 2017), frequency, and follow-up; and 4) outcome measures: intensity of headache (Headache Classification C, 2018) according to Visual Analog Scale (VAS) or similar tools and the statistical significance for both within-group and between-group analyses when available. The mean and standard deviations were extracted from the included studies for further statistical analysis. 2.5. Study selection Duplicates were removed and title/abstracts of all retrieved records were screened for eligibility by two researchers blinded to each other’s results (HvdM, CMS). The full texts of the remaining articles were ob tained and the full texts were assessed to see if the studies met the in clusion criteria for this review. In case of disagreement between the two reviewers, a third reviewer (RE) made the decision regarding inclusion of the article. ; , ; , The high co-morbidity between TMD and cervical impairments (Piekartz et al., 2020), as well as the neuroanatomical relationship be tween these two areas (Paparo, 2008; Castien and De Hertogh, 2019; Costa et al., 2017), may explain why physical therapy can have an effect on headache through treatment of the temporomandibular system (von Piekartz et al., 2016; Liang et al., 2019; Fernandez-de-las-Pe˜nas et al., 2007; Bragatto et al., 2016). Besides manual physical therapy and ex ercise therapy, some of the common approaches for treating TMD or headache are focusing on more general aspects of pain, like pain edu cation and counseling. This has also been found effective for patients with migraine (Kindelan-Calvo et al., 2014), TTH (Motoya et al., 2014) and TMD (Turner et al., 2006). As these physical therapy modalities may be an effective treatment for both TMD and headaches, it may be interesting to see how headache pain intensity responds to TMD-treatment. There is currently, however, no overview of the effect of physical therapy interventions for TMD on concomitant headache pain intensity. We hypothesized that TMD-treatment would have a positive effect on headache pain intensity, but that this may differ between different headaches types. Therefore, the aim of this study is to sys tematically evaluate the literature on the effectiveness of physical therapy interventions on concomitant headache pain intensity in pa tients with TMD. 2.3. Information sources (Dickerson et al., 2017; Butts et al., 2017; Paço et al., 2016; Martins et al., 2016; Armijo-Olivo et al., 2016; Calixtre et al., 2015). The most recent review and meta-analysis concluded that exercise therapy is effective in reducing TMD-pain. Even though headache is a common symptom in TMD (Di Paolo et al., 2017), headache pain intensity was not taken into consideration in these reviews. Literature searches to identify studies were performed in the elec tronic databases PubMed [1966–2020], Cochrane [1993–2020] and Physiotherapy Evidence Database (PEDro) [1999–2020]. The electronic search was supplemented by snowballing of full articles retrieved. The search was conducted on August 3, 2020. Patients with TMD report headache more frequently (68–85%) than the general population (50%) (Stovner and Andree, 2010; van der Meer et al., 2017a; Franco et al., 2010; de Leeuw and Klasser, 2013; National Center for Healt, 2016). Most common headaches in patients with TMD are Tension-Type Headache (TTH), migraine and headache attributed to TMD (van der Meer et al., 2017a; Franco et al., 2010). These headaches are classified by the International Classification for Headache Disorders 3rd edition (ICHD-3) (Headache Classification C, 2018). In contrast to the primary headaches TTH and migraine, headache attributed to TMD has a known cause for the headache complaints which is the TMD. This underlying TMD condition needs to be treated in order to decrease the headache complaints attributed to TMD. Another secondary headache that may be frequent in patients with TMD is a cervicogenic headache, as patients with TMD often report cervical dysfunction and patients with cervicogenic headache frequently report signs of a TMD (Mingels et al., 2019; Piekartz et al., 2020; von Piekartz et al., 2016). 3. Results Although all included studies were RCTs, there was variation in the applied protocols regarding the therapy modality and the type of control intervention. 3.3. Characteristics and results of individual studies 3.3. Characteristics and results of individual studies 2.9. Effect sizes were classified as small (<0.20), moderate (≥0.20 and ≤0.80) or large (>0.80), according to Cohen’s criteria (Cohen, 1988) In the five included RCTs (Costa et al., 2015; Maluf et al., 2010; Michelotti et al., 2004; Michelotti et al., 2012; Piekartz and Hall, 2013) the study population ranged from 28 to 54 persons (see Table 1). The follow-up period ranged from two weeks to six months. Four articles used the VAS for headache intensity as an outcome measure (Costa et al., 2015; Maluf et al., 2010; Michelotti et al., 2004; Michelotti et al., 2012). One article used a colored analog scale (CAS) to rate headache pain intensity (Piekartz and Hall, 2013). The frequency of the interventions ranged from daily for three months (Michelotti et al., 2004; Michelotti et al., 2012) to weekly for eight weeks (Maluf et al., 2010). The session time ranged from several minutes for home therapy (Michelotti et al., 2004; Michelotti et al., 2012) to full 30–40 min sessions with a therapist (Costa et al., 2015; Maluf et al., 2010; Piekartz and Hall, 2013). To assess the certainty of the findings, the GRADE recommendations were followed considering the following domains: trial design limita tions (using the RoB tool), inconsistency of results, indirectness, imprecision of results and publication bias (Slavin, 1995; Balshem et al., 2011; Andrews et al., 2013). The certainty was classified as one of the four levels: high, moderate, low, or very low. The details of this method have been reported previously (Calixtre et al., 2015; Atkins et al., 2004; Richards et al., 2013). 2.8. Synthesis of results and additional analyses For each study, between-group effect sizes were calculated according to the standardized mean difference (SMD) (Hedges and Olkin, 1985), 2 Musculoskeletal Science and Practice 50 (2020) 102277 H.A. van der Meer et al. 3.2. Risk of bias within studies 3.2. Risk of bias within studies using the follow-up data. When means and standard deviations were unavailable, the first author was contacted for the details. All contacted authors provided information needed. In case only one article studied an intervention, the between-group SMD was considered. When multiple studies were available, the outcomes were pooled using Review Man ager 5.3 and forest plots were provided (RevMan. Published online, 2014). All articles received were classified as a high risk of bias on blinding of participants and personnel, and unclear risk on selective reporting (Figs. 2 and 3). 3.1. Study selection The search strategy revealed 87 initial articles from PubMed, 52 from Pedro and 6 from Cochrane (see Fig. 1). After applying inclusion and exclusion criteria, five articles were included (Costa et al., 2015; Maluf et al., 2010; Michelotti et al., 2004; Michelotti et al., 2012; Piekartz and Hall, 2013). The list of excluded studies can be found in Appendix 2. 3.4.1. Counseling and exercise versus counseling and/or splint 3.4.1. Counseling and exercise versus counseling and/or splint 3.4.1. Counseling and exercise versus counseling and/or splint Th ti l t di d th ff t f lti d l i t ti g g p Three articles studied the effect of a multimodal intervention of counseling and exercises (Costa et al., 2015; Michelotti et al., 2004; Michelotti et al., 2012). The control interventions were splint therapy (i. e. a removable artificial occlusal surface placed on the upper or lower dental arch) (Wieckiewicz et al., 2015), counseling, or a combination of both counseling and splint therapy. 3.4.2. Static stretching versus global stretching 3.4.2. Static stretching versus global stretching One study (Maluf et al., 2010) compared static stretching techniques for the cervical spine, upper limbs and mandibular muscles with postural reeducation. This study showed a large between-group effect size in favor of static stretching (SMD: 0.91; 95%CI: 1.76, −0.06) on reducing headache pain intensity. There is a low certainty of evidence, downgraded by risk of bias and inconsistency, that static stretching of the cervical spine, upper limbs and mandibular muscles is more effective than global postural reeducation for headache pain intensity. 3.4.3. Orofacial and cervical manual therapy versus cervical manual therapy 3.4.3. Orofacial and cervical manual therapy versus cervical manual therapy One study (Piekartz and Hall, 2013) applied orofacial therapy (i.e. jaw muscle and -joint exercises) combined with cervical manual therapy and compared this to cervical manual therapy alone. The between-group effect size was large (SMD: 1.57; 95%CI: 2.26, −0.89) showing that the intervention was superior to control intervention on reducing headache pain intensity. The level of certainty regarding the evidence was mod erate, downgraded by inconsistency. 3.4. Effect of the interventions and level of evidence The GRADE criteria were applied for all therapy modalities. The studies were split in 3 subgroups, according to the intervention type and also analyzed together (Table 2 and 3). For two studies (Costa et al., 2015; Piekartz and Hall, 2013), additional data were collected from the Fig. 1. Prisma flow diagra. Fig. 1. Prisma flow diagra. 3 H.A. van der Meer et al. Musculoskeletal Science and Practice 50 (2020) 102277 Musculoskeletal Science and Practice 50 (2020) 102277 Fig. 2. Risk of bias graph. Fig. 2. Risk of bias graph. Fig. 3. Risk of bias summary. self-massage of the masticatory muscles, application of moist heat pads on the painful muscles, stretching, and coordination exercises. There was a moderate between-group effect size (SMD: 0.05; 95%CI: 0.61, 0.51). Fig. 4 shows the pooled mean difference, which is 0.15 (95%CI: 0.17, 0.46), indicating there is no difference in effect for counseling and ex ercises compared to counseling and/or splint therapy. Based on the GRADE recommendations (Table 3), we see that there is a low certainty of the found effects, downgraded by risk of bias and imprecision. 4.1. The influence of headache types in TMD-treatment authors to calculate the effect sizes (Table 2). authors to calculate the effect sizes (Table 2). When taking all included studies together as TMD physical therapy, this review shows that there is a very low level of certainty for TMD- treatment on reducing headache pain intensity, downgraded by high risk of bias, inconsistency and imprecision (Atkins et al., 2004). The pooled data analysis showed small overall effect (SMD: 0.12; 95%CI: 0.39, 0.16), in favor of TMD-focused physical therapy compared to control interventions (see also Fig. 5). 3.4.4. Overall effect on headache by physical therapy focused on TMD 3.4.4. Overall effect on headache by physical therapy focused on TMD authors to calculate the effect sizes (Table 2). 4. Discussion On the individual analysis, there was a small between-group effect size for Costa et al. (2015) (SMD: 0.42; 95%CI: 0.09, 0.51), showing no clinically relevant difference between the multimodal therapy or com bined counseling and splint therapy. The two other articles studied counseling and home exercises for three months (Michelotti et al., 2004; Michelotti et al., 2012). One study (Michelotti et al., 2012) compared education to splint therapy and showed small between-group effect size (SMD: 0.00; 95%CI: 0.59, 0.59). The other study (Michelotti et al., 2004) compared education and education combined with physical therapy for TMD. The physical therapy regimen contained self-relaxation exercises, The aim of this study was to systematically evaluate the literature on the effectiveness of physical therapy for TMD on concomitant headache pain intensity. The therapy modalities varied across the five included articles. The certainty of the findings was very low for the effectiveness of physical therapy for TMD on headache intensity. 4.1. The influence of headache types in TMD-treatment Two studies described a specific headache diagnosis based on the 4 H.A. van der Meer et al. Musculoskeletal Science and Practice 50 (2020) 102277 5 Table 1 Characteristics of included studies considering study design, subjects, interventions and outcome. Author, year Complaints Intervention group Control group Therapist Follow- up Headache VAS outcomes Between group p- value Population Intervention Population Control intervention Intervention group Control group N (%F) Age±SD Type Freq. N (%F) Age±SD Type Freq. B m (SD) FU m (SD) B m (SD) FU m (SD) Costa, 2015 Myofascial TMD and headache 30 (90) 36 ± 6.7 Counseling, relaxation exercises, stretching and auto-massage jaw muscles 3–5x/week 30min for 5mo. 30 (90) 27.5 ± 6.7 Counseling and occlusal appliance 1x Therapist (undefined) 5 months 7.6 (2.2) 4.4 (2.5) 6.5 (1.6) 3.4 (2.2) NS Maluf, 2010 TMD-pain 12 (100) 30.1 ± 7.1 Static stretching of cervical spine, upper limbs, and mandibular muscles 1x/week 40min for 2mo. 12 (100) 30.0 ± 4.3 Global posture reeducation 1x/week 40min for 2mo. Therapist (undefined) 8 weeks 65.7 (21.7) 16.4 (16.6) 73.5 (26.2) 39.2 (29.8) ? Michelotti, 2004 TMD-pain 26 (86.1) 28.2 ± 8.8 Education + home exercises 7x/week ? min for 3mo. 23 (91.2) 31.8 ± 13.0 Education 1x Dentist 3 months 26.1 (29.7) 11.2 (17.4) 13.3 (19.7) 12.1 (17.0) NS Michelotti, 2012 Myogenous TMD-pain 23 (82.6) 30.2 ± 13.0 Education + home exercises 7x/week ? min for 3mo. 4. Discussion Overall PT for TMD versus control interventions Outcome N patients (studies) Standardized Mean Difference (95%CI) Certainty of the evidence (GRADE quality) Headache pain - VAS 220 (5 RCTs) (Michelotti et al., 2004; Michelotti et al., 2012; Piekartz and Hall, 2013; Costa et al., 2015; Maluf et al., 2010) −0.12 (−0.39, 0.16) ⊕©©© VERY LOW Due to risk of bias, inconsistency and imprecision. Counseling and exercise versus counseling and/or splint therapy Outcome N patients (studies) Standardized Mean Difference (95%CI) GRADE quality Headache pain - VAS 153 (3 RCTs) (Michelotti et al., 2004; Costa et al., 2015; Michelotti et al., 2012) 0.15 (−0.17, 0.46) ⊕⊕©© LOW Due to risk of bias and imprecision. Static stretching versus global stretching Outcome N patients (studies) Standardized Mean Difference (95%CI) GRADE quality Headache pain - VAS 24 (1 RCT) (Maluf et al., 2010) −0.91 (−1.76, −0.06) ⊕⊕©© LOW Due to risk of bias and inconsistency. Orofacial and cervical manual therapy versus cervical manual therapy Outcome N patients (studies) Standardized Mean Difference (95%CI) GRADE quality Headache pain - CAS 43 (1 RCT) (Piekartz and Hall, 2013) −1.57 (−2.26, −0.88) ⊕⊕⊕© MODERATE Due to inconsistency. GRADE: Grades of Recommendation, Assessment, Development, and Evaluation; PT: physical therapy; TMD: temporomandibular disorder; n: number of participants; VAS: visual analog scale; CAS: colored analog scale. * Methodological quality limitations based on the Cochrane Risk of Bias tool (high risk: serious −1 or very serious −2; unclear risk; not serious or serious −1). GRADE Working Group grades of evidence High certainty: We are very confident that the true effect lies close to that of the estimate of the effect Moderate certainty: We are moderately confident in the effect estimate: The true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different Low certainty: Our confidence in the effect estimate is limited: The true effect may be substantially different from the estimate of the effect Very low certainty: We have very little confidence in the effect estimate: The true effect is likely to be substantially different from the estimate of effect. H.A. van der Meer et al. H.A. van der Meer et al. Table 2 Table 2 Between-group and within-group effect sizes for individual studies stratified for different types of physical therapy compared to control interventions. Overall PT for TMD versus control interventions Outcome Trial Overall PT Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - VAS Costa et al., 2015 30 1.34 30 1.59 0.05 Maluf et al., 2010 12 2.46 12 1.18 0.60 Michelotti et al., 2004 26 0.60 23 0.06 0.62 Michelotti et al., 2012 23 −0.02 21 0.01 −0.03 Piekartz and Hall, 2013 19 2.37 17 0.34 −2.42 Counseling and exercise versus counseling and/or splint therapy Outcome Trial Counseling + exercise Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - VAS Costa et al., 2015 30 1.34 30 1.59 0.05 Michelotti et al., 2004 26 0.60 23 0.06 0.62 Michelotti et al., 2012 23 −0.02 21 0.01 −0.03 Static stretching versus global stretching Outcome Trial Static stretching Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - VAS Maluf et al., 2010 12 2.46 12 1.18 0.60 Orofacial and cervical manual therapy versus cervical manual therapy Outcome Trial Orofacial + cervical manual therapy Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - CAS Piekartz and Hall, 2013 19 2.37 17 0.34 −2.42 PT: physical therapy; TMD: temporomandibular disorder; n: number of participants; ES: effect size; VAS: visual analog scale; CAS: colored analog scale; N/A: not applicable. Between-group and within-group effect sizes for individual studies stratified for different types of physical therapy compared to control interventions. Table 3 Table 3 Summary of findings table according to the GRADE recommendations for studies comparing different types of PT for TMD applied to patients with TMD and headache. Overall PT for TMD versus control interventions Outcome N patients (studies) Standardized Mean Difference (95%CI) Certainty of the evidence (GRADE quality) Headache pain - VAS 220 (5 RCTs) (Michelotti et al., 2004; Michelotti et al., 2012; Piekartz and Hall, 2013; Costa et al., 2015; Maluf et al., 2010) −0.12 (−0.39, 0.16) ⊕©©© VERY LOW Due to risk of bias, inconsistency and imprecision. Counseling and exercise versus counseling and/or splint therapy Outcome N patients (studies) Standardized Mean Difference (95%CI) GRADE quality Headache pain - VAS 153 (3 RCTs) (Michelotti et al., 2004; Costa et al., 2015; Michelotti et al., 2012) 0.15 (−0.17, 0.46) ⊕⊕©© LOW Due to risk of bias and imprecision. Static stretching versus global stretching Outcome N patients (studies) Standardized Mean Difference (95%CI) GRADE quality Headache pain - VAS 24 (1 RCT) (Maluf et al., 2010) −0.91 (−1.76, −0.06) ⊕⊕©© LOW Due to risk of bias and inconsistency. Orofacial and cervical manual therapy versus cervical manual therapy Outcome N patients (studies) Standardized Mean Difference (95%CI) GRADE quality Headache pain - CAS 43 (1 RCT) (Piekartz and Hall, 2013) −1.57 (−2.26, −0.88) ⊕⊕⊕© MODERATE Due to inconsistency. GRADE: Grades of Recommendation, Assessment, Development, and Evaluation; PT: physical therapy; TMD: temporomandibular disorder; n: number of participants; VAS: visual analog scale; CAS: colored analog scale. * Methodological quality limitations based on the Cochrane Risk of Bias tool (high risk: serious −1 or very serious −2; unclear risk; not serious or serious −1). GRADE Working Group grades of evidence High certainty: We are very confident that the true effect lies close to that of the estimate of the effect Moderate certainty: We are moderately confident in the effect estimate: The true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different Low certainty: Our confidence in the effect estimate is limited: The true effect may be substantially different from the estimate of the effect Very low certainty: We have very little confidence in the effect estimate: The true effect is likely to be substantially different from the estimate of effect. s for studies comparing different types of PT for TMD applied to patients with TMD and headache. 4. Discussion 21 (71.4) 30.3 ± 11.4 Splint 1x Mandibular surgeon 3 months 33.3 (19.6) 33.7 (19.2) 33.9 (21.1) 33.7 (19.2) NS Von Piekartz, 2013 CGH and TMD symptoms 22 (63.6) 34.7 ± 7.1 Jaw muscle and -joint exercises combined with cervical manual therapy 1–2x/week 30min for 3–6 weeks 21 (66.7) 36.1 ± 6.5 Cervical manual therapy 1–2x/week 30min for 3–6 weeks Physical therapist 6 months 7.4a (1.1) 3.5a (2.0) 7.1a (1.1) 6.7a (1.2) <.001 All included studies are Randomized Controlled Trials; TMD: temporomandibular disorder; CGH: cervicogenic headache; N: number of study participants; F: female; VAS: visual analog scale; m: mean; SD: standard deviation; N/A: not applicable; (?): missing; B: baseline; FU: follow-up; p: p-value; NS: not significant; mo.: months. a Musculoskeletal Science and Practice 50 (2020) 102277 Musculoskeletal Science and Practice 50 (2020) 102277 Table 2 Between-group and within-group effect sizes for individual studies stratified for different types of physical therapy compared to control interventions. Overall PT for TMD versus control interventions Outcome Trial Overall PT Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - VAS Costa et al., 2015 30 1.34 30 1.59 0.05 Maluf et al., 2010 12 2.46 12 1.18 0.60 Michelotti et al., 2004 26 0.60 23 0.06 0.62 Michelotti et al., 2012 23 −0.02 21 0.01 −0.03 Piekartz and Hall, 2013 19 2.37 17 0.34 −2.42 Counseling and exercise versus counseling and/or splint therapy Outcome Trial Counseling + exercise Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - VAS Costa et al., 2015 30 1.34 30 1.59 0.05 Michelotti et al., 2004 26 0.60 23 0.06 0.62 Michelotti et al., 2012 23 −0.02 21 0.01 −0.03 Static stretching versus global stretching Outcome Trial Static stretching Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - VAS Maluf et al., 2010 12 2.46 12 1.18 0.60 Orofacial and cervical manual therapy versus cervical manual therapy Outcome Trial Orofacial + cervical manual therapy Comparison Between-group ES n Within-group ES n Within-group ES Headache pain - CAS Piekartz and Hall, 2013 19 2.37 17 0.34 −2.42 PT: physical therapy; TMD: temporomandibular disorder; n: number of participants; ES: effect size; VAS: visual analog scale; CAS: colored analog scale; N/A: not applicable. Table 3 Summary of findings table according to the GRADE recommendations for studies comparing different types of PT for TMD applied to patients with TMD and headache. Table 3 For instance, one theory states that the increase in pain transmission from peripheral tissues, such as the masticatory system, to the trigeminal system nega tively interferes with the efficacy of the treatment (Porporatti et al., 2015). Central sensitization (CS) can also play a role in the efficacy of treatment, as patients with multiple complaints such as chronic TTH, migraine and TMD tend to show more signs of CS (Costa et al., 2017; Campi et al., 2017; Woolf, 2011; La Touche et al., 2018). Patients with signs of CS manifest pain hypersensitivity and hyperalgesia, for whom certain therapies may increase the pain rather than decrease it (Woolf, 2011; Louw et al., 2017). A combination of peripheral and central mechanisms can also contribute to the etiology and interference with therapy: input from the periphery (i.e. masticatory system) may turn to painful output due to CS (Woolf, 2011; La Touche et al., 2018). How ever, combining a TMD treatment with specific medication for migraine is found to be more effective as compared to a single treatment for either TMD or migraine (Goncalves et al., 2013). Thus, it is important to know which type of headache is the concomitant headache with the TMD complaints. relaxing the muscles and by that decreasing the TMD-pain and headache pain intensity (Maluf et al., 2010; Piekartz and Hall, 2013). Addition ally, both studies showed that combining treatment regarding the temporomandibular area (jaw, masseter muscle, temporal muscle) and cervical area (spine and muscles) are effective for headache intensity (Maluf et al., 2010; Piekartz and Hall, 2013). Other studies have also shown that exercises for the cervical spine can decrease both TMD complaints as well as headache complaints (Calixtre et al., 2015; Calixtre et al., 2019; Madsen et al., 2018; Castien et al., 2011). Three studies included applied home exercises as part of the physical therapy for TMD (Costa et al., 2015; Michelotti et al., 2004; Michelotti et al., 2012). However, it is unclear which specific home exercises were applied and if they were only addressed to the jaw or also the cervical spine. Furthermore, there is debate about the effectiveness of home exercises compared to supervised exercises. Table 3 For other disorders such as knee osteoarthritis, chronic neck pain and shoulder impinge ment, both types of exercise were effective, but when at least one su pervised training was done the effect increased and lasted longer (Deyle et al., 2005; Evans et al., 2012; Granviken and Vasseljen, 2015). Future studies should describe the types of exercises more elaborately and compare supervised exercises with home exercises to fully understand how to apply exercise therapy for best results. As patients with TMD pain often experience pain or dysfunction in the cervical spine (Piekartz et al., 2020; Visscher et al., 2001) and pa tients with headache also often experience neck problems (Ashina et al., 2015), it is important to not just look at the masticatory system in pa tients with TMD and headache, but also include the cervical spine and muscles (Costa et al., 2017). Bruxism may also play a role in this three-way association, as temporomandibular disorders, cervical impairment and headaches are all associated with bruxism (van der Meer et al., 2017a; Piekartz et al., 2020; Fernandes et al., 2013; Baad- Hansen et al., 2019). When patients are bruxing, not only their masti catory muscles are active but also muscles from the cervical spine (Gouw et al., 2020). Currently the exact working mechanism on these four as pects and how they influence each other and treatment outcomes re mains unclear. Therefore, more high-quality research is needed to establish the association between TMD, headache and cervical involvement and the effects of treating these complaints separately compared to treating them simultaneously. Table 3 VAS: visual analog scale; CAS: colored analog scale. * Methodological quality limitations based on the Cochrane Risk of Bias tool (high risk: serious −1 or very serious −2; unclear risk; not serious or serious −1). GRADE Working Group grades of evidence High certainty: We are very confident that the true effect lies close to that of the estimate of the effect Moderate certainty: We are moderately confident in the effect estimate: The true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different Low certainty: Our confidence in the effect estimate is limited: The true effect may be substantially different from the estimate of the effect Very low certainty: We have very little confidence in the effect estimate: The true effect is likely to be substantially different from the estimate of effect. Musculoskeletal Science and Practice 50 (2020) 1022 Fig. 4. Forest plot of comparison overall physical therapy versus control interventions. der Meer et al. Musculoskeletal Science and Practice 50 (2020) 102277 H.A. van der Meer et al. Fig. 4. Forest plot of comparison overall physical therapy versus control interventions. Fig. 4. Forest plot of comparison overall physical therapy versus control interventions. Fig. 5. Forest plot of comparison counseling and exercises versus counseling and/or splint therapy. Fig. 5. Forest plot of comparison counseling and exercises versus counseling and/or splint therapy. Fig. 5. Forest plot of comparison counseling and exercises versus counseling and/or splint therapy. ICHD-II, which were headache related to masticatory myofascial pain and cervicogenic headache (Costa et al., 2015; Piekartz and Hall, 2013; Headache Classification S, 2004). The other three studies did not describe the headache types (Maluf et al., 2010; Michelotti et al., 2004; Michelotti et al., 2012), but they may have been at least a part of patients with primary headache as these are very prevalent (10–63%) in patients with TMD (Di Paolo et al., 2017; Franco et al., 2010; van der Meer et al., 2017b; Goncalves et al., 2011). The presence of a primary headache may have negatively interfered with the efficacy of the different musculo skeletal TMD treatments on TMD complaints (Porporatti et al., 2015). This is possibly due to peripheral or central mechanisms. 4.3. Strengths and limitations of the study There are several strengths in this review. First, this review is the first study to approach the issue of the effect of treating TMD on headache pain intensity. By describing the limitations per study, a clear suggestion for future research can be made. Secondly, all steps within this review have been done by two researchers, blinded to each other’s results. As multiple factors play an important role in the etiology of both TMD and headache, it is important for therapists to define these factors before starting treatment (Schiffman et al., 2014; van der Meer et al., 2017a; Headache Classification C, 2018; Gaul et al., 2011; Dekker- Bakker et al., 2008). Because this review has not shown clear evidence for all physical therapy modalities, physical therapists must consider which treatment to apply based on the beforementioned factors. As orofacial physical therapy and cervical manual therapy do appear to be effective to reduce headache pain intensity, a specialized physical therapist should be part of the health care team for the treatment of TMD and headache, although they may not be available in all countries (Piekartz and Hall, 2013; Gaul et al., 2011; Dekker-Bakker et al., 2008). y , However, the results of this review must be interpreted considering some limitations. First, when interpreting the pooled results one should consider that these are based on a heterogeneity of interventions, patient populations, and therapists. Furthermore, most included studies scored a high or unclear risk of bias on allocation concealment. A meta- epidemiological study stated that this bias may exaggerate treatment effects (Armijo-Olivo et al., 2015b). As blinding of participants or therapists in physical intervention studies is near impossible to have, most studies scored poorly on these aspects. Also, the interventions studied in this review could all be given by a physical therapist and are part of the physical therapy modalities, but were sometimes given by undefined therapists (Costa et al., 2015; Maluf et al., 2010), by a dentist (Michelotti et al., 2004), or a mandibular surgeon (Michelotti et al., 2012). Physical therapists are experts in the musculoskeletal field and are equipped to apply interventions to promote movement, reduce pain, restore function and prevent disability, just as the interventions within this review (APTA, 2015). If these interventions would have been applied to the patients by physical therapists, the outcomes may have been different. 4) Acknowledgements Not applicable. 5). Registration Although it is impossible to blind the therapist in hands-on and counseling studies, intention-to-treat and blinding of subjects are possible but were not used in the included studies. We suggest that future studies should include placebo or sham groups as a comparison so the placebo effect of those therapies can be explored (de Morton, 2009; Castro, 2007). If this is not possible, future studies should compare two distinct interventions to establish the effect of one intervention compared to the other. Furthermore, intention-to-treat analysis should be used more consequently, to reduce bias and increase the quality of methodology and the level of evidence (Higgins et al., 2011; de Morton, 2009). Future studies should also report the headache diagnosis of the patients, as it is very likely that the effect of TMD-treatment on primary headaches is different as compared to the effect on secondary head aches. More research needs to be done to establish the role primary headache may have in the effectiveness of TMD treatment (van der Meer et al., 2017a; Franco et al., 2010; Goncalves et al., 2011). Additionally, the treatment protocol needs to be available so physical therapists can apply the treatment methods in the clinical practice when the therapy is effective. The review is registered on PROSPERO (registration number CRD420170624). Supplementary data to this article can be found online at https://doi. org/10.1016/j.msksp.2020.102277. Supplementary data to this article can be found online at https://doi. org/10.1016/j.msksp.2020.102277. 4.3. Strengths and limitations of the study Most preferably, a collaboration between different dis ciplines should be applied in the future for optimal results (Rocabado et al., 1982; Gaul et al., 2011). For the current review, the findings should be interpreted with caution as there was not a physical therapist involved in each study, but contained other disciplines or home exer cises, so a full conclusion of the effectiveness of physical therapy cannot be given. Furthermore, this review did not include chiropractic or osteopathic interventions in the search. Even though all three pro fessions work with musculoskeletal complaints and could therefore be applied within the same review, they all require different educational degrees and are therefore not interchangeable. Thus, they were not included in the review, but it may be interesting for future studies to look at the effectiveness of those interventions on headache pain in tensity in patients with TMD. Finally, there were only two studies that specified which headache type the patients were diagnosed with (Costa et al., 2015; Piekartz and Hall, 2013). As different headache types have different etiologies, treatments may have a different effect on each headache type (Headache Classification C, 2018). 5. Conclusion Due to the methodological shortcomings, diversity of interventions and inconsistency of findings, there is currently a very low certainty that there is an effect of physical therapy for TMD on concomitant headache intensity compared to control interventions. 4.2. The role of the muscles and the cervical spine In this review, two studied interventions (stretching and orofacial therapy) focused on myogenous problems rather than arthrogenous (Maluf et al., 2010; Piekartz and Hall, 2013). Headache is more preva lent in patients with muscle-related TMD than in patients with joint-related TMD (van der Meer et al., 2017a; Goncalves et al., 2011; Ballegaard et al., 2008), and some headaches, for example TTH, are similar to certain muscular referred pain patterns (Bendtsen and Fern´andez-De-La-Pe˜nas, 2011; Fern´andez-p´erez et al., 2012). This may explain the effectiveness of the muscle-oriented physical therapy for TMD on headache intensity. Both stretching and orofacial therapy aim at 7 H.A. van der Meer et al. Musculoskeletal Science and Practice 50 (2020) 102277 Conflicts of interest There is no conflict of interest within this study. Ethical approval Not applicable. Funding This study was funded by the Dutch Research Council (Nederlandse Organisatie voor Wetenschappelijk Onderzoek – NWO) [grant number 023.006.004]. References Andrews, J.C., Schünemann, H.J., Oxman, A.D., et al., 2013. GRADE guidelines: 15. 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https://openalex.org/W2586663470	https://www.mdpi.com/2079-9276/6/1/8/pdf?version=1486558194	English	null	The Impact of Pay-As-You-Throw Schemes on Municipal Solid Waste Management: The Exemplar Case of the County of Aschaffenburg, Germany	Resources	2,017	cc-by	11,483	Juergen Morlok 1, Harald Schoenberger 2,3, David Styles 2,4, Jose-Luis Galvez-Martos 2,5,* and Barbara Zeschmar-Lahl 6 1 County of Aschaffenburg, Aschaffenburg D-63739, Germany; juergen.morlok@lra-ab.bayern.de 2 E3 Environmental Consultants Ltd, Aberdeen AB118DY, UK; y g, g , y; j g y 2 E3 Environmental Consultants Ltd, Aberdeen AB118DY, UK; harald.schoenberger@iswa.uni-stuttgart.de (H.S.); d.styles@bangor.ac.uk (D.S.) 2 E3 Environmental Consultants Ltd, Aberdeen AB118DY, UK; harald.schoenberger@iswa.uni-stuttgart.de (H.S.); d.styles@bangor.ac.uk (D.S.) E Environmental Consultants Ltd, Aberdeen AB118DY, UK; harald.schoenberger@iswa.uni-stuttgart.de (H.S.); d.styles@bangor.ac.uk (D.S.) 3 Institut für Siedlungswasserbau, Wasserguete- und Abfallwirtschaft, Universitaet Stuttgart, Stuttgart D-70569, Germany g y 4 School of Environment, Natural Resources and Geography, Bangor University, Bangor, LL572UW, UK 5 School of Engineering, University of Aberdeen, King’s College, Old Aberdeen, Aberdeen AB243UE, UK 6 BZL Kommunikation und Projektsteuerung GmbH, Oyten D-28876, Germany; bzl@bzl-gmbh.de * Correspondence: jlgalvez@e3europa.eu; Tel.: +44-7562-842388 4 School of Environment, Natural Resources and Geography, Bangor University, Bangor, LL572UW, UK 5 School of Engineering, University of Aberdeen, King’s College, Old Aberdeen, Aberdeen AB243UE, UK 6 BZL Kommunikation und Projektsteuerung GmbH, Oyten D-28876, Germany; bzl@bzl-gmbh.de * Correspondence: jlgalvez@e3europa.eu; Tel.: +44-7562-842388 Academic Editor: Johannes Paul Received: 2 November 2016; Accepted: 23 January 2017; Published: 8 February 2017 J Received: 2 November 2016; Accepted: 23 January 2017; Published: 8 February 2017 Abstract: The “pay-as-you-throw” (PAYT) scheme is an economic instrument for waste management that applies the “polluter pays” principle by charging the inhabitants of municipalities according to the amount of residual, organic, and bulky waste they send for third-party waste management. When combined with well-developed infrastructure to collect the different waste fractions (residual waste, paper and cardboard, plastics, bio waste, green cuttings, and many recyclables) as well as with a good level of citizens’ awareness, its performance has frequently been linked to an increase in the collection rates of recyclables. However, the establishment and operation of PAYT systems can require signiﬁcant resource inputs from municipalities. In this paper, PAYT is analysed through a case study from the German County of Aschaffenburg, covering nearly 20 years of implementation across 32 municipalities with 173,000 inhabitants. Key performance indicators include temporal trends in the county’s recyclables collection rate, waste treatment fees for residents, and municipal waste management costs, benchmarked against German municipalities not implementing PAYT. We conclude that PAYT could make an important contribution towards material reuse and recycling objectives for the new circular economy. Keywords: waste management; economic instruments; Pay-As-You-Throw; municipal solid waste; recycling; environmental management; awareness raising resources resources The Impact of Pay-As-You-Throw Schemes on Municipal Solid Waste Management: The Exemplar Case of the County of Aschaffenburg, Germany Juergen Morlok 1, Harald Schoenberger 2,3, David Styles 2,4, Jose-Luis Galvez-Martos 2,5,* and Barbara Zeschmar-Lahl 6 resources resources 1.1. Fundamentals of Pay-As-You-Throw Waste management policies include a range of complementary measures such as regulatory, economic, educational, and informative instruments [1,2]. The aim of an economic instrument is to persuade waste producers to divert waste from landﬁll or incineration towards material recovery, in order to optimise the use of resources while contributing to the costs of the waste management service. Economic instruments are implemented through national or regional waste policies, such as waste disposal taxes (landﬁll tax, incineration tax, product levies, etc.), waste pricing (unit based, differential rates, variable rates, pay as you throw, etc.), deposit refund schemes, extended producer Resources 2017, 6, 8; doi:10.3390/resources6010008 www.mdpi.com/journal/resources 2 of 16 Resources 2017, 6, 8 responsibility, tradable permits, recycling subsidies, value-added tax (VAT) exemptions for repair and recycling activities, etc. Most of these measures fall outside the scope of local governments. However, in the context of municipal solid waste management, the “Pay-As-You-Throw” (PAYT) approach (also known as unit pricing [3] and differential and variable rate or variable fee charge systems [1,2]) is an economic instrument that applies the “polluter pays” principle at the municipal level by charging inhabitants according to the amount of waste they send for third party management [4]. Technical implementation of the PAYT approach is based on the following three pillars: identiﬁcation of the waste generator, measurement of the amount of waste sent for treatment, and unit pricing, e.g., per kg and/or per emptying. The experience gained so far reveals that the waste fee should not only depend on the amount of waste generated, but should be comprised of both a basic and a variable (service-based) fee [5]. On the one hand, this reﬂects the cost structure of waste disposal, which consists of ﬁxed and variable costs [6], and, on the other hand, the inclusion of a ﬁxed (basic) fee helps to avoid illegal disposal practices which can increase if fees are only levied on collected waste quantities [7,8]. Figure 1 shows the conventional structure of a waste fee, wherein single component fees refer to charges based only on weight. Resources 2017, 6, 8 2 of 16 responsibility, tradable permits, recycling subsidies, value-added tax (VAT) exemptions for repair and recycling activities, etc. Most of these measures fall outside the scope of local governments. 1.1. Fundamentals of Pay-As-You-Throw However, in the context of municipal solid waste management, the “Pay-As-You-Throw” (PAYT) approach (also known as unit pricing [3] and differential and variable rate or variable fee charge systems [1,2]) is an economic instrument that applies the “polluter pays” principle at the municipal level by charging inhabitants according to the amount of waste they send for third party management [4]. Technical implementation of the PAYT approach is based on the following three pillars: identification of the waste generator, measurement of the amount of waste sent for treatment, and unit pricing, e.g., per kg and/or per emptying. The experience gained so far reveals that the waste fee should not only depend on the amount of waste generated, but should be comprised of both a basic and a variable (service-based) fee [5]. On the one hand, this reflects the cost structure of waste disposal, which consists of fixed and variable costs [6], and, on the other hand, the inclusion of a fixed (basic) fee helps to avoid illegal disposal practices which can increase if fees are only levied on collected waste quantities [7,8]. Figure 1 shows the conventional structure of a waste fee, wherein single component fees refer to charges based only on weight Figure 1. Possible waste fee components (adapted from [5]). Figure 1. Possible waste fee components (adapted from [5]). Figure 1. Possible waste fee components (adapted from [5]). Figure 1. Possible waste fee components (adapted from [5]). The PAYT approach means that a substantial part of the overall fee is allocated according to the amount of waste collected in order to motivate waste prevention and recovery. Against this background, the PAYT approach can be implemented in different ways [7] depending on the waste accounting method: - Per user identifier: The PAYT approach means that a substantial part of the overall fee is allocated according to the amount of waste collected in order to motivate waste prevention and recovery. 1.1. Fundamentals of Pay-As-You-Throw Against this background, the PAYT approach can be implemented in different ways [7] depending on the waste accounting method:  Volume-b  Weight-b - Per user identiﬁer: - Per bin identifier (individually o  Volume based accounting ■ Volume-based accounting  Volume-based accounting  Weight-based accounting ■ Weight-based accounting - Pre-paid systems - Per bin identiﬁer (individually or collectively assigned bins) Pre paid sack  Tag, sticker, or token ■ Volume-based accounting (identiﬁcation system) ■ Weight-based accounting Pre-paid systems ■ Pre-paid sack ■ Tag, sticker, or token Resources 2017, 6, 8 3 of 16 Resources 2017, 6, 8 The most common forms of PAYT schemes are volume-based schemes (choice of container size); sack-based schemes (number of sacks set out for collection); weight-based schemes (the weight of the waste collected in a given container); and frequency-based schemes (the frequency with which a container is set out for collection) [9]. This last approach can be combined with volume- and weight-based schemes. Common volume-based schemes, in which citizens pay for a speciﬁc size of container, may result in payments not corresponding with weight because containers are often only partially ﬁlled upon collection. The pre-paid sack system is commonly considered to be a volume-based system, although sacks are usually ﬁlled so that the volume and weight of a sack is relatively constant in relation to the fee paid per sack. 1.2. The County of Aschaffenburg The County of Aschaffenburg consists of 32 municipalities and has about 173,000 inhabitants and a population density of 247 inhabitants per km2. Until the early 1990s, untreated waste was landﬁlled. As the landﬁll neared its capacity limit, a new site was sought. However, public acceptance of a new landﬁll was very low, resulting in protests. As a consequence, the county was forced to develop new options to divert waste from landﬁll, and introduced the separate collection of plastic waste in 1990, the incineration of residual waste in a neighbouring county, the separate collection of wood waste in 1994, increased fees to reduce commercial waste, a reduced collection frequency for bulky waste (to twice per year), and separate bio waste collection in 1994/1995 with a trial in the municipality of Stockstadt [10]. Thus, the county switched from waste disposal by landﬁlling to waste management with the target of preventing and recycling waste. Following initial trials during 1994 to 1996 in the municipality of Stockstadt, the county rolled out a PAYT system in 1997. This system has been working ever since, and today, the County of Aschaffenburg has one of the highest recorded rates of recyclables collection (86%) and one of the lowest recorded rates of residual waste generation (55 kg per capita per year (kg·cap−1·yr−1)). The success of the PAYT system in the County of Aschaffenburg is replicated in other Germany counties (e.g., the County of Landsberg am Lech, the County of Schweinfurt, the County of Calw, the County of Heidenheim, and about 75% of the municipalities in the County of Wetterau), in Italy [11–13], and in Belgium [14]. However, its early adoption and the availability of a long data time series on waste management performance makes the County of Aschaffenburg an excellent PAYT case study and benchmark, as described in this paper. 1.3. PAYT as a Best Environmental Management Practice 1.3. PAYT as a Best Environmental Management Practice A best environmental management practice, BEMP, is deﬁned by the European Commission Regulation EC No. 1221/2009 on Eco-Audit and Management Schemes (EMAS) [15], article 2 point 14, as the “most effective way to implement the environmental management system by organisations in a relevant sector and that can result in best environmental performance under given economic and technical conditions”. A BEMP should minimise the environmental impact of organisational operations in a technically feasible and economically viable manner that is widely applicable across relevant organisations. In this context, a benchmark of excellence is deﬁned as the performance of frontrunners implementing best practice, whereas key performance indicators are used to report the performance of BEMPs and to quantify benchmarks of excellence. In the case of PAYT schemes, two key performance indicators are proposed. - Collection rate of recyclable materials (%). This indicator is frequently reported as “recycling rate”, but, given the amount of rejects from existing sorting and recycling plants, the term recyclables collection rate is preferred to avoid its misinterpretation. PAYT schemes are designed to increase the amount of recovered recyclable materials from municipal solid waste, so their implementation should increase values for this indicator. - Residual waste (kg·cap−1·yr−1). This is the amount of waste that the system user disposes in the residual waste bin. For practical recording reasons, this deﬁnition excludes the amount of waste - Residual waste (kg·cap−1·yr−1). This is the amount of waste that the system user disposes in the residual waste bin. For practical recording reasons, this deﬁnition excludes the amount of waste Resources 2017, 6, 8 4 of 16 rejected in recycling or sorting plants from the separately collected recyclable waste fraction(s) or illegally disposed waste. During the elaboration of a background report for the European Commission on best environmental management practices for the waste management sector in Europe [16], we concluded that, under certain conditions, PAYT is a BEMP and that the performance of the system in Aschaffenburg represented a benchmark of excellence. Speciﬁcally, we concluded that BEMP is a PAYT system comprised of a weight-based door-to-door collection of residual, organic, and bulky waste. The successful implementation of an efﬁcient PAYT system requires well-developed infrastructure to collect different fractions of recyclable waste in a convenient manner for citizens, either in individual bins outside their homes or in conveniently located centralized or mobile collection points. 1.4. Aim While PAYT is well known and has been implemented in many municipalities around Europe during the last ten years, there is a lack of detailed case studies published in the literature. To address this gap, we describe the 20-year implementation of PAYT in the County of Aschaffenburg, providing full disclosure on the initial aims, operational details, environmental performance, and economic aspects. The primary intention of this paper is to demonstrate successful implementation of PAYT as a best practice under speciﬁc conditions of applicability, acknowledging that PAYT is not always the most appropriate option depending on pre-existing infrastructure and public awareness. 1.3. PAYT as a Best Environmental Management Practice Materials that must be catered for include paper/cardboard/board, organic waste, packaging waste, glass containers, ferrous metals, non-ferrous metals, end-of-life electrical and electronic equipment, refrigerators and other white goods, waste plastic, pure waste polystyrene, waste wood, green cuttings, non-commercial construction and demolition waste, waste tyres, exhausted printer cartridges, waste vegetable fat, waste textiles and shoes, cork, CDs/DVDs, etc. Awareness raising is also a key element for effective PAYT implementation; informed citizens understand and support the scheme. All the practices necessary to assure successful implementation of PAYT are described in the background report on best environmental management practices for the waste management sector prepared for the European Commission [16]. During the elaboration of a background report for the European Commission on best environmental management practices for the waste management sector in Europe [16], we concluded that, under certain conditions, PAYT is a BEMP and that the performance of the system in Aschaffenburg represented a benchmark of excellence. Speciﬁcally, we concluded that BEMP is a PAYT system comprised of a weight-based door-to-door collection of residual, organic, and bulky waste. Th f l i l t ti f fﬁi t PAYT t i ll d l d i f t t Past studies have shown that pre-paid sack schemes achieve good performance [1], but that volume-based systems using varying bin sizes achieve comparatively poor performance in terms of waste prevention and recycling [1,9]. The highest recycling rates and lowest residual waste quantities are achieved with weight-based systems when they are accompanied by well-developed infrastructure and supported by waste-aware citizens. This is the case in Aschaffenburg, which we now report in more detail as a best practice case study. 2. Implementation of the System Resources 2017, 6, 8 5 of 16 facility via telemetry in real time, where processing, accounting, and the billing of end users occurs. Aschaffenburg also uses the collected data to measure the economic efficiency of the system and to optimise the logistics of the system. Figure 2. Process chart for electronic identification and data transfer in a bin identification scheme (adapted from [4]). All bi i d i h hi h b d b d h h b d Figure 2. Process chart for electronic identiﬁcation and data transfer in a bin identiﬁcation scheme (adapted from [4]). reader (see Figure 3) is only for the delivery and return of the bins. An example of a bar code is given in Figure 3 and examples of the chips are shown in Figure 4. For densely populated areas and high- rise buildings, access to container systems is restricted to assigned users. The owner or operator of high-rise buildings can opt for 1100 L containers for the whole community or for individual bins for each household in the building. The choice would always depend on the available space for individual containers. The experience in Aschaffenburg is that high-rise buildings with individual bins produce significantly less waste compared to buildings with large shared bins. In the case of shared bins, locks for bins are an optional service offered by the County and are intended to avoid misuse of bins by unauthorized users. All waste bins are equipped with a chip that can be read by a transponder, whereas the bar code reader (see Figure 3) is only for the delivery and return of the bins. An example of a bar code is given in Figure 3 and examples of the chips are shown in Figure 4. For densely populated areas and high-rise buildings, access to container systems is restricted to assigned users. The owner or operator of high-rise buildings can opt for 1100 L containers for the whole community or for individual bins for each household in the building. The choice would always depend on the available space for individual containers. The experience in Aschaffenburg is that high-rise buildings with individual bins produce signiﬁcantly less waste compared to buildings with large shared bins. In the case of shared bins, locks for bins are an optional service offered by the County and are intended to avoid misuse of bins by unauthorized users. Figure 2. 2. Implementation of the System The County of Aschaffenburg implements a weight-based collection of residual waste, bio waste, and bulky waste, as well as the separate collection of paper from all households. In nearly all of its 32 municipalities the County operates collection centres (also known as “container parks” or “civic amenity sites”) to separately collect recyclable waste fractions such as glass and metals, and the County composts green cuttings. In addition, woody fractions are sent to biomass-ﬁred power plants, residual waste is incinerated according to Best Available Technique (BAT) standards, bio waste is anaerobically digested, and subsidies are provided to households for home composting and for using re-usable nappies, and to households with incontinent persons. The motivation for introducing a weight-based charging system for residual and bio waste collection centered on fairness (user and polluter pays principles) for domestic users (commercial and industrial enterprises were excluded), the need to introduce new bins with wheels to facilitate manual handling as per Directive 90/269/EEC [17], the introduction of centralized billing by the county in 1994 (previously carried out by municipalities), high incineration costs, limited composting capacity for bio waste, and ecological considerations. 5 of 16 Resources 2017, 6, 8 Resources 2017, 6, 8 Initiation of the Aschaffenburg PAYT system required considerable effort to acquire and process data for billing, accounting, and system optimisation purposes. The data collection and processing scheme employed is illustrated in Figure 2. All bins and containers need to be coded and collection trucks are equipped with a reading device and a weighing device. Data are transferred to a central facility via telemetry in real time, where processing, accounting, and the billing of end users occurs. Aschaffenburg also uses the collected data to measure the economic efﬁciency of the system and to optimise the logistics of the system. Resources 2017, 6, 8 5 of 16 facility via telemetry in real time, where processing, accounting, and the billing of end users occurs. Aschaffenburg also uses the collected data to measure the economic efficiency of the system and to optimise the logistics of the system p g y Figure 2. Process chart for electronic identification and data transfer in a bin identification scheme (adapted from [4]). All waste bins are equipped with a chip that can be read by a transponder whereas the bar code Figure 2. Process chart for electronic identiﬁcation and data transfer in a bin identiﬁcation scheme (adapted from [4]). 2. Implementation of the System Examples of microchips (a) for new bins; and (b) for retrofitting existing bins [16,18]. Fi 5 h t ll ti t k th t i i d ith t id tifi ti t d Figure 4. Examples of microchips (a) for new bins; and (b) for retroﬁtting existing bins [16,18]. (a) Standard chip for new bins (b) Chips for the retrofit of existing bins a weighing system, requiring frequent maintenance and calibration owing to vibrations during operation of the truck. Experience shows that increased maintenance costs are compensated by the increased rates of collection for recyclable fractions. Figure 5 shows a waste collection truck that is equipped with a waste identiﬁcation system and a weighing system, requiring frequent maintenance and calibration owing to vibrations during operation of the truck. Experience shows that increased maintenance costs are compensated by the increased rates of collection for recyclable fractions. Figure 4. Examples of microchips (a) for new bins; and (b) for retrofitting existing bins [16,18]. Figure 5 shows a waste collection truck that is equipped with a waste identification system and a weighing system, requiring frequent maintenance and calibration owing to vibrations during operation of the truck. Experience shows that increased maintenance costs are compensated by the increased rates of collection for recyclable fractions Figure 5. A waste collection truck equipped with a waste identification system [16,18]. The benefits arising from the implementation of a weight-based PAYT system are dependent on the existence of well-developed infrastructure to separately collect and process the multitude of separated waste fractions, including residual waste, paper/cardboard, glass, plastics, and bio waste, ll d d l f h d h f ll Figure 5. A waste collection truck equipped with a waste identification system [16,18]. Figure 5. A waste collection truck equipped with a waste identiﬁcation system [16,18]. he existence of well-developed infrastructure to separately collect and process the multitude o eparated waste fractions, including residual waste, paper/cardboard, glass, plastics, and bio waste Figure 5. A waste collection truck equipped with a waste identification system [16,18]. Figure 5. A waste collection truck equipped with a waste identiﬁcation system [16,18]. receive various other fractions (e.g., green cuttings, demolition waste, bulky waste, etc.). Also, socioeconomic factors and environmental awareness appear to be important for PAYT success within a specific locality. PAYT may increase the risk of illegal dumping, although this did not occur in Aschaffenburg [19]. 2. Implementation of the System Process chart for electronic identification and data transfer in a bin identification scheme (adapted from [4]). All waste bins are equipped with a chip that can be read by a transponder, whereas the bar code reader (see Figure 3) is only for the delivery and return of the bins. An example of a bar code is given in Figure 3 and examples of the chips are shown in Figure 4. For densely populated areas and high- rise buildings, access to container systems is restricted to assigned users. The owner or operator of high-rise buildings can opt for 1100 L containers for the whole community or for individual bins for each household in the building. The choice would always depend on the available space for individual containers. The experience in Aschaffenburg is that high-rise buildings with individual bins produce significantly less waste compared to buildings with large shared bins. In the case of shared bins, locks for bins are an optional service offered by the County and are intended to avoid misuse of bins by unauthorized users Figure 3. Example of the information automatically read by an identification system, taken from [16,18]. Figure 3. Example of the information automatically read by an identification system, taken from [16,18]. Figure 3. Example of the information automatically read by an identiﬁcation system, taken from [16,18]. Figure 3. Example of the information automatically read by an identification system, taken from [16,18]. Figure 3. Example of the information automatically read by an identiﬁcation system, taken from [16,18]. Figure 3. Example of the information automatically read by an identification system, taken from [16,18]. Figure 3. Example of the information automatically read by an identiﬁcation system, taken from [16,18]. 6 of 16 Resources 2017, 6, 8 (a) Standard chip for new bins (b) Chips for the retrofit of existing bins Figure 4. Examples of microchips (a) for new bins; and (b) for retrofitting existing bins [16,18]. Figure 5 shows a waste collection truck that is equipped with a waste identification system and Figure 4. Examples of microchips (a) for new bins; and (b) for retroﬁtting existing bins [16,18]. Resources 2017, 6, 8 6 of 16 (a) Standard chip for new bins (b) Chips for the retrofit of existing bins (a) Standard chip for new bins (b) Chips for the retrofit of existing bins (a) Standard chip for new bins Figure 4. 2. Implementation of the System the existence of well-developed infrastructure to separately collect and process the multitude of separated waste fractions, including residual waste, paper/cardboard, glass, plastics, and bio waste, collected directly from citizens’ homes, and on the provision of convenient collection centres to receive various other fractions (e.g., green cuttings, demolition waste, bulky waste, etc.). Also, socioeconomic factors and environmental awareness appear to be important for PAYT success within a specific locality. PAYT may increase the risk of illegal dumping, although this did not occur in Aschaffenburg [19]. The beneﬁts arising from the implementation of a weight-based PAYT system are dependent on the existence of well-developed infrastructure to separately collect and process the multitude of separated waste fractions, including residual waste, paper/cardboard, glass, plastics, and bio waste, collected directly from citizens’ homes, and on the provision of convenient collection centres to receive various other fractions (e.g., green cuttings, demolition waste, bulky waste, etc.). Also, socioeconomic factors and environmental awareness appear to be important for PAYT success within a speciﬁc locality. PAYT may increase the risk of illegal dumping, although this did not occur in Aschaffenburg [19]. 3.1. Environmental Performance Figure 6 clearly illustrates the drastic change in waste management performance within the county. The introduction of a weight-based PAYT system across Aschaffenburg in 1997 was followed by a 7 of 16 Resources 2017, 6, 8 signiﬁcant increase in the collection of recyclable waste and a large decrease in residual waste disposal (Figure 6). The county achieved an overall collection rate for recyclables of up to 86% [19], which is a signiﬁcant improvement on the average performance of PAYT systems, with typical recycling rates of around 70% [7]. The 86% recycling rate in Aschaffenburg is considered to be a benchmark of excellence for the waste management sector. The main differentiating factors in this speciﬁc case are: - the use of a weighing system - provision of an extensive infrastructure for the collection of recyclable waste streams (see Table 1) - a high level of environmental awareness and active support from the citizens Table 1. Existing infrastructure for the collection of recyclable waste streams in Aschaffenburg [20]. Waste Type Collection System Residual waste Residual waste bin: collection rate every 14 days, with weighing and identiﬁcation; bin sizes: 120, 240, 660, or 1100 L, available upon request and with a lock Reloading station: direct delivery for a fee Bio waste Bio waste bin: collection rate every 14 days (every 7 days in the months of June, July, and August) with weighing and identiﬁcation, bin sizes: 60 or 120 L Garden waste/green cuttings Household collection rounds twice per year in each municipality on advertised dates. Delivery to municipal collection and shredding sites, or to the district recycling centre Waste paper Paper bin (blue bin), which has been introduced throughout the entire county, four-week collection; bin sizes: 240, or 1100 L Collection by a non-proﬁt association, infrequent in every municipality 30 collection centres (also known as “container parks” or “civic amenity sites”) Sales packaging Yellow recycling bags for light packaging: monthly collection Metals: depot containers (180 locations) Glass: depot containers for white, green, and brown glass (180 locations) Bulky waste for disposal Collection on call, written registration required, fee by weight Reloading station: direct delivery for a fee Bulky waste for recycling Waste wood, scrap metal, and electrical appliances (white goods, refrigerators, and display units) are collected twice a year at the kerbside with ﬁxed collection schedules. 3.1. Environmental Performance Due to the lack of reliable data, commercial waste similar to municipal solid waste (MSW) is not included. separately collected recyclable waste) on a per capita basis from 1995 to 2013 for the County of Aschaffenburg and for Germany overall. In 2013, the amount of residual waste arising in Aschaffenburg was 55 kg per capita per year, compared with 165 kg in 1995 and 220 kg·cap−1·yr−1 for Germany overall (down from 380 kg in 1995). The timing and magnitude of the decrease in residual waste in Aschaffenburg strongly suggest that the implementation of PAYT was a major driving force. However, the influences of other mechanisms implemented at local level, such as awareness raising and the development of better infrastructure for waste sorting and recycling, will also have been significant and were not an inherent component of PAYT per se. In any case, the stabilisation of residual waste quantities at a low level confirms the enduring effect of PAYT and associated infrastructure, given that initial awareness raising activities following PAYT implementation were not sustained. It is important to remark that, as with many other best practices in waste management, it is not possible to isolate the impact of a single technique from accompanying measures implemented simultaneously. Nonetheless, the specific case of Aschaffenburg is striking in the magnitude and immediacy of the impact following PAYT implementation, making it an outstanding exemplar of (probable) PAYT performance. Comparison with the German average performance over the same period indicates that wider socioeconomic conditions and societal awareness of waste management may have made a small contribution to reduced residual waste quantities in County Aschaffenburg. The total waste generated and the residual waste quantities were considerably smaller for Aschaffenburg than for Germany overall in 1995, before PAYT had been introduced. In order to estimate the “PAYT effect” in terms of residual waste avoidance, we considered the period 1995 to 2000, during which PAYT was rolled out across Aschaffenburg—covering the large reduction (71%) in residual waste generation (Figure 7). Over the same period, German average residual waste quantity declined by 19%. We assumed that residual waste generation in Aschaffenburg would have Figure 7 visualises the change in quantities of residual waste and total waste (disposed plus separately collected recyclable waste) on a per capita basis from 1995 to 2013 for the County of Aschaffenburg and for Germany overall. 3.1. Environmental Performance In 2013, the amount of residual waste arising in Aschaffenburg was 55 kg per capita per year, compared with 165 kg in 1995 and 220 kg·cap−1·yr−1 for Germany overall (down from 380 kg in 1995). The timing and magnitude of the decrease in residual waste in Aschaffenburg strongly suggest that the implementation of PAYT was a major driving force. However, the inﬂuences of other mechanisms implemented at local level, such as awareness raising and the development of better infrastructure for waste sorting and recycling, will also have been signiﬁcant and were not an inherent component of PAYT per se. In any case, the stabilisation of residual waste quantities at a low level conﬁrms the enduring effect of PAYT and associated infrastructure, given that initial awareness raising activities following PAYT implementation were not sustained. It is important to remark that, as with many other best practices in waste management, it is not possible to isolate the impact of a single technique from accompanying measures implemented simultaneously. Nonetheless, the speciﬁc case of Aschaffenburg is striking in the magnitude and immediacy of the impact following PAYT implementation, making it an outstanding exemplar of (probable) PAYT performance. Comparison with the German average performance over the same period indicates that wider socioeconomic conditions and societal awareness of waste management may have made a small contribution to reduced residual waste quantities in County Aschaffenburg. declined by an equivalent percentage under a counterfactual “business as usual (BAU)” scenario, against which the actual 1995 to 2000 reduction was benchmarked to estimate the additional PAYT effect (Table 2). Using this approach, we estimate that PAYT reduced residual waste by 84 The total waste generated and the residual waste quantities were considerably smaller for Aschaffenburg than for Germany overall in 1995, before PAYT had been introduced. In order to estimate the “PAYT effect” in terms of residual waste avoidance, we considered the period 1995 to 2000, during which PAYT was rolled out across Aschaffenburg—covering the large reduction (71%) in residual waste generation (Figure 7). Over the same period, German average residual waste quantity declined by 19%. We assumed that residual waste generation in Aschaffenburg would have declined by an equivalent percentage under a counterfactual “business as usual (BAU)” scenario, against which the actual 1995 to 2000 reduction was benchmarked to estimate the additional PAYT effect (Table 2). 3.1. Environmental Performance Special waste (hazardous waste in small quantities) Mobile collection, twice a year in each municipality (46 stopping points) Year-round acceptance of small quantities in the district recycling yard Collection centres Waste metal, waste wood, ﬂat glass, cans, hollow glass, waste paper, rubble, electrical appliances (IT and entertainment devices), non-ferrous metals, CDs, corks, used cooking oils, PU foam cans, and textiles provision of an extensive infrastructure for the collection of recyclable waste streams (see Table 1 a high level of environmental awareness and active support from the citizens 8 of 16 Resources 2017, 6, 8 Figure 6. Quantity of the different waste fractions of the County of Aschaffenburg from 1989 to 2013; the quantities are given in kg·cap−1·yr−1 [19]. Due to the lack of reliable data, commercial waste similar to municipal solid waste (MSW) is not included. Figure 7 visualises the change in quantities of residual waste and total waste (disposed plus Figure 6. Quantity of the different waste fractions of the County of Aschaffenburg from 1989 to 2013; the quantities are given in kg·cap−1·yr−1 [19]. Due to the lack of reliable data, commercial waste similar to municipal solid waste (MSW) is not included. Figure 6. Quantity of the different waste fractions of the County of Aschaffenburg from 1989 to 2013; the quantities are given in kg·cap−1·yr−1 [19]. Due to the lack of reliable data, commercial waste similar to municipal solid waste (MSW) is not included. Fi 7 i li th h i titi f id l t d t t l t (di d l Figure 6. Quantity of the different waste fractions of the County of Aschaffenburg from 1989 to 2013; the quantities are given in kg·cap−1·yr−1 [19]. Due to the lack of reliable data, commercial waste similar to municipal solid waste (MSW) is not included. Figure 6. Quantity of the different waste fractions of the County of Aschaffenburg from 1989 to 2013; the quantities are given in kg·cap−1·yr−1 [19]. Due to the lack of reliable data, commercial waste similar to municipal solid waste (MSW) is not included. Fi 7 i li th h i titi f id l t d t t l t (di d l Figure 6. Quantity of the different waste fractions of the County of Aschaffenburg from 1989 to 2013; the quantities are given in kg·cap−1·yr−1 [19]. 3.1. Environmental Performance Quantities of waste fractions arising in Aschaffenburg in 1995 and 2000, before and after the implementation of PAYT, and estimated counterfactual business-as-usual (BAU) quantities of waste fractions arising in 2000, based on the average reduction in residual waste in Germany over that period. Table 2. Quantities of waste fractions arising in Aschaffenburg in 1995 and 2000, before and after the implementation of PAYT, and estimated counterfactual business-as-usual (BAU) quantities of waste fractions arising in 2000, based on the average reduction in residual waste in Germany over that period. Waste type 1995 (A) 2000 PAYT (B) 2000 BAU (C) Absolute PAYT Effect Table 2. Quantities of waste fractions arising in Aschaffenburg in 1995 and 2000, before and after the implementation of PAYT, and estimated counterfactual business-as-usual (BAU) quantities of waste fractions arising in 2000, based on the average reduction in residual waste in Germany over that period. Table 2. Quantities of waste fractions arising in Aschaffenburg in 1995 and 2000, before and after the implementation of PAYT, and estimated counterfactual business-as-usual (BAU) quantities of waste fractions arising in 2000, based on the average reduction in residual waste in Germany over that period. Waste type 1995 (A) 2000 PAYT (B) 2000 BAU (C) Absolute PAYT Effect Table 2. Quantities of waste fractions arising in Aschaffenburg in 1995 and 2000, before and after the implementation of PAYT, and estimated counterfactual business-as-usual (BAU) quantities of waste fractions arising in 2000, based on the average reduction in residual waste in Germany over that period. g ( ) ( ) kg·cap−1·yr−1 Residual waste 163.0 48.0 132.0 −115.0 −84.0 Aluminium 0.2 0.1 0.2 −0.1 −0.1 Paper and cardboard 80.0 101.0 85.7 +21.0 +15.3 Mixed plastics 14.0 21.0 15.9 +7.0 +5.1 Glass 34.0 32.0 33.5 −2.0 −1.5 Steel & iron 21.0 20.0 20.7 −1.0 −0.7 Food waste 4.0 24.0 9.4 +20.0 +14.6 Green waste 75.0 74.0 74.7 −1.0 −0.7 Woody waste 24.0 20.0 22.9 −4.0 −2.9 Textiles 2.6 2.4 2.5 −0.2 −0.1 Construction 28.0 45.0 32.6 +17.0 +12.4 Hazardous 1.5 0.6 1.3 −0.9 −0.7 Bulky 27.0 2.7 20.5 −24.3 −17.8 Commercial 0.0 10.0 10.0 +10.0 +10.0 Change in residual waste not accounted for by increase in major separated fractions * −50.0 −36.5 * Calculated as the reduction in residual waste plus sum of increases in paper and cardboard, mixed plastics, and food waste. 3.1. Environmental Performance Using this approach, we estimate that PAYT reduced residual waste by 84 kg·cap−1·yr−1 and signiﬁcantly increased the separation (recycling) of paper and cardboard, mixed plastics, food waste, and construction materials (Table 2). Small reductions in the quantities of separated aluminium, glass, 9 of 16 Resources 2017, 6, 8 R steel and iron, woody waste, textiles, and hazardous waste may reﬂect the reduced generation of these fractions (waste prevention), given that total waste generation declined from 475 to 387 kg·cap−1·yr−1 during the same period. In fact, the decrease in the residual waste fraction is 50 kg·cap−1·yr−1 greater than the sum of increased waste separation over the period (Table 2), implying that a signiﬁcant proportion of residual waste reduction was attributable to waste prevention. kg·cap−1·yr−1 and significantly increased the separation (recycling) of paper and cardboard, mixed plastics, food waste, and construction materials (Table 2). Small reductions in the quantities of separated aluminium, glass, steel and iron, woody waste, textiles, and hazardous waste may reflect the reduced generation of these fractions (waste prevention), given that total waste generation declined from 475 to 387 kg·cap−1·yr−1 during the same period. In fact, the decrease in the residual waste fraction is 50 kg·cap−1·yr−1 greater than the sum of increased waste separation over the period (Table 2), implying that a significant proportion of residual waste reduction was attributable to waste prevention. Figure 7. Development of the quantities of residual waste and recyclables collection from municipal solid waste for 1995–2013 for (a) the County of Aschaffenburg [21] and (b) Germany [22]. Figure 7. Development of the quantities of residual waste and recyclables collection from municipal solid waste for 1995–2013 for (a) the County of Aschaffenburg [21] and (b) Germany [22]. Figure 7. Development of the quantities of residual waste and recyclables collection from municipal solid waste for 1995–2013 for (a) the County of Aschaffenburg [21] and (b) Germany [22]. Figure 7. Development of the quantities of residual waste and recyclables collection from municipal solid waste for 1995–2013 for (a) the County of Aschaffenburg [21] and (b) Germany [22]. Table 2. Quantities of waste fractions arising in Aschaffenburg in 1995 and 2000, before and after the implementation of PAYT, and estimated counterfactual business-as-usual (BAU) quantities of waste fractions arising in 2000, based on the average reduction in residual waste in Germany over that period. Waste type 1995 (A) 2000 PAYT (B) 2000 BAU (C) Absolute PAYT Effect Table 2. 3.1. Environmental Performance residual waste was based on residual waste composition recorded by the German waste management organisation Abfallzweckverband (AZV) Suedniedersachsen [23]. The AZV processes the residual and bulky wastes of the Counties of Goettingen, Northeim, and Osterode am Harz, and of the City of Goettingen, with about 490,000 inhabitants in total. Net GHG savings from additional recycling were calculated by multiplying the additional quantities of each material recycled by (i) the respective emission factors for recycling (burdens) and (ii) the respective GHG emissions for avoided raw material production (credits) [24]. It was assumed that food waste recycling occurred via anaerobic digestion, which does not derive an avoided food production credit, but does derive an energy credit, calculated based on a net electricity output of 0.21 MJ/kg food waste [25,26] and a German electricity GHG intensity of 0.65 kg CO2e per kWh [23]. Net GHG savings from avoided waste incineration were calculated by multiplying the entire quantity incinerated by an incineration emission factor [24] and subtracting an energy credit to represent the substitution of grid electricity generation with electricity generated from the waste incineration plant, assuming a lower heating value (LHV) of 10 MJ/kg for municipal solid waste at 65% dry matter, 30% net energy conversion efﬁciency, and a German electricity GHG intensity of 0.65 kg CO2e per kWh [24]. GHG emissions arising from the extraction of the raw materials needed to replace incinerated materials were calculated by multiplying speciﬁc residual waste fractions [19] by embodied carbon factors [24]. To avoid double counting with additional recycling credits, and to reﬂect the apparent waste prevention component of residual waste reduction, these calculations were only made for the change in residual waste not already accounted for by the increase in separated fractions (Table 2). Figure 8 presents the resulting GHG savings, expressed per capita per year. Considering the aforementioned factors, we conservatively estimate that PAYT implementation reduced GHG emissions by 91 kg·CO2e·cap−1·yr−1. At the municipality level (173,000 inhabitants), this translates into a GHG saving of 15,716 tonnes CO2e per year. of Goettingen, with about 490,000 inhabitants in total. Net GHG savings from additional recycling were calculated by multiplying the additional quantities of each material recycled by (i) the respective emission factors for recycling (burdens) and (ii) the respective GHG emissions for avoided raw material production (credits) [24]. 3.1. Environmental Performance In order to estimate the greenhouse gas (GHG) savings associated with PAYT implementation, we conservatively reflected on only the following effects, using the quantities presented in the last column of Table 2; the reduction in residual waste incineration and the increased recycling rates of paper and cardboard, mixed plastics, food waste, and construction materials. We did not consider ff b d h h d ff l f d f Waste Type 1995 (A) 2000 PAYT (B) 2000 BAU (C) Absolute Change (B−A) PAYT Effect (B−C) kg·cap−1·yr−1 Residual waste 163.0 48.0 132.0 −115.0 −84.0 Aluminium 0.2 0.1 0.2 −0.1 −0.1 Paper and cardboard 80.0 101.0 85.7 +21.0 +15.3 Mixed plastics 14.0 21.0 15.9 +7.0 +5.1 Glass 34.0 32.0 33.5 −2.0 −1.5 Steel & iron 21.0 20.0 20.7 −1.0 −0.7 Food waste 4.0 24.0 9.4 +20.0 +14.6 Green waste 75.0 74.0 74.7 −1.0 −0.7 Woody waste 24.0 20.0 22.9 −4.0 −2.9 Textiles 2.6 2.4 2.5 −0.2 −0.1 Construction 28.0 45.0 32.6 +17.0 +12.4 Hazardous 1.5 0.6 1.3 −0.9 −0.7 Bulky 27.0 2.7 20.5 −24.3 −17.8 Commercial 0.0 10.0 10.0 +10.0 +10.0 Change in residual waste not accounted for by increase in major separated fractions * −50.0 −36.5 * Calculated as the reduction in residual waste plus sum of increases in paper and cardboard, mixed plastics, and food waste. In order to estimate the greenhouse gas (GHG) savings associated with PAYT implementation, we conservatively reﬂected on only the following effects, using the quantities presented in the last column of Table 2; the reduction in residual waste incineration and the increased recycling rates of paper and cardboard, mixed plastics, food waste, and construction materials. We did not consider any waste prevention effect beyond this, in part owing to the difﬁculty of deﬁning appropriate quantities and counterfactual fates of individual fractions (e.g., what happens to bulky wastes, green wastes, and woody residues not collected as waste?). Owing to lack of early data on commercial waste, we could not accurately represent any change in this waste stream. The composition of residual waste was based on residual waste composition recorded by the German waste management organisation Resources 2017, 6, 8 wastes, and wo t ld 10 of 16 mercial iti f Abfallzweckverband (AZV) Suedniedersachsen [23]. The AZV processes the residual and bulky wastes of the Counties of Goettingen, Northeim, and Osterode am Harz, and of the City of Goettingen, with about 490,000 inhabitants in total. 3.1. Environmental Performance It was assumed that food waste recycling occurred via anaerobic digestion, which does not derive an avoided food production credit, but does derive an energy credit, calculated based on a net electricity output of 0.21 MJ/kg food waste [25,26] and a German electricity GHG intensity of 0.65 kg CO2e per kWh [23]. Net GHG savings from avoided waste incineration were calculated by multiplying the entire quantity incinerated by an incineration emission factor [24] and subtracting an energy credit to represent the substitution of grid electricity generation with electricity generated from the waste incineration plant, assuming a lower heating value (LHV) of 10 MJ/kg for municipal solid waste at 65% dry matter, 30% net energy conversion efficiency, and a German electricity GHG intensity of 0.65 kg CO2e per kWh [24]. GHG emissions arising from the extraction of the raw materials needed to replace incinerated materials were calculated by multiplying specific residual waste fractions [19] by embodied carbon factors [24]. To avoid double counting with additional recycling credits, and to reflect the apparent waste prevention component of residual waste reduction, these calculations were only made for the change in residual waste not already accounted for by the increase in separated fractions (Table 2). Figure 8 presents the resulting GHG savings, expressed per capita per year. Considering the aforementioned factors, we conservatively estimate that PAYT implementation reduced GHG emissions by 91 kg·CO2e·cap−1·yr−1. At the municipality level (173,000 inhabitants), this translates into a GHG saving of 15,716 tonnes CO2e per year. Figure 8. GHG savings attributable to the reduced incineration of residual waste and the additional recycling of separated waste fractions between 1995 and 2000 in the County of Aschaffenburg, based on the absolute changes in waste volumes and changes relative to estimated business-as-usual (Table 2), accounting for raw material substitution effects Figure 8. GHG savings attributable to the reduced incineration of residual waste and the additional recycling of separated waste fractions between 1995 and 2000 in the County of Aschaffenburg, based on the absolute changes in waste volumes and changes relative to estimated business-as-usual (Table 2), accounting for raw material substitution effects. Figure 8. 3.2. Economic Implications 3.2. Economic Implications Waste fees before and after introducing the weight-based system in Aschaffenburg are publicly available [20,27]. In 2013, the waste management fee in Aschaffenburg was lower than the fee before 1997 (Figure 9)—despite the additional activities and equipment associated with PAYT implementation (including the separate collection of the different fractions, the erection of facilities to recycle or to recover waste streams, weighing equipment, etc.). This fee reduction proves that weight-based PAYT is not necessarily a more expensive waste management option than others, contrary to some studies [28]. However, waste management costs vary greatly from one municipality to another and over time, based on a multitude of factors. The sharp decrease in residual waste sent for incineration between 1994 and 1997 reduced the disposal cost by 46% (Figure 9), though incineration costs were particularly high at that time (EUR 232 t−1 in 1997, compared with EUR 103.60 t−1 in 2014). In 1999, the waste management fee had to be increased by 10%, as the management costs until that time had not considered the whole county. In 2000, there was another increase of 10% to pay for sanitation measures on the legacy landﬁll. But from 2002 to 2013, the fee signiﬁcantly decreased by about 23%, even though the county further invested in the anaerobic digestion of bio waste, collection centres, weighed collection of green cuttings, and other measures. In 2015, the waste fee again decreased by 10%. Waste fees before and after introducing the weight-based system in Aschaffenburg are publicly available [20,27]. In 2013, the waste management fee in Aschaffenburg was lower than the fee before 1997 (Figure 9)—despite the additional activities and equipment associated with PAYT implementation (including the separate collection of the different fractions, the erection of facilities to recycle or to recover waste streams, weighing equipment, etc.). This fee reduction proves that weight-based PAYT is not necessarily a more expensive waste management option than others, contrary to some studies [28]. However, waste management costs vary greatly from one municipality to another and over time, based on a multitude of factors. The sharp decrease in residual waste sent for incineration between 1994 and 1997 reduced the disposal cost by 46% (Figure 9), though incineration costs were particularly high at that time (EUR 232 t−1 in 1997, compared with EUR 103.60 t−1 in 2014). 3.1. Environmental Performance GHG savings attributable to the reduced incineration of residual waste and the additional recycling of separated waste fractions between 1995 and 2000 in the County of Aschaffenburg, based on the absolute changes in waste volumes and changes relative to estimated business-as-usual (Table 2), accounting for raw material substitution effects Figure 8. GHG savings attributable to the reduced incineration of residual waste and the additional recycling of separated waste fractions between 1995 and 2000 in the County of Aschaffenburg, based on the absolute changes in waste volumes and changes relative to estimated business-as-usual (Table 2), accounting for raw material substitution effects. Figure 8. GHG savings attributable to the reduced incineration of residual waste and the additional recycling of separated waste fractions between 1995 and 2000 in the County of Aschaffenburg, based on the absolute changes in waste volumes and changes relative to estimated business-as-usual (Table 2), accounting for raw material substitution effects Figure 8. GHG savings attributable to the reduced incineration of residual waste and the additional recycling of separated waste fractions between 1995 and 2000 in the County of Aschaffenburg, based on the absolute changes in waste volumes and changes relative to estimated business-as-usual (Table 2), accounting for raw material substitution effects. 11 of 16 11 of 16 Resources 2017, 6, 8 R 2017 6 8 11 of 16 11 of 16 3.2. Economic Implications 3.2. Economic Implications ources 2017, 6, 8 12 of 16 Table 3 Waste treatment fee breakdown in the County of Aschaffenburg for the example of Figure 9 [20] Table 3. Waste treatment fee breakdown in the County of Aschaffenburg for the example of Figure 9 [20]. rces 2017, 6, 8 12 of Table 3 Waste treatment fee breakdown in the County of Aschaffenburg for the example of Figure 9 [20] Type of Fee 1997 2012 Basic fee 32% 47.0% Collection fee 17% 18.5% Weight fee 51% 34.5% breakdown in the County of Aschaffenburg for t Type of Fee 1997 2012 Basic fee 32% 47.0% Collection fee 17% 18.5% Weight fee 51% 34.5% The percentage for the weight component decreased, but it still appears sufﬁcient to motivate waste prevention/recycling. Figure 10 shows an example of the annual bill for a household in Aschaffenburg, indicating the basic fee, the service charge to collect the waste (collection fee) at a given frequency, and the weight fee, separated for residual waste and bio waste (at a basic fee of zero to encourage its separation). The percentage for the weight component decreased, but it still appears sufficient to motivate waste prevention/recycling. Figure 10 shows an example of the annual bill for a household in Aschaffenburg, indicating the basic fee, the service charge to collect the waste (collection fee) at a given frequency, and the weight fee, separated for residual waste and bio waste (at a basic fee of zero to encourage its separation). Figure 10. Example of an annual bill for waste services for a four-person household in Aschaffenb ith t bi f id l t (120 L) bi t (60 L) d / db d Figure 10. Example of an annual bill for waste services for a four-person household in Aschaffenb Figure 10. Example of an annual bill for waste services for a four-person household in Aschaffenburg with separate bins for residual waste (120 L), bio waste (60 L), and paper/cardboard. Figure 10. Example of an annual bill for waste services for a four-person household in Aschaffenburg with separate bins for residual waste (120 L), bio waste (60 L), and paper/cardboard. When comparing waste management costs across municipalities or cities, it is best to compare so-called “unrecovered costs”, representing the difference between total costs and total revenues. These are the costs that usually have to be passed on to citizens in the form of annual waste fees. 3.2. Economic Implications 3.2. Economic Implications In 1999, the waste management fee had to be increased by 10%, as the management costs until that time had not considered the whole county. In 2000, there was another increase of 10% to pay for sanitation measures on the legacy landfill. But from 2002 to 2013, the fee significantly decreased by about 23%, even though the county further invested in the anaerobic digestion of bio waste, collection centres, weighed collection of green cuttings, and other measures. In 2015, the waste fee again decreased by 10%. Figure 9. Evolution of waste fees in the County of Aschaffenburg from 1997 (the year the PAYT system for residual waste was established) to 2015 for an average four-person household with and without a bin for organic waste (data from [27]). h f h f A h ff b f h b f h ll f Figure 9. Evolution of waste fees in the County of Aschaffenburg from 1997 (the year the PAYT system for residual waste was established) to 2015 for an average four-person household with and without a bin for organic waste (data from [27]). Figure 9. Evolution of waste fees in the County of Aschaffenburg from 1997 (the year the PAYT system for residual waste was established) to 2015 for an average four-person household with and without a bin for organic waste (data from [27]). Figure 9. Evolution of waste fees in the County of Aschaffenburg from 1997 (the year the PAYT system for residual waste was established) to 2015 for an average four-person household with and without a bin for organic waste (data from [27]). 12 of 16 Resources 2017, 6, 8 Resources 2017, 6, 8 The fee in the County of Aschaffenburg consists of the basic fee, the collection fee (to empty the bins), and the weight fee. Between 1997 and 2012, the average fee breakdown changed as shown in Table 3. Table 3. Waste treatment fee breakdown in the County of Aschaffenburg for the example of Figure 9 [20]. ources 2017, 6, 8 12 of 16 Table 3 Waste treatment fee breakdown in the County of Aschaffenburg for the example of Figure 9 [20] Table 3. Waste treatment fee breakdown in the County of Aschaffenburg for the example of Figure 9 [20]. 3.2. Economic Implications 3.2. Economic Implications In the evaluation of total costs, the following costs are usually considered: • costs for collecting the different waste fractions (e.g., residual waste, bio waste, and paper) • costs for collecting the different waste fractions (e.g., residual waste, bio waste, and paper • costs for the treatment/disposal of residual waste (e.g., incineration) and the recycling/energy recovery of waste fractions, distinguishing between municipality-owned and third-party plants • costs for the operation, closure, and management of legacy landﬁlls (leachate treatment, landscaping, etc.) • costs for staff and administration related to waste management • miscellaneous costs Resources 2017, 6, 8 In addition, the total costs can include the costs for services provided: costs for staff and administration related to waste management  miscellaneous costs • by private waste management companies on behalf of the municipality • by the municipality itself In addition, the total costs can include the costs for services provided:  by private waste management companies on behalf of the municipality • by municipalities providing services for another municipality y p g p  by the municipality itself In the evaluation of revenues from recycling/recovery activities, the following income streams can be considered:  by municipalities providing services for another municipality In the evaluation of revenues from recycling/recovery activities, the following income streams b id d In the evaluation of revenues from recycling/recovery activities, the following income streams can be considered:  by municipalities providing services for another municipality In the evaluation of revenues from recycling/recovery activities, the following income streams can be considered • selling electricity or/and heat from the incineration of refuse derived fuels, residual w and biogas from the anaerobic digestion of bio waste or from landﬁlls • selling biogas from anaerobic digestion • selling separately collected or separated paper/cardboard • selling separately collected packaging • selling separately collected or separated scrap metal • selling compost • fees charged to businesses for waste collection and disposal  selling electricity or/and heat from the incineration of refuse derived fuels, residual waste biogas from the anaerobic digestion of bio waste or from landfills  selling biogas from anaerobic digestion  selling separately collected or separated paper/cardboard  selling separately collected packaging  selling separately collected or separated scrap metal  selling compost  fees charged to businesses for waste collection and disposal After the considerable efforts and measures implemented by the County of Aschaffenburg, the unrecovered costs are kept relatively low, compared with other counties and cities, at 44.5 EUR·cap−1·yr−1 in 2013 (Figure 11) [29]. 3.2. Economic Implications 3.2. Economic Implications In When comparing waste management costs across municipalities or cities, it is best to compare so-called “unrecovered costs”, representing the difference between total costs and total revenues. 13 of 16 Resources 2017, 6, 8 These are the costs that usually have to be passed on to citizens in the form of annual waste fees. 4. Discussion The implementation of PAYT in the County of Aschaffenburg is representative of a best environmental management practice, as the weight-based system produced particularly high collection rates of recyclable materials. Improving the efﬁciency of recyclables separation and collection is a priority for member states of the European Union to comply with overarching targets to recycle 65% of municipal waste and 75% of packaging waste by 2030, according to new objectives in the proposed directive on waste [30]. The implementation of PAYT schemes, along with the development of appropriate infrastructure, awareness raising, and other Circular Economy policies, would help to achieve such demanding objectives. Other types of waste collection have so far failed to achieve the same levels of landﬁll diversion and waste recycling. For instance, the amount of residual waste produced in Germany per capita per year has been very stable for the last ten years. The widespread adoption of proven approaches such as PAYT is urgently required to further improve the efﬁciency of waste management [16]. It is acknowledged within Circular Economy policies that local waste management organisations are crucial to the realisation of ambitious waste recovery objectives. Some recent examples of PAYT implementation in Italy have been reported to have achieved high recycling rates and low residual waste quantities. In the Treviso region, a residual waste quantity of only 55 kg·cap−1·yr−1 was reported for 2015 [11,12], and, in the municipality of Trento, a residual waste quantity of 102 kg·cap−1·yr−1 was achieved [13]. In Flanders, Belgium, pre-paid sacks were used in a simpliﬁed weight-based PAYT system, signiﬁcantly increasing the recycling rate to 71% and reducing the residual waste quantity to 149 kg·cap−1·yr−1 [14]. Pre-paid sack systems show a signiﬁcant decrease in the quantity of residual waste, but the achievable ﬁgures are lower compared to optimum weight-based systems, such as that implemented in Aschaffenburg; i.e., 53.5% in Switzerland, 46% in the County of Schweinfurt (Germany), 44% in Ghent and Destelbergen (Belgium), and 38% in Torelles de Llobregat (Spain) [1]. The applicability of PAYT is likely to be affected by geographical considerations. For instance, in a country with a hot climate, the collection frequency for bio waste has to be higher for hygiene reasons, which may be associated with higher collection costs. However, in that case, if a high degree of bio waste separation is achieved, the collection frequency for residual waste would be lower, compensating partially for the increase of costs. 3.2. Economic Implications 3.2. Economic Implications Thus, high environmental performance is not necessarily associated with high unrecovered costs. After the considerable efforts and measures implemented by the County of Aschaffenburg, the unrecovered costs are kept relatively low, compared with other counties and cities, at 44.5 EUR∙cap−1∙yr−1 in 2013 (Figure 11) [29]. Thus, high environmental performance is not necessarily associated with high unrecovered costs. Figure 11. Unrecovered costs in 33 counties and 11 cities in Germany in 2013, based on [28]. Figure 11. Unrecovered costs in 33 counties and 11 cities in Germany in 2013, based on [28]. Figure 11. Unrecovered costs in 33 counties and 11 cities in Germany in 2013, based on [28]. Figure 11. Unrecovered costs in 33 counties and 11 cities in Germany in 2013, based on [28]. Resources 2017, 6, 8 14 of 16 14 of 16 4. Discussion Under certain circumstances, the legislative framework is not compatible with the development of PAYT schemes. This particular case arises in the United Kingdom, where a debate continues about the applicability of PAYT at local level [31]. A well-studied alternative to PAYT in the UK that avoids legal barriers is the implementation of recycling incentive schemes. They consist of payments or rewards given to users to encourage people to recycle more, typically consisting of vouchers paid to individuals or communities, or waste management fee refunds paid back to individuals. The behavioural aspect is important here; while PAYT addresses a whole range of citizens with different awareness levels, recycling incentive schemes have the most impact on users with a high level of awareness, who act responsively and increase their recyclables collection rate accordingly [9]. For instance, Bracknell Forest increased the amount of recyclables collected by 91 kg·cap−1·yr−1 with a new incentive scheme that is popular among citizens and has improved public perceptions and acceptance of recycling. Other exemplary approaches have been demonstrated in The Netherlands, where recycling incentive schemes have reduced residual waste by 37% [16]. The experience gained with recycling incentive schemes shows that they can be considered a BEMP, due to their performance and costs, but cannot be benchmarked against PAYT due to their different scope and applicability. Finally, in the example of the County of Aschaffenburg, it is remarkable that, despite the very low quantities of residual waste achieved, the implementation of PAYT did not appear to have a signiﬁcant effect on the long-term total amount of waste generated and managed by the county. This has been recorded previously for other examples of PAYT. In other words, signiﬁcant waste prevention cannot be achieved through PAYT implementation, but requires other policies developed at the national or regional level (e.g., product policies, waste prevention plans, and tax regulations [32]) and more 15 of 16 Resources 2017, 6, 8 targeted actions at the local level, such as awareness raising campaigns, reuse initiatives, second hand markets, repair cafes, etc. targeted actions at the local level, such as awareness raising campaigns, reuse initiatives, second hand markets, repair cafes, etc. Acknowledgments: The Joint Research Centre of the European Commission is acknowledged for the funding received through contract 154367 2014 A08 DE. Author Contributions: J. 4. Discussion Morlok is responsible for municipal waste management in the County of Aschaffenburg and has provided the data shown in the paper and provided feedback on the text. H. Schoenberger, D. Styles, and J.L. Galvez-Martos have drafted the text and performed the analysis of the received data and the literature review. B. Zeschmar-Lahl has coordinated and led the work, contributed to the text, provided feedback, and proof-read the article before submission. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Impacts on Unit-Based Waste Collection Charges. Available online: http://www1.oecd.org/officialdocuments/ publicdisplaydocumentpdf/?cote=ENV/EPOC/WGWPR(2005)10/FINAL&docLanguage=En (accessed on 5 September 2016). 2. Van Beukering, P.J.H.; Bartelings, H.; Linderhof, V.G.M.; Oosterhuis, F.H. Effectiveness of unit-based pricing of waste in the Netherlands: Applying a general equilibrium model. Waste Manag. 2009, 29, 2892–2901. [CrossRef] [PubMed] 3. Dijkgraaf, E.; Gradus, R.H.J.M. Cost savings in unit-based pricing of household waste: The case of the Netherlands. Resour. 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Available online: http://ec.europa.eu/ environment/waste/pdf/ﬁnal_report_10042012.pdf (accessed on 5 September 2016). 10. County of Aschaffenburg. Proposal of the Waste Fee Dated 09.08.1995, submitted to the Council of the County (In German). 1995. Available online: http://www.landkreis-aschaffenburg.de (accessed on 5 September 2016). 11. Conto, P. Contarina Spa. Verso l’obiettivo dei 10 kg/ab All’anno di Riﬁuti Residui nel Trevigiano. 2015. Available online:http://www.forumriﬁuti.it/ﬁles/forumriﬁuti/docs/conto.pdf (accessed on 15 November 2015). 12. Contarina Spa. Integrated Waste Management. 2015. Available online: http://www.contarina.it/ﬁles/en/ ppt.pdf (accessed on 5 December 2015). 13. Fedrizzi, S. Progetto di Riduzione dei Riﬁuti nel Comune di Trento—Strategie di Prevenzione dei Riﬁuti. 2015. Available online: http://blank.ecomondo.com/upload_ist/AllegatiProgrammaEventi/Fedrizzi_2508495. pdf (accessed on 5 December 2015). 14. Regions for Recycling (R4R). 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https://openalex.org/W2516707762	https://europepmc.org/articles/pmc5517581?pdf=render	English	null	Serum NGAL and copeptin levels as predictors of acute kidney injury in asphyxiated neonates	Clinical and experimental nephrology	2,016	cc-by	5,323	& Piotr Surmiak piotr.surmiak@sum.edu.pl Clin Exp Nephrol (2017) 21:658–664 DOI 10.1007/s10157-016-1320-6 Clin Exp Nephrol (2017) 21:658–664 DOI 10.1007/s10157-016-1320-6 ORIGINAL ARTICLE ORIGINAL ARTICLE Keywords Asphyxia Acute kidney injury LCN2 protein Copeptins Osmolality Keywords Asphyxia Acute kidney injury LCN2 protein Copeptins Osmolality Results During the study period, 8 of asphyxiated nenates (18.6 %) suffered from AKI, while 35 newborns have no signs of AKI and 30 healthy infants. We did not observe any differences in creatinine and copeptin levels, as well as serum osmolality in all three investigated groups (AKI, no- AKI, and controls) in cord blood, and 24 h after birth. Serum NGAL levels in umbilical cord blood were signiﬁcantly higher in the AKI group (174.3 ng/mL) compared with no- AKI (88.5 ng/mL, p = 0.01) and control groups (28.5 ng/ mL, p \ 0.001), and 24 h after birth (respectively, AKI 152.5 ng/mL vs no-AKI 74.9 ng/mL, p = 0.02 vs controls 39.1 ng/mL, p \ 0.001). NGAL concentration showed a strong negative correlation to umbilical artery pH (Rho = -0.42, p = 0.04), base excess (Rho = -0.31, p = 0.03), and Apgar score in 1st min (Rho = -0.41, 1 Department of Neonatology, School of Medicine in Katowice, Medical University of Silesia, Medyko´w 14 Street, 40-752 Katowice, Poland Serum NGAL and copeptin levels as predictors of acute kidney injury in asphyxiated neonates Małgorzata Baumert1 • Piotr Surmiak1 • Andrzej Wie˛cek2 • Zoﬁa Walencka1 Received: 24 March 2016 / Accepted: 4 August 2016 / Published online: 2 September 2016 The Author(s) 2016. This article is published with open access at Springerlink.com p = 0.02) and 5th min of life (Rho = -0.20, p = 0.001). ROC curve analysis demonstrated a good predictive value for NGAL levels ([140.7 ng/mL) which allows to diagnose AKI in asphyxiated patients with 88.9 % sensitivity (95 % CI 75–95 %) and 95.0 % speciﬁcity (95 % CI 76–99 %). Conclusion NGAL seems to be a promising marker, even in subclinical AKI in neonates, due to its high speciﬁcity, but copeptin did not meet expectations. p = 0.02) and 5th min of life (Rho = -0.20, p = 0.001). ROC curve analysis demonstrated a good predictive value for NGAL levels ([140.7 ng/mL) which allows to diagnose AKI in asphyxiated patients with 88.9 % sensitivity (95 % CI 75–95 %) and 95.0 % speciﬁcity (95 % CI 76–99 %). 2 Department of Nephrology, Endocrinology and Metabolic Diseases, School of Medicine in Katowice, Medical University of Silesia, Francuska 20/24 Street, 40-027 Katowice, Poland & Piotr Surmiak piotr.surmiak@sum.edu.pl Abstract Background Acute kidney injury (AKI) is the most common complication of perinatal asphyxia. Recent research indi- cates that serum neutrophil gelatinase-associated lipocalin (NGAL) is an early marker for AKI, but there are the lacks of data about its use in term neonates with perinatal asphyxia. Methods A prospective cohort study was conducted on 43 term neonates. Umbilical cord blood and 24 h after birth serum NGAL, copeptin, creatinine, and molality were measured in all asphyxiated and controls neonates. Background Acute kidney injury (AKI) is the most common complication of perinatal asphyxia. Recent research indi- cates that serum neutrophil gelatinase-associated lipocalin (NGAL) is an early marker for AKI, but there are the lacks of data about its use in term neonates with perinatal asphyxia. Methods A prospective cohort study was conducted on 43 term neonates. Umbilical cord blood and 24 h after birth serum NGAL, copeptin, creatinine, and molality were measured in all asphyxiated and controls neonates. Results During the study period, 8 of asphyxiated nenates (18.6 %) suffered from AKI, while 35 newborns have no signs of AKI and 30 healthy infants. We did not observe any differences in creatinine and copeptin levels, as well as serum osmolality in all three investigated groups (AKI, no- AKI, and controls) in cord blood, and 24 h after birth. Serum NGAL levels in umbilical cord blood were signiﬁcantly higher in the AKI group (174.3 ng/mL) compared with no- AKI (88.5 ng/mL, p = 0.01) and control groups (28.5 ng/ mL, p \ 0.001), and 24 h after birth (respectively, AKI 152.5 ng/mL vs no-AKI 74.9 ng/mL, p = 0.02 vs controls 39.1 ng/mL, p \ 0.001). NGAL concentration showed a strong negative correlation to umbilical artery pH (Rho = -0.42, p = 0.04), base excess (Rho = -0.31, p = 0.03), and Apgar score in 1st min (Rho = -0.41, Keywords Asphyxia Acute kidney injury LCN2 protein Copeptins Osmolality Introduction Acute kidney injury (AKI) is a complex disorder with clinical manifestations ranging from mild dysfunction to complete anuric kidney failure. Renal failure is characterized by increased serum levels of creatinine and nitrogenous waste products, decreased in glomerular ﬁltration rate and imbalance in water and electrolyte homeostasis [1]. Diagnosis of AKI in neonates is difﬁcult as many of them have non-oliguric renal failure, especially in premature infants [2]. Moreover, serum creatinine concentration during the ﬁrst few days after birth reﬂects the mother’s and not the infant’s kidney function [3]. There are many reasons for development of AKI in newborns. Renal failure may have a prenatal onset in con- genital disease, especially in genetic diseases, and in the postnatal period maybe related to hypoxic ischemic injury, intubation at birth, respiratory distress syndrome, toxic insults, etc. [1, 4]. Some authors reported the high occurence of AKI in neonates with perinatal asphyxia [5, 6]. In a study by Abu-Haweleh, perinatal asphyxia, with a prevalence of 42 %, was the most common predisposing factor for AKI in neonates and thus, associated with high mortality [7]. As 123 3 Clin Exp Nephrol (2017) 21:658–664 659 neonates, with AKI, are at risk for developing chronic kid- ney disease and hypertension in the adult life, we search for novel markers that can improve the diagnosis of AKI within the ﬁrst hours of an insult allowing the implementation of an effective prophylactic actions and/or treatment. neonates, with AKI, are at risk for developing chronic kid- ney disease and hypertension in the adult life, we search for novel markers that can improve the diagnosis of AKI within the ﬁrst hours of an insult allowing the implementation of an effective prophylactic actions and/or treatment. newborns of mothers suffering from diabetes mellitus, hypertension, pre-eclampsia, and children of multiple pregnancies and metabolic disorders, and with evidence of congenital infections, as well as those that were born to mothers with clinical chorioamnionitis. Patient care and monitoring were performed as part of the hospital’s standard protocol. Neonates after the inci- dence of perinatal asphyxia were transferred to NICU, and acid–base balance was obtained 1 h after delivery. Needs for institution of hypothermia protocol was determined according to the recommendations from ‘‘Total Body Hypothermia for Perinatal Asphyxia (TOBY)’’ study group [16]; however, none of neonates from asphyxiated group met the criteria for whole body cooling. Methods Blood samples were drawn immediately after delivery of the child from the umbilical artery from the placental side of the cord and at 24 h after birth from the peripheral neonatal vessels. Blood samples were used for measure- ment of acid–base balance and serum NGAL, copeptin, creatinine, lactate levels as well as serum osmolality.After centrifugation, cord blood serum was frozen in tubes at -80 C. Serum copeptin concentration was determined using the immunoluminometric assay (Brahms CT-pro AVP LIA, Brahms GmbH, Hennigsdorf, Germany). The detection limit of the assay was 0.4 pmol/L. Introduction One of these novel biomarkers of AKI is neutrophil gelatinase-associated lipocalin (NGAL). It is known that serum and urinary levels of NGAL are elevated in neonates with AKI after cardiac surgery [8], and in a critically ill pediatric population [9]. In our previous studies, we demonstrated that NGAL can be a valuable biomarker of acute incidence of perinatal asphyxia in neonates [10, 11]. Hence, we want to test our hypothesis by demonstrating that elevated serum NGAL concentration is also an invaluable marker of AKI resulting from hypoxia.Moreover, we would also like to determine whether copeptin plays an important role in the evaluation of AKI. Copeptin is released in an equimolar ratio to vasopressin (AVP); however, it is more stable in circula- tion and easy to measure. Serum copeptin levels have been found to closely resemble the production of AVP, which is strongly related to serum osmolality [12]. Serum copeptin levels are increased in response to increased osmolality and dehydratation [13]. Some authors noticed that serum copeptin concentration was strongly related to factors associated with perinatal stress, such as asphyxia [14]. Material Serum NGAL concentration was determined using the sandwich enzyme immunoassay for the quantitative mea- surement of human lipocalin-2 (BioVendor- Labolatorni medicina a.s. Brno, Czech Republic). The detection limit of the assay was 0.02 ng/mL. Blood gas analyses were established using a model Rapidlab 865 Blood Gas Ana- lyzer (Siemens Medical Solutions Diagnostics, Bad Nau- heim, Germany). Serum creatinine concentration was measured by a kinetic colorimetric Jaffe method (Modular P Analyzer; Roche Almere, the Netherlands). Serum osmolality was determined using a cryoscopic method establishing its freezing temperature with the use of the Vapor Pressure Osmometer 5520. Among 1,673 infants, born at the University Hospital in Katowice, Poland between January 2012 to May 2014, 43 term neonates were enrolled in a prospective study, after obtaining informed written consent from their parents. This study was approved by the Human Ethics Committee of the Medical University of Silesia. The collection of umbilical cord blood and blood samples 24 h after birth was required for this research. Indicators of acute perinatal asphyxia included: the presence of a hypoxic event immediately prior or during delivery, history of fetal distress (bradycardia, late decel- erations), metabolic acidosis (base deﬁcit C-16 mmol/l) in the arterial umbilical cord blood, and pH B7.0. Statistical analysis Categorization of the enrolled asphyxiated neonates into asphyxia subgroups (AKI, no-AKI) was based on the Acute Kidney Injury Network (AKIN) criteria, as persistently increased serum creatinine ([1.5 mg/dL) for at least 24 h or rising values [0.3 mg/dL from the baseline [15]. Continuous variables among the groups were compared using the Kruskal–Wallis and Mann–Whitney U tests, while categorical variables were compared using Fisher’s exact or Levene’s tests. Quantitative variables were pre- sented as mean or median and 95 % conﬁdence intervals, whereas qualitative variables were shown as percentages. Spearman correlation coefﬁcients were calculated between NGAL, copeptin levels, and clinical variables. We also Thirty apparently healthy neonates of comparable ges- tational age born after an uncomplicated pregnancy and labor comprised the control group. In our study, we excluded preterm neonates, with con- genital abnormalities or chromosomal anomalies, 12 3 660 Clin Exp Nephrol (2017) 21:658–664 performed ROC curve analysis to determine the cutoff values of biochemical parameters as diagnostic markers of acute kidney injury in asphyxiated neonates. Statistical analysis was performed using standard procedures avail- able in STATISTICA 10 (Statsoft Polska Inc.) and Med- Calc Software Version 12.7.4. Statistical inferences were based on the level of signiﬁcance p \ 0.05. performed ROC curve analysis to determine the cutoff values of biochemical parameters as diagnostic markers of acute kidney injury in asphyxiated neonates. Statistical analysis was performed using standard procedures avail- able in STATISTICA 10 (Statsoft Polska Inc.) and Med- Calc Software Version 12.7.4. Statistical inferences were based on the level of signiﬁcance p \ 0.05. There were also no signiﬁcant differences in serum copeptin levels and serum osmolality in all three groups (AKI, no-AKI, and controls) in cord blood, and 24 h after birth, as shown in Table 2. There were also no differences in serum creatinine in umbilical cord blood and after 24 h in our population arranged by gestational age and birth weight, shown in Table 3. Although median serum copeptin concentrations were elevated within the AKI group (660.1 pg/mL) as compared with no-AKI (520.8 pg/mL) and controls (566.7 pg/mL), these differences were not signiﬁcant (p = 0.32). Result p value from Kruskal– Wallis or Fisher’s exact tests Table 2 Serum levels of creatinine (mg/dL), copeptin (pg/mL), NGAL (ng/mL), and serum osmolality (mmol/kg H2O) in cord blood and in serum after 24 h of life in asphyxiated (AKI and no-AKI) newborns and in the control group Asphyxiated n = 43 Controls n = 30 p value AKI n = 8 No-AKI n = 35 p value Umbilical creatinine (mg/dL) 0.8 (0.7–0.8) 0.9 (0.8–1.0) 0.56 0.9 (0.7–1.1) 0.8 (0.8–0.9) 0.26 Serum creatinine after 24 h (mg/dL) 0.8 (0.8–0.9) 0.9 (0.8–1.0) 0.62 1.0 (0.8–1.2) 0.8 (0.8–0.9) 0.09 Umbilical NGAL (ng/mL) 146.4 (105.8–187.0) 28.5 (21.9–35.0) \0.001 174.3 (117.8–230.7) 88.5 (59.1–118.0) \0.001 Serum NGAL after 24 h (ng/ mL) 113.5 (85.6–141.4) 39.1 (25.2–53.1) 0.06 152.5 (80.1–224.8) 74.9 (55.5–94.4) \0.001 Umbilical copeptin (pg/mL) 512.9 (429.2–596.6) 566.7 (452.4–681.1) 0.78 660.1 (273.5–1026.6) 520.8 (456.3–585.4) 0.32 Serum copeptin after 24 h (pg/ mL) 468.6 (396.7–540.5) 429.1 (342.8–515.5) 0.41 439.9 (310.3–569.5) 455.1 (394.8–515.3) 0.51 Umbilical serum osmolality (mmol/kg H2O) 284.5 (282.9–285.1) 284.1 (281.7–286.5) 0.72 286.4 (282.8–289.9) 284.1 (282.7–285.5) 0.29 Serum osmolality after 24 h (mmol/kg H2O) 283.8 (281.9–285.0) 284.0 (282.3–285.0) 0.35 283.1 (280.6–285.9) 284.1 (282.8–285.4) 0.76 Results are shown as medians and (95 % conﬁdence intervals). p value from Kruskal–Wallis and Levene’s tests NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury Table 1 Demographic and perinatal characteristics of asphyxiated and control neonates Asphyxiated neonates (n = 43) Control group (n = 30) p value Results are presented as medians and (95 % conﬁdence intervals) or percentages. Statistical analysis No differ- ences were observed between umbilical serum osmolality in all three investigated groups (AKI 286.4 mmol/kg H2O, no- AKI 284.1 mmol/kg H2O vs controls 284.1 mmol/kg H2O, p = 0.32, respectively), and 24 h after birth (AKI 283.1 mmol/kg H2O, no-AKI 284.1 mmol/kg H2O vs con- trols 284.0 mmol/kg H2O, p = 0.51, respectively)—Fig. 1. However, we observed signiﬁcant difference in serum osmolality between umbilical cord blood and venous blood in AKI group (p = 0.02). Result During the study period, 43 neonates were born asphyxi- ated-8 of them (18.6 %) developed AKI stage 1 based on serum creatinine AKIN criteria, while 35 newborns did not meet diagnostic criteria for AKI. Renal replacement ther- apy was not required. During the hospitalization, we observed in neonates with AKI creatinine levels normal- ization average on 3rd day of life. The demographic and clinical characteristics of the studied neonates are listed in Table 1. There were no sig- niﬁcant differences in these groups, except for the Apgar score (p \ 0.001). The median serum NGAL levels in umbilical cord blood were signiﬁcantly higher in the AKI group (174.3 ng/mL) Variable Asphyxiated neonates (n = 43) Control group (n = 30) p value Mother’s age (years) 27 (25–31) 28 (24–31) 0.48 Cesarean section (%) 85 71 0.18 Gender male/female (%) 54/46 61/39 0.58 Weeks of gestation (weeks) 37 (36–37) 38 (37–39) 0.11 Apgar 1st min (pts) 4 (3–6) 8 (8, 9) \0.001 Apgar 5th min (pts) 6 (5–7) 9 (8, 9) \0.001 Results are presented as medians and (95 % conﬁdence intervals) or percentages. p value from Kruskal– Wallis or Fisher’s exact tests Table 1 Demographic and perinatal characteristics of asphyxiated and control neonates Variable Asphyxiated neonates (n = 43) Control group (n = 30) p value Mother’s age (years) 27 (25–31) 28 (24–31) 0.48 Cesarean section (%) 85 71 0.18 Gender male/female (%) 54/46 61/39 0.58 Weeks of gestation (weeks) 37 (36–37) 38 (37–39) 0.11 Apgar 1st min (pts) 4 (3–6) 8 (8, 9) \0.001 Apgar 5th min (pts) 6 (5–7) 9 (8, 9) \0.001 Results are presented as medians and (95 % conﬁdence intervals) or percentages. NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury Result p value from Kruskal–Wallis and Levene’s tests NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury Asphyxiated n = 43 Controls n = 30 p value AKI n = 8 12 3 Clin Exp Nephrol (2017) 21:658–664 661 Table 3 Serum creatinine values in umbilical cord blood and after 24 h in our population arranged by gestational age and birth weight Variable Gestational age p value Birth weight p value 37–38 39–40 \2500 g [2500 g Creatinine level (mg/dL) Umbilical cord blood 0.74 (0.48–1.10) 0.68 (0.37–1.05) 0.41 0.81 (0.56–1.21) 0.65 (0.34–1.18) 0.18 24 h after 0.68 (0.36–1.25) 0.75 (0.44–1.15) 0.36 0.78 (0.39–1.09) 0.72 (0.42–1.27) 0.35 Results are presented as median values and (minimum and maximum values). p value from Mann–Whitney U test ne values in umbilical cord blood and after 24 h in our population arranged by gestational age and birth weight compared with no-AKI (88.5 ng/mL, p = 0.01) and con- trol groups (28.5 ng/mL, p \ 0.001), and 24 h after birth (respectively AKI 152.5 ng/mL vs no-AKI 74.9 ng/mL, p = 0.02 vs controls 39.1 ng/mL, p \ 0.001). A strong negative correlation between NGAL cord blood concentration and umbilical arterial pH (Rho = -0.42, ROC curve analysis demonstrated a critical level of NGAL in the umbilical cord blood[67.5 ng/mL, and this allows with 55.6 % sensitivity (95 % CI 28–79 %) and 89.5 % speciﬁcity (95 % CI 72–99 %) to predict AKI in newborns. However, serum NGAL concentration, 24 h after birth, with a value [140.7 ng/mL allows to diagnose AKI in asphyxiated Fig. 1 NGAL, copeptin and creatinine levels, and serum osmolality in neonates with acute kidney injury (AKI) and newborns no-AKI in umbilical cord blood and 24 h after birth. Results are shown as medians and 95 % conﬁdence intervals. NGAL neutrophil gelatinase- associated lipocalin, AKI acute kidney injury medians and 95 % conﬁdence intervals. NGAL neutrophil gelatinase- Fig. 1 NGAL, copeptin and creatinine levels, and serum osmolality in neonates with acute kidney injury (AKI) and newborns no-AKI in umbilical cord blood and 24 h after birth. Results are shown as medians and 95 % conﬁdence intervals. Result p value from Kruskal– Wallis or Fisher’s exact tests Table 2 Serum levels of creatinine (mg/dL), copeptin (pg/mL), NGAL (ng/mL), and serum osmolality (mmol/kg H2O) in cord blood and in serum after 24 h of life in asphyxiated (AKI and no-AKI) newborns and in the control group reatinine (mg/dL), copeptin (pg/mL), NGAL (ng/mL), and serum osmolality (mmol/kg H2O) in cord blood and in asphyxiated (AKI and no-AKI) newborns and in the control group Table 2 Serum levels of creatinine (mg/dL), copeptin (pg/mL), NGAL (ng/mL), and serum osmolality (mmol/kg erum after 24 h of life in asphyxiated (AKI and no-AKI) newborns and in the control group Table 2 Serum levels of creatinine (mg/dL), copeptin (pg/mL), NGAL (ng/mL), and serum osmolality (mmol/kg H2O) in cord blood and in serum after 24 h of life in asphyxiated (AKI and no-AKI) newborns and in the control group Asphyxiated n = 43 Controls n = 30 p value AKI n = 8 No-AKI n = 35 p value Umbilical creatinine (mg/dL) 0.8 (0.7–0.8) 0.9 (0.8–1.0) 0.56 0.9 (0.7–1.1) 0.8 (0.8–0.9) 0.26 Serum creatinine after 24 h (mg/dL) 0.8 (0.8–0.9) 0.9 (0.8–1.0) 0.62 1.0 (0.8–1.2) 0.8 (0.8–0.9) 0.09 Umbilical NGAL (ng/mL) 146.4 (105.8–187.0) 28.5 (21.9–35.0) \0.001 174.3 (117.8–230.7) 88.5 (59.1–118.0) \0.001 Serum NGAL after 24 h (ng/ mL) 113.5 (85.6–141.4) 39.1 (25.2–53.1) 0.06 152.5 (80.1–224.8) 74.9 (55.5–94.4) \0.001 Umbilical copeptin (pg/mL) 512.9 (429.2–596.6) 566.7 (452.4–681.1) 0.78 660.1 (273.5–1026.6) 520.8 (456.3–585.4) 0.32 Serum copeptin after 24 h (pg/ mL) 468.6 (396.7–540.5) 429.1 (342.8–515.5) 0.41 439.9 (310.3–569.5) 455.1 (394.8–515.3) 0.51 Umbilical serum osmolality (mmol/kg H2O) 284.5 (282.9–285.1) 284.1 (281.7–286.5) 0.72 286.4 (282.8–289.9) 284.1 (282.7–285.5) 0.29 Serum osmolality after 24 h (mmol/kg H2O) 283.8 (281.9–285.0) 284.0 (282.3–285.0) 0.35 283.1 (280.6–285.9) 284.1 (282.8–285.4) 0.76 Results are shown as medians and (95 % conﬁdence intervals). Result NGAL neutrophil gelatinase- associated lipocalin, AKI acute kidney injury compared with no-AKI (88.5 ng/mL, p = 0.01) and con- trol groups (28.5 ng/mL, p \ 0.001), and 24 h after birth (respectively AKI 152.5 ng/mL vs no-AKI 74.9 ng/mL, p = 0.02 vs controls 39.1 ng/mL, p \ 0.001). ROC curve analysis demonstrated a critical level of NGAL in the umbilical cord blood[67.5 ng/mL, and this allows with 55.6 % sensitivity (95 % CI 28–79 %) and 89.5 % speciﬁcity (95 % CI 72–99 %) to predict AKI in newborns. However, serum NGAL concentration, 24 h after birth, with a value [140.7 ng/mL allows to diagnose AKI in asphyxiated patients with 88.9 % sensitivity (95 % CI 75–95 %) and 95.0 % speciﬁcity (95 % CI 76–99 %)—Fig. 2. On the basis of the ROC curve analysis, a critical value was established for creatinine[1.09 mg/dL in serum after 24 h in prediction of A strong negative correlation between NGAL cord blood concentration and umbilical arterial pH (Rho = -0.42, p = 0.04), umbilical artery base excess (Rho = -0.31, p = 0.03), and Apgar score in 1st min (Rho = -0.41, p = 0.02) and 5th min of neonatal life (Rho = -0.20, p = 0.001). 12 3 662 Clin Exp Nephrol (2017) 21:658–664 AUC SE 95% CI P value* Umbilical NGAL level 0.66 0.14 0.46 – 0.83 0.01 NGAL after 24 hours 0.93 0.05 0.77 – 0.99 Fig. 2 Comparison of ROC curve analysis for prediction of AKI based on umbilical and 24-h NGAL levels in asphyxiated neonates. Comparison of ROC curves (Z statistic = 2.14). AUC area under ROC curve, SE standard error, 95 % conﬁdence interval for SE, NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury Fig. 2 Comparison of ROC curve analysis for prediction of AKI based on umbilical and 24-h NGAL levels in asphyxiated neonates. Comparison of ROC curves (Z statistic = 2.14). AUC area under ROC curve, SE standard error, 95 % conﬁdence interval for SE, NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury AKI in neonates with 71.4 % sensitivity (95 %CI 65–85 %) and 78.8 % speciﬁcity (95 %CI 63–90 %). However, area under ROC curve for umbilical creatinine level was not sig- niﬁcant in prediction of AKI—Fig. 3. Result We investigated NGAL, copeptin, creatinine levels, and serum osmolality in neonates after acute perinatal asphyxia with and without acute kidney injury, and in healthy controls. In our studies, we determined a signiﬁcant difference in NGAL concentration both in umbilical cord and venous blood after 24 h of life between asphyxiated neonates with AKI and without AKI. The ROC curve analysis revealed that serum NGAL concentration, at a cut-off value of 140.7 mg/dL after 24 h, could predict the development of AKI with high sensitivity (88.9 %) and speciﬁcity (95.0 %), while umbilical NGAL concentration character- ized with low sensitivity (55.6 %), but high speciﬁcity (89.5 %) for the prediction of AKI. We suggest that the elevation of serum NGAL concentration after 24 h may play a pivotal role as an indicator of hemodynamic insta- bility and serve as an important predictor of AKI. These data are in line with Raggal et al., who evaluated the NGAL concentration in asphyxiated neonates, 6 h after birth [21]. They demonstrated that a cut-off value of 157 ng/mL for serum NGAL could detect AKI in asphyxiated neonates with a sensitivity of 83.3 % and speciﬁcity of 94.4 %.El-Farghali et al. showed that serum Discussion There are many different causes of acute renal failure in newborns [1]. In this study, we have focused on asphyx- iated neonates. It is very important to recognize AKI as earlier as possible, hours after an insult occurred in comparison with days it may take serum creatinine to rise. However, validation of novel AKI biomarkers is impaired by the lack of a high quality, sensitive, and speciﬁc def- inition of AKI in neonates [17, 18]. In umbilical cord blood, normal creatinine values are unknown; however, there are some papers about creatinine values in neonates [19]. In our study, based on AKIN criteria in 8 neonates (18.6 %) of asphyxiated group diagnosed AKI and, simi- larly, on the faith of neonatal AKI KIDO classiﬁcation, 8 neonates developed AKI stage 1 [20]. 123 123 123 Clin Exp Nephrol (2017) 21:658–664 663 AUC SE 95% CI P value* Umbilical creatinine level 0.56 0.12 0.37 - 0.74 <0.001 Creatinine after 24hours 0.97 0.03 0.82 - 0.99 Fig. 3 Comparison of ROC curve analysis for prediction of AKI based on umbilical and 24-h creatinine levels in asphyxiated neonates. Comparison of ROC curves (Z statistic = 3.50). AUC area under ROC curve, SE standard error, 95 % conﬁdence interval for SE. NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury Fig. 3 Comparison of ROC curve analysis for prediction of AKI based on umbilical and 24-h creatinine levels in asphyxiated neonates. Comparison of ROC curves (Z statistic = 3.50). AUC area under ROC curve, SE standard error, 95 % conﬁdence interval for SE. NGAL neutrophil gelatinase-associated lipocalin, AKI acute kidney injury NGAL may also be serve as a clinically useful marker for early detection of AKI in critically ill neonates with sepsis [22]. The kidney responds to sepsis by upregulating NGAL production [23]. NGAL is one of the most strikingly upregulated genes and is an overexpressed protein in kid- neys after ischemia [18]. which results in the increase in serum copeptin concen- tration [26]. In our study, serum copeptin concentration did not reveal signiﬁcant diagnostic elements as a marker of osmolality. We have observed a signiﬁcantly lower serum osmolality, in the umbilical cord than in venous blood after 24 h, in neonates with AKI, but there were no signiﬁcant differences in serum copeptin concentra- tion. This could be as a result of possible subclinical kidney injury. Discussion Our ﬁndings indicate that copeptin cannot to be a pre- dictor of early AKI. We did not observe any signiﬁcant differences of serum copeptin levels in the groups with and without AKI, as well as the control group. Serum copeptin concentrations are characterized by a low sensitivity and speciﬁcity both in umbilical cord and venous blood after 24 h. These data are, however, different than obtained by Schlapbach et al. [24]. The authors indicated the highest copeptin cord blood concentrations in neonates with peri- natal asphyxia. They showed also that umbilical copeptin concentrations above 400 pmol/l had a high sensitivity and speciﬁcity for asphyxia.Copeptin pre-pro-vasopressin split product is also known as a marker of hydration status. Physiologically, when plasma osmolality is higher, copeptin levels are also increased [25]. Some authors demonstrated that in adults, during a hypertonic saline infusion and thirsting, there is a rise of serum osmolality A non-signiﬁcant decrease in copeptin concentration 24 h after birth has also been shown in the group with no- AKI and control group, however, without any differences in serum osmolality. Due to the absence of differences in copeptin concentration in asphyxiated infants (AKI group and no-AKI) and healthy children, we suggest that copeptin cannot be a valuable, early marker of asphyxia and AKI in neonates. However, Benzing et al. disagree with this statement [27]. The authors postulated that cord blood copeptin is highly sensitive albeit minimally speciﬁc marker of fetal/neonatal distress. Most likely the small number of investigated infants contributed to the difference between the results. 3 3 664 Clin Exp Nephrol (2017) 21:658–664 9. Zappitelli M, Washbum KK, Arikan AA, et al. Urine neutrophil gelatinase-associated lipocalin is an early marker of acute kidney injury in critically ill children: a prospective cohort study. Crit Care. 2007;11:173. Compliance with ethical standards 12. Mellor AJ, Boos CJ, Ball S, et al. Copeptin and arginine vaso- pressin at high altitude: relationship to plasma osmolality and perceived exertion. Eur J Appl Physiol. 2015;115:91–8. Conﬂict of interest The authors have declared that no conﬂict of interest exists. Conﬂict of interest The authors have declared that no conﬂict of interest exists. 13. Morgenthaler NG, Struck J, Jochberger S, et al. Copeptin: clinical use of a new biomarker. Trends Endocrinol Metab. 2008;19:43–9. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee at which the studies were con- ducted (Medical University of Silesia in Katowice no. KNW/022/ KB1/35/12) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 14. Schlapbach LJ, Frey S, Bigier S, et al. Copeptin concentration in cord blood in infants with early-onset sepsis, chorioamnionitis and perinatal asphyxia. BMC Pediatr. 2011;11:38. 15. Mehta RL, Kellum JA, Shah SV, et al. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit Care. 2007;11:R31. 16. Azzopardi DV, Strohm B, Edwards AD, TOBY Study Group, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. N Engl J Med. 2009;361:1349–58. Informed consent Informed consent was obtained from all individ- ual participants included in the study. 17. Sweetmann DU, Molloy EJ. Biomarkers of acute kidney injury in neonatal encephalopathy. Eur J Pediatr. 2013;172:305–16. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.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. 18. Mishra J, Ma Q, Prada A, et al. Identiﬁcation of NGAL as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol. 2003;14:2534–43. 19. Bateman DA, Thomas W, Parravicini E, Polesana E, Locatelli C, Lorenz JM. Serum creatinine concentration in very-low-birth- weight infants from birth to 34–36 wk postmenstrual age. Pediatr Res. 2015;77:696–702. 20. Selewski DT, Charlton JR, Jetton JG, Guillet R, Mhanna MJ, Askenazi DJ, Kent AL. Neonatal acute kidney injury. Pediatrics. 2015;136:e463–73. Conclusions Elevated serum NGAL concentration seems to be a promising, novel marker, even in subclinical forms of AKI in asphyxiated neonates, due to its high speciﬁcity, but copeptin did not meet such expectations. 10. Surmiak P, Baumert M, Fiala M, et al. Umbilical cord blood NGAL concentration as an early marker of perinatal asphyxia in neonates. Ginekol Pol. 2014;85:424–7. 11. Fiala M, Baumert M, Surmiak P, et al. Umbilical markers of perinatal hypoxia. Ginekol Pol. 2016;87:200–4. References 21. Raggal NE, Khafagy SM, Mahmoud NH, et al. Serum NGAL as a marker of acute kidney injury in asphyxiated neonates. Indian Pediatr. 2013;50:459–62. 1. Andreoli SP. Acute renal failure in the newborn. Semin Perinatol. 2004;28:112–3. 2. Gupta BD, Sharma P, Bagla J, et al. Renal failure in asphyxiaed neonates. Indian Pediatr. 2005;42:928–34. 22. El-Farghali OG, El-Raggal NM, Mahmoud NH, et al. Serum neutrophil gelatinase- associated lipocalin as a predictor of acute kidney in cricitally-ill neonates. Pak J Biol Sci. 2012;15:231–7. 3. Askenazi DJ, Ambalavanan N, Goldstein SL. Acute kidney injury in critically ill newborns: what do we know? What do we need to learn? Pediatr Nephrol. 2009;24:265–74. 23. Parracini E, Nemerofsky SL, Michelson KA, et al. Urinary neu- trophil gelatinase- associated lipocalin is a promising biomarker for late onset culture- positive sepsis in very low birth weight infants. Pediatr Res. 2010;67:636–40. 4. Cataldi L, Leone R, Morett U, et al. Potential risk factors for the development of acute failure in preterm newborn infants: a case– control study. Arch Dis Child Fetal Neonatal Ed. 2005;90:514–9. 24. Meijer E, Bakker SJ, Halbesman N, et al. Copeptin, a surrogate marker of vasopressin, is associated with microalbuminuria in large population cohort. Kidney Int. 2010;77:29–36. 5. Portman RJ, Carter BS, Gaylord MS, et al. Predicting neonatal morbidity after perinatal asphyxia: a scoring system. Am J Obstet Gynecol. 1990;162:174–82. 25. Szinnai G, Morgenthaler NG, Berneis K, et al. Changes in plasma copeptin, the C-terminal portion of arginine vasopressin during water deprivation and excess in healthy subjects. J Clin Endo- crinol Metab. 2007;92:972–3978. 6. Karlo J, Bhat BV, Koner BC, et al. Evaluation of renal function in term babies with perinatal asphyxia. Indian J Pediatr. 2014;81:243–7. 6. Karlo J, Bhat BV, Koner BC, et al. Evaluation of renal function in term babies with perinatal asphyxia. Indian J Pediatr. 2014;81:243–7. 7. Abu-Haweleh AF. Acute renal failure in newborn: etiology and mortality rate in Jordan patients. Saudi J Kidney Dis Transplant. 1998;9:18–21. 26. Benzing J, Wellmann S, Achini F, et al. Plasma copeptin in preterm infants: a highly sensitive marker of fetal and neonatal stress. J Clin Endocrinol Metab. 2011;96:E982–5. 8. Mishra J, Dent C, Tarabinshi R, et al. Neutrophil gelatinase- associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet. 2005;365:1231–8. 12 123
https://openalex.org/W3045347208	https://www.e3s-conferences.org/10.1051/e3sconf/202018001009/pdf	English	null	Numerical modeling of the thermal conduction process in water-air convector’s fins	E3S web of conferences	2,020	cc-by	4,442	* Corresponding author: zkolev@uni-ruse.bg Numerical modeling of the thermal conduction process in water-air convector’s fins Zhivko Kolev1,*, and Seher Kadirova 2 1University of Ruse, Department of Heat, Hydraulics and Environmental Engineering, 7017 Ruse, Bulgaria 2University of Ruse, Department of Electronics, 7017 Ruse, Bulgaria 2University of Ruse, Department of Electronics, 7017 Ruse, Bulgaria Abstract. The paper presents results from numerical modelling of thermal conduction process in ABAQUS. The computing mesh of the model has been verified. Based on the simulation results, the average temperature of the heat exchanger’s external surface and the external heat convection coefficient have been determined. The process of thermal conduction in the fins is modelled on the basis of boundary conditions, which values are obtained by simulation of the heat convection process in the convector’s pipes. The average heat parameters have been determined relative to the first, middle and last fins, by developing a geometric model in ABAQUS. The computing mesh has been verified by increasing the number of cells and nodes. The external heat convection coefficient is calculated by Newton's law. © 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/). E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 1 Introduction The processes of thermal conduction and the determination of their parameters have been studied in various fields. In [1] the thermal conductivity of building composite materials has been investigated. The thermal conductivity coefficient of the materials has been determined by TCi Mathis Thermal Conductivity Analyzer for non-destructive measurements. The mass specific heat capacity of the materials has been also determined. The paper presents investigation of thermal-physical characteristics of environmentally friendly building materials made by clay, sand and straw. Paper [2] presents determination of the heat capacity, thermal conductivity and thermal diffusivity of linalool - ethanol - water solutions. The purpose is determination of physicochemical and thermal properties of solutions with different ethanol concentrations, in order to describe the stability of the system. Heat transfer in thin plates with temperature dependent conductivity, has been considered in [3]. Due to the small thickness of the plates, two-dimensional modeling has been used. Graphical dependences of the thermal conductivity coefficient on the temperature of the plates are built. Investigation [4] presents numerical study of transient heat transfer process in longitudinal fins with different profiles. In the numerical models, linear or nonlinear functions of the temperature for the heat transfer coefficient and thermal conductivity coefficient have been used. E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 Many papers present investigations of heat transfer processes in finned tube exchangers. Many papers present investigations of heat transfer processes in finned tube exchangers. In [5] heat transfer and flow characteristics of air over flat finned tubes, have been determined. Non-perforated fins and perforated with different shapes fins, have been investigated. The aim is determination of effective passive techniques for thermal performance enhancement of finned heat exchangers. Paper [6] presents experimental study of heat transfer in cross-flow heat exchanger with smooth tubes or integral finned tubes. The heat flow, heat transfer coefficient and overall heat transfer coefficient, have been determined at different velocities of the water flow in tubes, and different velocities of the air flow, which crosses the tubes. Experimental investigation of spiral finned tube heat exchanger has been considered in [7]. Different fin pitch, fin height, transverse tube pitch, and longitudinal tube pitch, have been investigated. The heat transfer Nusselt number and the flowing resistance Euler number, have been determined at different fluid flowing Reynolds number. Another research presents determination of the heat transfer coefficient of longitudinal finned tubes [8]. 1 Introduction The local heat transfer coefficient and the temperature distribution have been determined for various Reynolds numbers of the fluid flow. In [9] a fin and tube heat exchanger has been studied by numerical modeling method. The gas flowing over the fin side is simulated at constant internal tube wall temperature, and the temperature distribution in the fins and the fluid velocity profile, have been determined. Paper [10] presents experimental study of the convective heat transfer process in finned tube counter flow heat exchanger. The convection heat transfer coefficient has been determined for plain tube, two finned tube and three finned tube. The investigation has been accomplished by different compositions of the liquid flowing in the pipes, at different Reynolds numbers. Numerical modeling of cross flow heat exchanger with louvered fins has been considered in [11]. A numerical model has been developed based on the thermal resistance concept and the finite difference method. Investigation [12] presents simulation study of a finned-tube latent heat thermal energy storage system. Numerical method based on heat capacity formulation, has been used for investigation of the influence of the convection heat transfer on the processes in the storage system. In [13] numerical simulation of the heat transfer in finned tube heat recovery unit, is considered. A method based on fluid-solid coupling is used for calculation of some parameters for the heat transfer process between the tube and the fluids. Paper [14] presents numerical study of circular tube bank fin heat exchanger with vortex generators. The fin’s temperature distribution and the fluid’s velocity distribution have been determined by simplified 3D model. CFD simulation investigation of the heat transfer in the pipes of water-air convector is considered in [15]. Pipe’s sections with reduced heat transfer intensity, have been investigated. The numerical modeling of heat transfer processes is related to determination of the temperature field [15-17]. ABAQUS is used in [15] and [16], while in [17] the used software product is ANSYS FLUENT. The investigation of heat transfer processes in the heat consumers of heat pump installations is important, in terms of the wide-ranging use of the heat pumps for different needs [15, 18-21]. An experimental reversible water-to-water heat pump installation for air- conditioning is considered in [15] and [18]. The operation of air-to-water heat pump system for building’s heating, has been investigated in [19]. 1 Introduction In [20] and [21] mechanical vapor recompression heat pump for treatment of industrial wastewater, has been investigated. For determination of certain parameters, in some cases it is necessary to study a sequence of heat transfer processes. The paper presents investigation of thermal conduction processes in the fins of water-air convector. The study has been accomplished in heating regime of the laboratory heat pump installation. The convector is used as a heat consumer. The temperature field in several fins of the convector’s heat exchanger has been modelled, based on the results obtained from numerical modelling of heat transfer process in the 2 2 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 convector’s pipes [15]. The temperature distribution results in the convector’s fins have been used for determination of the heat convection coefficient (  c ex t ernal h , W/m2.K) between the heat exchanger’s external surface and the air flow. For this purpose, the average temperature of the surface ( wt , ºC) has been calculated. The calculations are on the base of two types of simulation results. One of them is the temperature field along the internal surface of the pipes. The other one is the temperature results for the heat exchange surface of three fins along the heat exchanger. The investigated fins are the first (№1), the middle (№79) and the last one (№157), respectively. 2.1 Principal scheme of heat exchanger Figure 1 presents a principal scheme of the convector’s heat exchanger. It is two-pipe staggered finned pipe sheaf. When the heat pump installation operates in heating mode, the heat flow is transferred from the water flow in the pipes to the ambient air. The first row of finned pipes in the direction of air flow has been investigated. Fig. 1. Principal scheme of heat exchanger. Fig. 1. Principal scheme of heat exchanger. Fig. 1. Principal scheme of heat exchanger. 2.3 Verification of computing mesh The verification of computing mesh has been performed using two temperatures (t1 and t2, ºC), defined for computing nodes. They are located on two of the edges of the middle fin (Fig. 3). Since the thermal resistance of the fin’s wall is negligible, it is assumed that these are the values of the temperatures of the edges’ nodes, located on the fin’s opposite side. Fig. 3. Temperature points for verification of computing mesh. Fig. 3. Temperature points for verification of computing mesh. Fig. 3. Temperature points for verification of computing mesh. Fig. 3. Temperature points for verification of computing mesh. Fig. 3. Temperature points for verification of computing mesh. 2.2 Schematic diagram of the simulation model Figure 2 presents a schematic diagram of the numerical model for simulation of the temperature field in the convector’s fins. For this purpose, the heat flux (q, W/m2) from the heat exchange surface to the environment, and the average temperatures of the fins’ internal 3 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 cylindrical surfaces (tw-1(I) ÷ tw-4(X), ºC), have been set as boundary conditions. The temperature values have been determined from the simulation of the heat transfer process in the convector’s pipes. Fig. 2. Schematic diagram of the simulation model. Fig. 2. Schematic diagram of the simulation model. 2.4 Calculation of the heat convection coefficient The heat convection coefficient between the heat exchanger’s external surface and the air flow has been calculated by (1): c ex t ernal w heated air q h t t    (1) (1) (1) where heated air t is the average temperature of the air, heated by the exchanger’s external surface, ºC. This temperature has been calculated on the base of the basic heat equation, using experimentally determined parameters of the heat pump installation. https://doi.org/10.1051/e3sconf/202018001009 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 E3S Web of Conferences 180, 01009 (2020) 3.1 Results from determination of the boundary conditions Figures 4 - 6 present results obtained for the temperature field in the convector’s pipes. They are base for calculation of the average temperature of the external cylindrical surfaces of the pipes between the fins, and the temperatures tw-1(I) ÷ tw-4(X). The temperatures tw-1(I) ÷ tw-4(X) are boundary conditions for simulation the thermal conduction process in the three investigated fins. Fig. 4. Temperature field of the fluid – cross-section in the middle of the first fin. Fig. 5. Temperature field of the fluid – cross-section in the middle of the middle fin. Fig. 6. Temperature field of the fluid – cross-section in the middle of the last fin. In these simulations, „Tet” shape of the computing cells is set. The approximate size of the cells is 0.005 m. The number of computing cells is 69293, and the number of computing Fig 4 Temperature field of the fluid cross section in the middle of the first fin Fi 4 T t fi ld f th fl id ti i th iddl f th fi t fi Fig. 4. Temperature field of the fluid – cross-section in the middle of the first fin. Fig. 4. Temperature field of the fluid – cross-section in the middle of the first fin. Fig. 5. Temperature field of the fluid – cross-section in the middle of the middle fin. Fig. 5. Temperature field of the fluid – cross-section in the middle of the middle fin. Fig. 6. Temperature field of the fluid – cross-section in the middle of the last fin. Fig. 6. Temperature field of the fluid – cross-section in the middle of the last fin. In these simulations, „Tet” shape of the computing cells is set. The approximate size of the cells is 0.005 m. The number of computing cells is 69293, and the number of computing 5 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 nodes is 18384. The used turbulent model is „Spalart–Allmaras”. The obtained temperature field of the fluid presents the heat transfer intensity in different sections of the pipes. Tables 1 – 3 present the values of the temperatures, used as boundary conditions for simulation the thermal conduction process in the three fins. The first row of the tables gives information about the number of the pipe’s cross-sections. The average heat flux q is 1108.79 W/m2, and is with constant value for all fins. 3.1 Results from determination of the boundary conditions The presented temperatures are obtained from the average values in the respective computing nodes in the internal surface of the pipes. The temperatures are highest in the first cross-section, because there the temperature of the fluid flowing in the pipe is the highest. 3.1 Results from determination of the boundary conditions The value of heat flux has been determined by calculations of the heat transfer process in the convector’s pipes. nodes is 18384. The used turbulent model is „Spalart–Allmaras”. The obtained temperature field of the fluid presents the heat transfer intensity in different sections of the pipes. determined by calculations of the heat transfer process in the convector s pipes. Table 1. Temperature values for simulation of the first fin. I II III IV V VI VII VIII IX X tw-1, оС 48.109 45.511 44.990 44.188 44.271 44.061 44.169 44.162 43.918 44.293 tw-2, оС 47.912 46.242 46.005 44.500 44.723 44.641 45.009 44.533 44.929 44.237 tw-3, оС 47.582 45.714 46.300 44.111 44.879 44.405 44.883 44.321 44.936 44.218 tw-4, оС 47.862 45.392 45.578 44.183 44.275 44.042 44.188 44.123 44.166 44.079 Table 2. Temperature values for simulation of the middle fin. I II III IV V VI VII VIII IX X tw-1, оС 47.559 46.387 45.444 44.411 44.317 44.479 44.685 44.425 44.220 44.207 tw-2, оС 46.516 46.364 45.536 44.767 44.301 44.572 44.292 44.940 44.231 44.059 tw-3, оС 46.943 45.807 45.240 44.514 44.239 44.098 44.169 44.167 44.057 44.099 tw-4, оС 47.569 45.947 44.910 44.505 44.181 44.167 44.462 43.705 43.969 44.197 Table 3. Temperature values for simulation of the last fin. I II III IV V VI VII VIII IX X tw-1, оС 46.879 46.150 44.999 44.139 44.279 43.848 44.111 43.744 44.557 43.883 tw-2, оС 47.174 46.694 45.164 45.207 44.267 43.556 44.344 44.389 44.369 44.718 tw-3, оС 46.859 47.222 45.056 45.338 44.193 44.618 44.392 44.614 44.592 44.908 tw-4, оС 46.498 46.076 45.096 44.553 44.415 43.956 44.373 43.612 44.507 44.745 The presented temperatures are obtained from the average values in the respective computing nodes in the internal surface of the pipes. The temperatures are highest in the first cross-section, because there the temperature of the fluid flowing in the pipe is the highest. Table 1. Temperature values for simulation of the first fin. I II III IV V VI VII VIII IX X tw-1, оС 48.109 45.511 44.990 44.188 44.271 44.061 44.169 44.162 43.918 44.293 tw-2, оС 47.912 46.242 46.005 44.500 44.723 44.641 45.009 44.533 44.929 44.237 tw-3, оС 47.582 45.714 46.300 44.111 44.879 44.405 44.883 44.321 44.936 44.218 tw-4, оС 47.862 45.392 45.578 44.183 44.275 44.042 44.188 44.123 44.166 44.079 Table 2. Temperature values for simulation of the middle fin Table 1. Temperature values for simulation of the first fin. 3.2 Results from verification of computing mesh 6 6 6 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 Table 4 presents the results from verification of computing mesh. The differences in temperature values have been calculated by subtracting of the down row values from the up row values in each column of the table. The selected shape of computing cells is “Hex (sweep)”. Table 4. Results from verification of computing mesh. Table 4. Results from verification of computing mesh. Mesh Cell’s size, m Number of cells t1, оС Difference of t1, оС t2, оС Difference of t2, оС I 0.01000 220 30.1896 - 30.1470 - II 0.00800 238 30.1365 0.0531 29.9749 0.1721 III 0.00700 258 30.1168 0.0197 29.9467 0.0282 IV 0.00600 282 29.7362 0.3806 29.6725 0.2742 V 0.00500 320 29.7321 0.0041 29.6640 0.0085 VI 0.00450 356 29.5580 0.1741 29.4934 0.1706 VII 0.00400 463 29.3519 0.2061 29.2850 0.2084 VIII 0.00350 517 29.1894 0.1625 29.1871 0.0979 IX 0.00335 613 29.3248 -0.1354 29.2689 -0.0818 From the data presented in the table it can be seen that the tendency is to increase the values of temperatures t1 and t2, until reaching of computing mesh IХ. The change of temperatures with increasing mesh density is presented. On the base of the obtained results, computing mesh VIII has been selected. In Fig. 7 can be seen the number of cells and their specific shapes. The number of the nodes is 1312. From the data presented in the table it can be seen that the tendency is to increase the values of temperatures t1 and t2, until reaching of computing mesh IХ. The change of temperatures with increasing mesh density is presented. On the base of the obtained results, computing mesh VIII has been selected. In Fig. 7 can be seen the number of cells and their specific shapes. The number of the nodes is 1312. From the data presented in the table it can be seen that the tendency is to increase the values of temperatures t1 and t2, until reaching of computing mesh IХ. The change of temperatures with increasing mesh density is presented. On the base of the obtained results, computing mesh VIII has been selected. In Fig. 7 can be seen the number of cells and their specific shapes. The number of the nodes is 1312. Fig. 7. View of selected computing mesh. 3.2 Results from verification of computing mesh 3.3 Simulation results for the temperature distribution in fins Figures 8 - 10 present the simulation temperature fields obtained for the three fins. The presented temperature distribution expresses the heat transfer intensity between the respective zones of the fins and the air flow. It can be seen that the temperatures of the fins’ areas, located at the greatest distance from the pipes, are the lowest. Fig. 7. View of selected computing mesh. Fig. 7. View of selected computing mesh. Fig. 7. View of selected computing mesh. Fig. 7. View of selected computing mesh. 7 7 From the data presented in the table it can be seen that the tendency is to increase the values of temperatures t1 and t2, until reaching of computing mesh IХ. The change of temperatures with increasing mesh density is presented. On the base of the obtained results, computing mesh VIII has been selected. In Fig. 7 can be seen the number of cells and their specific shapes. The number of the nodes is 1312. 3.3 Simulation results for the temperature distribution in fins Figures 8 - 10 present the simulation temperature fields obtained for the three fins. The presented temperature distribution expresses the heat transfer intensity between the respective zones of the fins and the air flow. It can be seen that the temperatures of the fins’ areas, located at the greatest distance from the pipes, are the lowest. Figures 8 - 10 present the simulation temperature fields obtained for the three fins. The presented temperature distribution expresses the heat transfer intensity between the respective zones of the fins and the air flow. It can be seen that the temperatures of the fins’ areas, located at the greatest distance from the pipes, are the lowest. 7 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 Fig. 8. Veiw of the temperature field in the first fin. Fig. 9. Veiw of the temperature field in the middle fin. Fig. 10. Veiw of the temperature field in the last fin. Fig. 8. Veiw of the temperature field in the first fin. Fig. 8. Veiw of the temperature field in the first fin. Fig. 8. Veiw of the temperature field in the first fin. Fig. 8. Veiw of the temperature field in the first fin. Fig 9 Veiw of the temperature field in the middle fin Fig. 9. Veiw of the temperature field in the middle fin. Fig. 9. Veiw of the temperature field in the middle fin. Fig. 9. Veiw of the temperature field in the middle fin. Fig. 10. Veiw of the temperature field in the last fin. Fig. 10. Veiw of the temperature field in the last fin. 3.4 Results for the average temperatures and heat convection coefficient Table 5 presents the calculated results for the following parameters: average temperatures of the heat exchange surfaces of the fins, average temperature of the heat exchanger’s external surface, and external heat convection coefficient. Table 5. Results for the calculated parameters. w first _ fin t  , оС w middle _ fin t  , оС w last _ fin t  , оС w t , оС heated air t , оС  c ex t ernal h , W/(m2.K) 38.492 38.319 38.387 39.386 19.662 56.215 Table 5. Results for the calculated parameters. 8 E3S Web of Conferences 180, 01009 (2020) TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018001009 The ambient temperature during the investigation has been 14.2 oC. The ambient temperature during the investigation has been 14.2 oC. The relatively small difference in the average temperatures of the studied fins is an indicator of a relatively small difference in the amount of heat they exchange with the air flow crossing them. g For comparison of the results presented in Table 5 with results obtained by another method, the heat convection coefficient of the two-pipe sheaf is calculated, based on a criterion equation. The value obtained is 40.737 W/m2.K. It should be noted that the data in Table 5 refer to the first row of the sheaf. 4 Conclusions A methodology for determination of the external heat convection coefficients of finned water-air heat exchangers has been developed. The methodology is consist of numerical modeling of the heat convection processes in the ducts and subsequent numerical modeling of the thermal conduction processes in the fins. Schematic diagram of the simulation model has been built in ABAQUS. The model has been developed, and the computing mesh has been verified. The temperature field of the pipes’ external heat exchange surface has been obtained by ignoring the thermal resistance of the pipes walls. The average temperature of the convector’s external heat exchange surface has been calculated based on the temperature field of the pipes’ external surface in the zones between the fins, as well as based on the fins’ temperature field. The average external heat convection coefficient has been calculated on the base of the average temperature of the heat exchanger’s external surface. The big number of temperatures used as boundary conditions to simulate the process of thermal conductivity in the fins is a prerequisite for accurate determination of the simulation temperature field. The developed methodology allows the results obtained in the first simulation to be used as boundary conditions for the implementation of the second simulation. Thus, the temperature field in the fins of the heat exchanger can be obtained, given the existing difficulties in measuring the maximum number of temperatures and its experimental building. The precise determination of the external heat convection coefficient is very important, given the fact that it has a higher impact on the overall heat transfer coefficient of the investigated heat exchanger. References 1. R. Petkova-Slipets, P. Zlateva, Thermal insulating properties of straw-filled environmentally friendly building materials, De Gruyter, 52 (2017) 2. S. Tasheva, V. Gandova, K. Dobreva, I. Dincheva, V. Prodanova-Stefanova, A. Stoyanova, Studies of physicochemical and thermal properties of linalool-ethanol- water system, Research Journal of pharmaceutical, Biological and Chemical Sceince, 10 (6), 220 (2019) 3. R. M. Saldanha da Gama, F. B. de Freitas Rachid, M. L. 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https://openalex.org/W2515830325	https://dash.harvard.edu/bitstream/1/41555785/1/91409%2010180.full.pdf	English	null	Semantic representations in the temporal pole predict false memories	Proceedings of the National Academy of Sciences of the United States of America	2,016	cc-by	10,408	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41555785 Citation Chadwick, Martin J., Raeesa S. Anjum, Dharshan Kumaran, Daniel L. Schacter, Hugo J. Spiers, and Demis Hassabis. 2016. “Semantic Representations in the Temporal Pole Predict False Memories.” Proceedings of the National Academy of Sciences 113 (36): 10180–85. https:// doi.org/10.1073/pnas.1610686113. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Contributed by Daniel L. Schacter, July 4, 2016 (sent for review May 23, 2016; reviewed by Guillen Fernandez and Kathleen B. McDermott) Contributed by Daniel L. Schacter, July 4, 2016 (sent for review May 23, 2016; reviewed by Guillen Fernandez and Kathleen B. McDermott) Recent advances in neuroscience have given us unprecedented insight into the neural mechanisms of false memory, showing that artificial memories can be inserted into the memory cells of the hippocampus in a way that is indistinguishable from true memories. However, this alone is not enough to explain how false memories can arise naturally in the course of our daily lives. Cognitive psy- chology has demonstrated that many instances of false memory, both in the laboratory and the real world, can be attributed to semantic interference. Whereas previous studies have found that a diverse set of regions show some involvement in semantic false memory, none have revealed the nature of the semantic represen- tations underpinning the phenomenon. Here we use fMRI with representational similarity analysis to search for a neural code consistent with semantic false memory. We find clear evidence that false memories emerge from a similarity-based neural code in the temporal pole, a region that has been called the “semantic hub” of the brain. We further show that each individual has a partially unique semantic code within the temporal pole, and this unique code can predict idiosyncratic patterns of memory errors. Finally, we show that the same neural code can also predict vari- ation in true-memory performance, consistent with an adaptive perspective on false memory. Taken together, our findings reveal the underlying structure of neural representations of semantic knowledge, and how this semantic structure can both enhance and distort our memories. Despite the well-characterized cognitive mechanisms involved in the DRM effect (7), its neural basis is currently not well un- derstood. Previous neuroimaging and patient studies have provided robust evidence that a core network of regions in the medial and lateral temporal lobe, as well as frontal and parietal regions (8–16), is involved when encoding or retrieving semantic false memories. However, a mechanistic understanding of how these regions gen- erate false memories is lacking. In particular, although it is known that the semantic relatedness between the different words drives the illusion (3, 6, 7), little is known about the neural basis of this semantic relatedness. Contributed by Daniel L. Schacter, July 4, 2016 (sent for review May 23, 2016; reviewed by Guillen Fernandez and Kathleen B. McDermott) Computational models of semantic cognition propose that concepts are represented by a similarity-based code in an amodal “semantic hub,” situated in the apex of the ventral processing stream in the temporal pole (TP) (17, 18). Although other regions, such as the temporo-parietal cortex (19), have also been linked to the representation of abstract conceptual knowl- edge, the TP is most consistently implicated in both patient and neuroimaging studies (17, 20, 21). These computational models therefore make clear predictions about the expected neural basis of semantic false memory. Namely, the TP semantic hub should contain a similarity-based code, such that the neural representations of DRM words reflect the known semantic relatedness between those words. Further- more, the likelihood that a given word list will generate a false memory should be directly related to the degree of neural overlap. This prediction has not previously been investigated, despite the clear implications for understanding both false memory and the structure of semantic knowledge. Here we used Author contributions: M.J.C., D.K., D.L.S., H.J.S., and D.H. designed research; M.J.C. and R.S.A. performed research; M.J.C. and R.S.A. analyzed data; and M.J.C., D.K., D.L.S., H.J.S., and D.H. wrote the paper. false memory \| semantic \| temporal pole \| fMRI \| pattern similarity E ach of us has a vast store of semantic knowledge that we apply to incoming sensory data to extract meaning from the world around us. Semantic representations are capable of capturing important structural features of the world at many different levels of abstraction, which allows for rapid and flexible responses to a diverse array of environmental challenges. This preexisting knowledge structure guides ongoing cognition, which usually aids performance, but under some circumstances can lead us into error (1–3). A striking example is the widely studied DRM (Deese, Roediger, and McDermott) false-memory illusion (4, 5). In a typical DRM task, subjects are asked to memorize a set of words such as “snow,” “winter,” “ice,” and “warm.” After a delay, sub- jects will typically falsely remember having seen the semantically related word “cold.” It is widely agreed that this memory illusion is driven by the semantic relatedness between words contained in the encoding list (e.g., “snow”) and falsely remembered words that were not actually presented (e.g., “cold”). As such, it is thought that each list item automatically, but weakly, activates the se- mantically related concept (Fig. 1A). This activation leads to memory confusion, either through a cumulative priming of the related lure (5, 6) or the encoding of the semantic overlap as a “gist” memory (3), resulting in a false memory unless the error is detected by some internal monitoring process (7). As such, the DRM effect provides a powerful method for investigating the nature of false memories, as well as the structure of semantic knowledge and its effects on cognition. E 10180–10185 \| PNAS \| September 6, 2016 \| vol. 113 \| no. 36 Semantic representations in the temporal pole predict false memories i h d i ka b 1 j b h h a i l h c d 1 i b 2 Martin J. Chadwicka,b,1, Raeesa S. Anjumb, Dharshan Kumarana, Daniel L. Schacterc,d,1, Hugo and Demis Hassabisa,2 aGoogle DeepMind, London EC4A 3TW, United Kingdom; bDivision of Psychology and Language Sciences, Department of Experimental Psychology, Institute of Behavioural Neuroscience, University College London, London WC1H 0AP, United Kingdom; cDepartment of Psychology, Harvard University, Cambridge, MA 02138; and dCenter for Brain Science, Harvard University, Cambridge, MA 02138 Significance (B) We hypothesize that the neural repre- sentation of DRM list items should overlap with the neural representation of the related lure concept. The extent of overlap should directly reflect the se- mantic relatedness and false-memory likelihood of each DRM lure concept. To assess neural overlap for a DRM list, we measured the fMRI voxel pattern similarity between the lure concept and the four related list items. encoding the semantic relatedness between thematically related words (Fig. 2). Furthermore, this result shows that the precise level of neural overlap in the TP predicts the probability that a false memory will be constructed for a given DRM list. This result is therefore fully consistent with the computational ac- counts of semantic cognition, and demonstrates that a simi- larity-based code in the TP is capable of generating false memories. Strikingly, our measure of false-memory likelihood is a canonical measure taken from an independent set of sub- jects (26, 27), yet we can nevertheless successfully predict this information based purely on the neural data of our group of subjects. This result clearly demonstrates a robust level of agreement across different individuals in the neural represen- tation of the concepts contained within the DRM lists. Thus, it appears that the TP is responsible for representing a shared conceptual space, which is a vital component of successful communication. For completeness, we also looked for regions displaying a negative correlation between neural overlap and false-memory likelihood, although it is not clear that any such correlation is theoretically meaningful. This analysis revealed a single significant cluster in the right superior frontal gyrus [peak Montreal Neurological Institute (MNI) coordinates: 24, 20, 50; T = 4.71; cluster extent = 163 voxels]. A B B ] To ensure that TP neural data are really capturing meaningful semantic representations, we ran an additional set of control analyses based on the functionally defined TP region of interest (ROI). We examined four issues. First, if the neural data are capturing semantic relatedness between the lure and list items, we should find that each lure is more similar to its own list than any other list, regardless of any differences in false-memory strength. To assess this hypothesis, we directly compared the neural similarity within and across the 40 DRM sets. As expected, this analysis revealed a significant within-set increase in simi- larity (Z = 3.11, P < 0.001). Results We used a representational similarity analysis approach, which uses the neural pattern similarity between pairs of stimuli to infer the representational similarity (22). This method is therefore well- suited for assessing neural overlap between semantic representa- tions (23–25), as the degree of overlap should be directly reflected in the representational similarity. We used this approach to mea- sure the degree of neural overlap between each set of DRM words and their related lure word (Fig. 1). Crucially, each DRM list is known to have a different probability of inducing a false memory, with some much greater than others (26, 27). If our prediction is correct, then we should find a brain region displaying a direct correspondence between the degree of neural overlap and false- memory likelihood across the different DRM word lists. To measure the neural representations of the DRM word lists, 18 participants viewed 40 separate four-word DRM lists, along with the 40 associated lure words (Table S1), while we collected fMRI data. While viewing the words, subjects performed an incidental categorization task (man-made or natural) to ensure that all words were processed at the semantic level. We used the canonical false- recognition scores reported in ref. 27 as our measure of false- memory likelihood, and applied a searchlight analysis (28) across the whole brain to establish whether any brain region displayed the predicted positive correlation between neural overlap and false-memory likelihood. Temporal pole neural overlap B Canonical false memory likelihood A 0.5 0.6 0.7 0.8 0.15 0.20 0.25 Fig. 2. Neural overlap correlates with canonical false-memory likelihood in the left TP. (A) A whole-brain searchlight analysis revealed a significant cluster in the left TP (peak MNI coordinates: −51, 17, −25; T = 5.33; cluster extent = 92 voxels), with no other region displaying any significant in- formation. Results are displayed on a cortical surface map using BrainNet Viewer (50). (B) To visualize the relationship between neural overlap and false memory, we plot the group average neural overlap for each of the 40 DRM lists against canonical false-memory likelihood, using a cross-validation procedure over subjects to avoid artificial inflation of the effect size. There is a clear positive correlation between the two [r(39) = 0.40, P = 0.012], showing that the degree of semantic relatedness in the neural data predicts variation in false-memory strength across the DRM lists. Significance False memories can arise in daily life through a mixture of fac- tors, including misinformation and prior conceptual knowledge. This can have serious consequences in settings, such as legal eyewitness testimony, which depend on the accuracy of mem- ory. We investigated the brain basis of false memory with fMRI, and found that patterns of activity in the temporal pole region of the brain can predict false memories. Furthermore, we show that each individual has unique patterns of brain activation that can predict their own idiosyncratic set of false-memory errors. Together, these results suggest that the temporal pole may be responsible for the conceptual component of illusory memories. False memories can arise in daily life through a mixture of fac- tors, including misinformation and prior conceptual knowledge. This can have serious consequences in settings, such as legal eyewitness testimony, which depend on the accuracy of mem- ory. We investigated the brain basis of false memory with fMRI, and found that patterns of activity in the temporal pole region of the brain can predict false memories. Furthermore, we show that each individual has unique patterns of brain activation that can predict their own idiosyncratic set of false-memory errors. Together, these results suggest that the temporal pole may be responsible for the conceptual component of illusory memories. Author contributions: M.J.C., D.K., D.L.S., H.J.S., and D.H. designed research; M.J.C. and R.S.A. performed research; M.J.C. and R.S.A. analyzed data; and M.J.C., D.K., D.L.S., H.J.S., and D.H. wrote the paper. Reviewers: G.F., Radboud University Nijmegen Medical Centre; and K.B.M., Washington University in St. Louis. The authors declare no conflict of interest. The authors declare no conflict of interest. Freely available online through the PNAS open access option. 1To whom correspondence may be addressed. Email: mjchadwick@google.com or dls@ wjh.harvard.edu. 2H.J.S. and D.H. contributed equally to this work. 2H.J.S. and D.H. contributed equally to this work. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1610686113/-/DCSupplemental. 10180–10185 \| PNAS \| September 6, 2016 \| vol. 113 \| no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1610686113 A B Fig. 1. Neural predictions arising from the DRM false-memory illusion. (A) For three DRM lists, we illustrate the semantic relatedness between pre- sented list items on the periphery, and the unseen related concept at the center. Beneath each, we show the likelihood that this word list will produce a false memory for the related lure concept. As semantic relatedness increases, so does the false-memory likelihood. Significance Second, to ensure that the neural effects were not driven by extraneous factors, such as the word frequency or the visual similarity of the lure and list words, we investigated whether either of these factors correlated with the TP neural overlap. Neither of these variables significantly pre- dicted the neural data (word frequency: Z = 0.02, P = 0.98; visual similarity: Z = 1.72, P = 0.085), suggesting that they are not significant drivers of neural similarity in this region. Third, the task performed in the scanner while subjects viewed the words was a semantic category judgment task (man-made or natural). Although the categorical nature of the encoding task was Fig. 1. Neural predictions arising from the DRM false-memory illusion. (A) For three DRM lists, we illustrate the semantic relatedness between pre- sented list items on the periphery, and the unseen related concept at the center. Beneath each, we show the likelihood that this word list will produce a false memory for the related lure concept. As semantic relatedness increases, so does the false-memory likelihood. (B) We hypothesize that the neural repre- sentation of DRM list items should overlap with the neural representation of the related lure concept. The extent of overlap should directly reflect the se- mantic relatedness and false-memory likelihood of each DRM lure concept. To assess neural overlap for a DRM list, we measured the fMRI voxel pattern similarity between the lure concept and the four related list items. fMRI to measure the neural overlap between DRM lists and related lures, allowing us to directly test this prediction. fMRI to measure the neural overlap between DRM lists and related lures, allowing us to directly test this prediction. Chadwick et al. Results To investigate this possibility, we derived a subject-specific measure of task category similarity between the lure and list of each DRM set based on the pattern of responses to each word (Methods). We found no evidence for a correlation between this variable and the neural data (Z = 0.46, P = 0.65), suggesting that task-driven categorical representations are not present in the TP. Finally, we investigated whether the correlation between neural overlap and canonical false-memory strength was still present after controlling for the three additional variables (word frequency, visual similarity, and categorical representation). Using a cross-validated ROI approach to avoid issues of statistical circularity (29), we found clear evidence for a significant corre- lation between neural overlap and canonical false memory even after partialling out the control variables (Z = 2.03, P = 0.021). Thus, our result cannot be explained by extraneous factors, such as word frequency or visual similarity. We further explored each of these three control variables using a searchlight analysis across the whole brain, but none of these analyses revealed any significant results. Given that our measure of neural overlap is in each case based on the average pattern expressed over four list items, this will greatly reduce the power of any analysis that is not explicitly based on some shared representation, such as the semantic gist. Thus, it is not surprising that these additional analyses did not find any significant results. The main focus of this study was to investigate the neural basis of false memory. However, an adaptive perspective on false memory (1) would suggest that our semantic knowledge should aid cogni- tion under most circumstances (31), rather than purely acting as a source of memory distortion. This hypothesis would therefore suggest that we ought to also find a positive correlation between neural overlap and true-memory performance for the list items that were actually presented during encoding. As predicted, we found a Neural-Behavioral Correlation −0.10 −0.05 0.00 0.05 0.10 0.15 * S3 Between Subject Within Subject A B Fig. 4. The TP contains subject-specific neural information that predicts false memories. (A) For each subject, we calculated the correlation between their TP neural overlap and their pattern of false memories. As a baseline, we calculated the average between-subject correlation. This procedure is illustrated schematically. Results 0.00 0.25 0.50 0.75 1.00 Hits False Alarms False Memories Proportion of each response type A e Canonical false memory likelihood Group false memory strength 0.45 0.55 0.65 0.75 0.85 0.25 0.50 0.75 B Proportion of each response type A the pattern of false-memory errors, over and above any shared semantic representations common to all subjects. To assess this issue we used an individuation analysis (30), comparing the within-subject neural-behavioral correlations (unique semantic in- formation) to the between-subject neural-behavioral correlations (shared semantic information), in each case controlling for the canonical false-memory strength to remove additional shared se- mantic information. This analysis revealed a significantly higher within- than between-subject correlation (Z = 2.63, P = 0.0042). This result was still significant after additionally partialling out the influence of the three control variables discussed above (Z = 2.55, P = 0.0054). This result provides clear evidence that each indi- vidual has a partially unique set of semantic representations within the TP that have a direct impact on memory distortions (Fig. 4). Such a result cannot be explained by incidental differences in TP physiology or anatomy alone (30), as these more basic properties would not predict each subject’s false-memory behavior. 1 A Group false memory strength Fig. 3. DRM recognition memory results. (A) Group mean hit rate, false-alarm rate, and false-memory rate are displayed for all responses regardless of confi- dence (blue), and for high-confidence responses (red). Error bars represent 95% confidence intervals, adjusted for within-subject data (51). False-memory rates were significantly greater than false-alarm rates (Z = 3.72, P < 0.001). This is clear evidence for the expected false-memory illusion, which is robust even for high- confidence responses (Z = 3.72, P < 0.001). (B) The group level false-memory likelihood across the 40 DRM lists correlated positively [r(39) = 0.48, P = 0.0016] with canonical false-recognition rates, even after removing one potential outlier [r(39) = 0.48, P = 0.0018]. All plots are based on a sample size of 18. p j y Importantly, the fMRI and behavioral data for each subject were collected in separate sessions separated by many weeks, which demonstrates that the structure of neural overlap must be stable over at least this length of time, and plausibly for much longer than this. This long delay also minimized any possible influence of the initial behavioral session on the neural representations expressed during scanning. Results This correlation remains [r(39) = 0.45, P = 0.005] after removing two potential outliers. A Temporal pole neural overlap B Canonical false memory likelihood 0.5 0.6 0.7 0.8 0.15 0.20 0.25 B A Temporal pole neural overlap 0.15 0.20 0. Fig. 2. Neural overlap correlates with canonical false-memory likelihood in the left TP. (A) A whole-brain searchlight analysis revealed a significant cluster in the left TP (peak MNI coordinates: −51, 17, −25; T = 5.33; cluster extent = 92 voxels), with no other region displaying any significant in- formation. Results are displayed on a cortical surface map using BrainNet Viewer (50). (B) To visualize the relationship between neural overlap and false memory, we plot the group average neural overlap for each of the 40 DRM lists against canonical false-memory likelihood, using a cross-validation procedure over subjects to avoid artificial inflation of the effect size. There is a clear positive correlation between the two [r(39) = 0.40, P = 0.012], showing that the degree of semantic relatedness in the neural data predicts variation in false-memory strength across the DRM lists. This correlation remains [r(39) = 0.45, P = 0.005] after removing two potential outliers. y This analysis revealed a significant cluster in the left TP, with no other significant information anywhere else in the brain. This re- sult provides evidence that this specific region is responsible for PNAS \| September 6, 2016 \| vol. 113 \| no. 36 \| 10181 0.00 0.25 0.50 0.75 1.00 All Responses High Confidence Hits False Alarms False Memories Canonical false memory likelihood Proportion of each response type Group false memory strength 0.45 0.55 0.65 0.75 0.85 0.25 0.50 0.75 A B Fig. 3. DRM recognition memory results. (A) Group mean hit rate, false-alarm rate, and false-memory rate are displayed for all responses regardless of confi- dence (blue), and for high-confidence responses (red). Error bars represent 95% confidence intervals, adjusted for within-subject data (51). False-memory rates were significantly greater than false-alarm rates (Z = 3.72, P < 0.001). This is clear evidence for the expected false-memory illusion, which is robust even for high- confidence responses (Z = 3.72, P < 0.001). (B) The group level false-memory likelihood across the 40 DRM lists correlated positively [r(39) = 0.48, P = 0.0016] with canonical false-recognition rates, even after removing one potential outlier [r(39) = 0.48, P = 0.0018]. All plots are based on a sample size of 18. Results By comparing the within-subject (red arrow) and average between-subject (blue arrows) correlations we can determine whether there is any subject-specific mapping between the neural and be- havioral data. (B) The group average within- and between-subject correla- tions are displayed for the false-memory data. Error bars display 95% confidence intervals on a one-way t test, corrected for within-subjects sta- tistical testing (51). The asterisk denotes a significant difference at P < 0.05. The plots are based on a sample size of 18. S3 A Neural-Behavioral Correlation −0.10 −0.05 0.00 0.05 0.10 0.15 * Between Subject Within Subject B B A y g Although our initial analysis focused on shared semantic rep- resentations, it is also likely that each of us forms some idiosyn- cratic semantic associations through our own individual experience. Such quirks of experience could lead to measurable differences in neural overlap in the TP, and consequently to unique patterns of false-memory errors. To investigate this possibility, our subjects participated in a DRM false-memory recognition task in a sepa- rate session that took place several weeks before the scanning session. The same 40 DRM word lists were used in both the be- havioral and scanning sessions, which allowed us to directly compare each individual’s neural data to their behavioral data. As expected, the subjects displayed the typical false-memory effect, and committed a large number of high-confidence false alarms to the critical lure stimuli (Fig. 3). As a further quality-control check, we explored the consistency of our group’s behavioral data compared with the canonical false-memory data (26, 27). The group false-memory likelihood correlated positively with both the canonical data r(39) = 0.53, P < 0.001 and with the neural data (Z = 1.68, P = 0.047), demonstrating that this subject group’s data conform to the canonical data, as expected. Fig. 4. The TP contains subject-specific neural information that predicts false memories. (A) For each subject, we calculated the correlation between their TP neural overlap and their pattern of false memories. As a baseline, we calculated the average between-subject correlation. This procedure is illustrated schematically. By comparing the within-subject (red arrow) and average between-subject (blue arrows) correlations we can determine whether there is any subject-specific mapping between the neural and be- havioral data. (B) The group average within- and between-subject correla- tions are displayed for the false-memory data. Results To further ensure that there was no such influ- ence, we leveraged the wide range of intersession delay lengths across subjects (minimum = 21 d, maximum = 239 d) that emerged as a consequence of differences in subject availability. If there were an effect of the behavioral session due to memory for the items experienced in this session, we would expect this effect to degrade over time. We would therefore expect a negative correlation be- tween the length of delay and the strength of neural-behavioral mapping. In fact, we find a nonsignificant positive correlation in- stead (r = 0.26, P = 0.30), which clearly shows that memory is not enhancing the neural overlap data. incidental to the effect of interest, it is nevertheless possible that neural representations related to the semantic categories are present in the TP. To investigate this possibility, we derived a subject-specific measure of task category similarity between the lure and list of each DRM set based on the pattern of responses to each word (Methods). We found no evidence for a correlation between this variable and the neural data (Z = 0.46, P = 0.65), suggesting that task-driven categorical representations are not present in the TP. Finally, we investigated whether the correlation between neural overlap and canonical false-memory strength was still present after controlling for the three additional variables (word frequency, visual similarity, and categorical representation). Using a cross-validated ROI approach to avoid issues of statistical circularity (29), we found clear evidence for a significant corre- lation between neural overlap and canonical false memory even after partialling out the control variables (Z = 2.03, P = 0.021). Thus, our result cannot be explained by extraneous factors, such as word frequency or visual similarity. We further explored each of these three control variables using a searchlight analysis across the whole brain, but none of these analyses revealed any significant results. Given that our measure of neural overlap is in each case based on the average pattern expressed over four list items, this will greatly reduce the power of any analysis that is not explicitly based on some shared representation, such as the semantic gist. Thus, it is not surprising that these additional analyses did not find any significant results. incidental to the effect of interest, it is nevertheless possible that neural representations related to the semantic categories are present in the TP. 10182 \| www.pnas.org/cgi/doi/10.1073/pnas.1610686113 Discussion Our study demonstrates that the left TP contains partially overlapping neural representations of related concepts, and that the extent of this neural overlap directly reflects the degree of semantic similarity between the concepts. Furthermore, the neural overlap between sets of related words predicts the like- lihood of making a false-memory error. Together, these findings provide support for neural network models of semantic cognition that posit that the TP uses a similarity-based coding scheme to sustain amodal-distributed representations of individual con- cepts and their relationships within an abstract semantic space (17, 18). The similarity-based coding of concepts has significant computational advantages in allowing efficient generalization of existing knowledge to novel situations, while still allowing for the “grounding” of each concept in a full set of domain-specific cortical regions mediated by the hub-like connectivity of the TP (17, 18), consistent with previous studies showing that domain- specific semantic features are indeed distributed across a wide range of cortical regions (32). Nevertheless, our results suggest that the type of coding scheme underpinning the representation of concepts within the TP has a potential cost, specifically the emergent property of false memories. Overall Task Structure. All participants took part in two separate experimental sessions separated by several weeks (mean 65 d, minimum = 21, maximum = 239). The first session was a behavioral session involving a standard DRM recognition paradigm, providing subject-specific false-memory data. The second session was a fMRI session, where subjects viewed words taken from the DRM lists during an incidental task. Both sessions are explained in more detail below. We elected to run the behavioral session before the scanning session to ensure that repeated exposure to the DRM words during scanning did not impact the behavioral false- memory effects. This procedure therefore provided us with a “pure” measure of individual false memories. However, we acknowledge that this design could still have the reverse problem, in that there could be carryover effects from the behavioral to the fMRI session. The long delay between sessions was built in to minimize any such issues, and further control analyses were conducted to further rule this out as a problem (Results). Although the focus of our experiment was on the structure of semantic representations, it is likely that interactions between regions in the medial temporal lobe and the TP are critical to the generation of false memories. Discussion This suggestion is supported by a meta-analysis that contrasted regions involved in abstract versus concrete words, and that found clear evidence that the TP was more active in response to abstract words, whereas ventral temporal regions showed a preference for concrete words (20). Finally, our results show that each individual’s unique TP representations predict idiosyncratic patterns of false-memory errors. Given that we rely on shared semantic representation to communicate with one another, this individual variation is per- haps surprising. However, it does converge with other recent reports of individual differences in semantic (30) and episodic representation (38), and suggests that divergent personal expe- rience is sufficient to create individually unique representations p ( ) Although we found clear evidence for a semantic code within the left TP, other neighboring regions have also been found to contain semantic-like representations, particularly the anterior ventral temporal cortex (23, 25). We suggest that these differing locations are likely a result of the level of semantic abstraction, as the set of studies with results located in anterior ventral temporal cortex all used highly concrete stimuli (either concrete words, or direct use of pictures). In contrast, our set of words included many abstract concepts, such as “justice” and “desire” (see Table S1 for full list). This suggestion is supported by a meta-analysis that contrasted regions involved in abstract versus concrete words, and that found clear evidence that the TP was more active in response to abstract words, whereas ventral temporal regions showed a preference for concrete words (20). Behavioral DRM Analysis. The recognition-memory test data were analyzed to determine whether the subjects displayed the expected false-memory effect. To do this analysis, for each subject we calculated the proportion of words categories as “old” for each of the three conditions (old items, new items, and lure items). This approach provided us with a measure of the hit rate, false- alarm rate, and for the related concept lure items, false-memory rate. To assess whether the expected false-memory effects were present, we conducted a series of planned pairwise comparisons between conditions using two-tailed Wilcoxon signed rank tests. First, we tested for a basic recognition memory effect by comparing the hit rate to the false-alarm rate. Next, we investigated whether our subjects displayed the expected false-memory effect, by com- paring the false-memory rate with the false-alarm rate. Results Error bars display 95% confidence intervals on a one-way t test, corrected for within-subjects sta- tistical testing (51). The asterisk denotes a significant difference at P < 0.05. The plots are based on a sample size of 18. g p p However, the key question was whether there might be a subject-specific mapping between the TP neural overlap and Chadwick et al. significant mapping between TP neural overlap and true-memory strength (Z = 2.33, P = 0.0099), which remained significant after partialling out the three control variables (Z = 2.29, P = 0.011). We also investigated the possibility that subject-specific TP neural coding might predict true memory performance, using an in- dividuation analysis (30). This analysis demonstrated a significant individuation effect in the TP for true memories (Z = 2.16, P = 0.016) as well as false memories, and this result remained signif- icant after partialling out the three control variables (Z = 2.11, P = 0.017). These results provide clear evidence that the semantic similarity code within this region can be beneficial for memory systems, as well as potentially leading to memory distortions. in higher-level semantic regions. This striking finding suggests that it will be important to further characterize both the shared and individually unique aspects of semantic cognition to better understand the nature of conceptual knowledge. Methods Participants. Eighteen participants (11 female; mean age 22, range 18–29) took part in this study. All were right-handed native English speakers and had nor- mal or corrected-to-normal vision. The study was approved by the University College London Research Ethics Committee, and all participants gave written consent to take part in the study. Stimuli. For both testing sessions, the stimuli were drawn from 40 standard DRM lists (26, 27). Because of time-constraints in the fMRI scanning session, we used only four list items from each DRM list (see Fig. 3B for a comparison with pre- vious results based on the full set of 15 list items). Where possible, these were four list items with the highest associative strength with the lure word. However, this full set of 40 lists contains some words that were repeated across lists, and some words that we considered to be culturally specific in semantic relatedness to the related concept, such as “United States” in the “army” list. Any unsuitable list words were therefore excluded, and alternative, lower list associates were included instead. The full set of stimuli used is displayed in Table S1. In total, the stimulus set included 40 DRM related concept lures and 160 associated DRM list items. For the behavioral DRM task, we also used an additional 160 unrelated novel words, which were matched to the DRM lists in average concreteness and frequency. Chadwick et al. Discussion Although our data cannot speak directly to this issue, there are two clear lines of evidence that are suggestive. First, results from both rodent (33) and human re- search (34) demonstrate that false memories can be established as memory “engrams” within the hippocampus through artificial manipulation or reconsolidation processes. Second, recent studies have shown that recognition memory performance can be driven by a dynamic similarity-based computation within the medial temporal lobe at retrieval (35, 36). It is likely that both sets of processes interact with the semantic representations in the TP to produce semantic false memories (3, 6, 37). Behavioral DRM Task. The first testing session was purely behavioral and in- volved a standard DRM recognition paradigm. During an encoding phase, subjects were presented with 40 sets of four-word DRM lists. Participants were instructed to memorize as many of the presented words as possible. For each list, the four words were presented consecutively for 500 ms each, with a 3-s interval between each list. The order of the 40 lists was randomized across subjects. Subjects were then required to perform an incidental visual discrimination task for 15 min, to minimize explicit rehearsal of the list words. Following this dis- traction period, subjects were given a recognition memory test for the previously presented words. All 160 DRM list words were presented, along with the 40 related concept lure words and 160 unrelated novel words. This set of 360 words was presented one at a time, in a randomized order. Subjects were required to decide whether they thought the word was old or new, along with a confidence judgment (sure or unsure). The task was self-paced, with no time limit. p ( , , ) Although we found clear evidence for a semantic code within the left TP, other neighboring regions have also been found to contain semantic-like representations, particularly the anterior ventral temporal cortex (23, 25). We suggest that these differing locations are likely a result of the level of semantic abstraction, as the set of studies with results located in anterior ventral temporal cortex all used highly concrete stimuli (either concrete words, or direct use of pictures). In contrast, our set of words included many abstract concepts, such as “justice” and “desire” (see Table S1 for full list). Discussion The precise sequences used were further optimized to reduce signal dropout in ventral anterior regions, including the TP. All MRI data were collected using a Siemens Avanto 1.5 Tesla MRI scanner with a 32-channel head coil at the Birkbeck-UCL Centre for Neu- roimaging in London. The functional data were acquired using a gradient-echo echo-planar imaging sequence in an ascending sequence, with a slice thickness of 2 mm and a 1-mm gap, TR = 85 ms, TE = 50 ms, slice tilt = −30°, field-of-view 192 mm, and matrix size 64 × 64. The whole brain was acquired with 40 slices, leading to a volume acquisition time of 3.4 s. The precise slice tilt was chosen as a compromise between sensitivity, coverage, and speed (40, 41). Following functional imaging, an anatomical image was acquired for each participant (T1-weighted FLASH, TR = 12 ms, TE = 5.6 ms, 1-mm3 resolution). TP ROI. To further explore the representations contained within the TP, a ROI was created based on the initial searchlight results. The ROI included all voxels within the TP that passed the searchlight cluster threshold of t > 3. The resulting ROI consisted of 92 voxels. Correlation Between Canonical False Memory and Neural Overlap. In Fig. 2B we report the correlation between the group average TP neural overlap for each DRM list and the canonical false-memory likelihood (26, 27), to illustrate the strength of the relationship. To avoid an artificial inflation of the effect size estimate due to nonindependence in the choice of ROI, we used a leave-one- subject-out cross-validation approach (29). On a given cross-validation fold, we took the searchlight maps for 17 of 18 subjects, averaged the maps, and selected the top 200 voxels. We then used these voxels as an ROI to measure the neural overlap in the 40 DRM lists for the remaining subject (note that in this case we did not normalize the neural overlap score by subtracting the between-list correlation, as we consider the raw correlation values to be descriptively more informative). This procedure was repeated 18 times, each time leaving out a different subject. This analysis resulted in neural overlap data for all 18 subjects based on an independently selected ROI, thereby avoiding statistical “double- dipping” (29). We averaged the neural overlap across the subjects to create a single summary neural overlap score for each of the 40 DRM lists. Discussion This score was then correlated with the canonical false-memory scores, as reported in Fig. 2B. An additional correlation was conducted after removing two potential outliers, identified using a bootstrapped Mahalanobis distance, and a threshold of Ds > 6. Note that whereas this cross-validation approach is guaranteed to provide an unbiased correlation, it is not guaranteed that the voxels used will be based on the same TP region as reported in the searchlight results. We therefore in- vestigated the number of voxels falling with the TP region [defined using the Harvard–Oxford Atlas (47)] for each fold of the cross-validation. Every single fold included at least some voxels within this region, ranging from 5 to 42, with an average of 25.5. Thus, a good proportion of the neural information going into this analysis was indeed based on the TP. fMRI Preprocessing. The first six functional volumes were discarded to allow for T1 equilibration. The remaining data were slice-time corrected and spatially realigned. Each participant’s structural image was coregistered to the first functional image. The structural images were segmented, and the deforma- tions estimated during this step were applied to both the structural and func- tional images to normalize them into MNI space. All preprocessing steps were conducted using SPM12. Default parameters were chosen for each step. Pattern Estimation. We were interested in investigating neural overlap between the neural representations of each set of DRM list words and their related concept. Our hypothesis was that each DRM list word should have a neural representation that overlaps with that related concept. To assess this hypothesis, we estimated two patterns for each DRM set: one pattern for the related concept itself, and another for all four of the list words combined. This latter pattern captured the neural pattern that was common across all four list items, which should therefore capture the representational overlap. If our hypothesis is correct, then this pattern should correlate with the related concept pattern. To estimate this set of patterns, we used the GLMdenoise toolbox (42), which implements a de-noising step in addition to estimating the β-weights for each regressor. Each pattern was estimated using an event-related regressor indicating the onset of the related concept/set of list words across the four functional sessions. This procedure resulted in a set of 80 β-weight images (40 DRM list patterns and 40 related lure concept patterns). Discussion For this analysis, 10,000 permutations were applied with 10-mm variance smoothing, and a standard cluster threshold of T > 3 was used to assess statistical significance (46). Only regions that are significant at P < 0.05 with family-wise error correction are reported. the fact that we used only the first four list associates from each DRM list, as opposed to the full set of 15. All behavioral results are reported in Fig. 3. the fact that we used only the first four list associates from each DRM list, as opposed to the full set of 15. All behavioral results are reported in Fig. 3. fMRI Task. Several weeks later, the same subjects came in for a second testing session in the fMRI scanner. During each of four functional runs, the 40 DRM related concepts and 160 DRM list words were presented one at a time for 3 s each. Thus, in total, each word was presented four times, to allow a more stable es- timate of the neural pattern. The order of presentation was randomized, with the additional constraint that words from the same DRM set were never presented consecutively. The behavioral task involved a semantic category decision for each presented word. Specifically, subjects had to decide whether they thought each word was more related to the category of “man-made” or “natural,” and in- dicate this by pressing the relevant button. Because we were simply interested in measuring the neural patterns expressed for each DRM concept, the task itself was incidental. However, we reasoned that a semantic categorization task would require the subjects to fully process the semantic meaning of each word. The fact that each word would be repeated four times during scanning introduces a possible source of noise because of novelty effects on the first presentation. We therefore allowed the subjects one practice block before entering the scanner to reduce any novelty signals in the subsequent scanning session. MRI Scan Details. It is well known that fMRI of the TPs can be problematic because of susceptibility artifacts (signal dropout) in this region, which can substantially reduce blood-oxygen level-dependent (BOLD) sensitivity (39). We therefore elected to use a 1.5 Tesla MRI scanner, which suffers from less-pro- nounced dropout in this region, and therefore can actually have greater BOLD sensitivity than higher field-strength scanners (40). Discussion Following this process, we compared false-memory and false-alarm rate using just high-confidence trials to determine whether the task induced robust false memories. Finally, we investigated whether the pattern of false-memory errors across the 40 DRM lists correlated with the false-memory rate reported by ref. 27, despite Finally, our results show that each individual’s unique TP representations predict idiosyncratic patterns of false-memory errors. Given that we rely on shared semantic representation to communicate with one another, this individual variation is per- haps surprising. However, it does converge with other recent reports of individual differences in semantic (30) and episodic representation (38), and suggests that divergent personal expe- rience is sufficient to create individually unique representations PNAS \| September 6, 2016 \| vol. 113 \| no. 36 \| 10183 data by subtracting the mean Pearson correlation between the DRM list and each unrelated lure concept. This procedure removed any general effects of similarity that were not driven by semantic relatedness, and resulted in a vector of 40 neural overlap scores for the 40 DRM lists. Our prediction is that the degree of neural overlap within the TP should reflect semantic relatedness, and therefore predict false-memory likelihood across the 40 DRM lists. We used the canonical false-recognition scores reported in (26, 27), as our measure of false- memory likelihood for the 40 DRM lists. We used a searchlight approach (28) to search across the whole brain for regions containing a neural code consistent with our predictions. This approach involves stepping through each voxel in the brain, and in each case running a representational similarity analysis on the cluster of voxels surrounding that central voxel (for all analyses, we used a spherical searchlight with 10-mm radius). We used a variation of this approach, where the value at each voxel was the average value of all searchlight analyses that included that voxel. This information-averaging approach more accurately reflects the multivariate nature of the analysis, and results in a smoother image (45). For computational efficiency, we restricted our analysis to a whole-brain gray-matter mask, created by averaging the normalized, segmented gray-matter images, and applying a threshold of 0.5. This searchlight approach was applied to the analysis described above, using a Fisher-transformed Pearson correlation to assess the mapping between neural overlap and false-memory likelihood in each searchlight. 10184 \| www.pnas.org/cgi/doi/10.1073/pnas.1610686113 Discussion This approach was repeated for all subjects, and statistical significance at each voxel was assessed at the group level using a nonparametric permutation approach (46). This procedure provides a means of applying strict family-wise error correction for multiple comparisons without any parametric assumptions. For this analysis, 10,000 permutations were applied with 10-mm variance smoothing, and a standard cluster threshold of T > 3 was used to assess statistical significance (46). Only regions that are significant at P < 0.05 with family-wise error correction are reported. data by subtracting the mean Pearson correlation between the DRM list and each unrelated lure concept. This procedure removed any general effects of similarity that were not driven by semantic relatedness, and resulted in a vector of 40 neural overlap scores for the 40 DRM lists. Our prediction is that the degree of neural overlap within the TP should reflect semantic relatedness, and therefore predict false-memory likelihood across the 40 DRM lists. We used the canonical false-recognition scores reported in (26, 27), as our measure of false- memory likelihood for the 40 DRM lists. We used a searchlight approach (28) to search across the whole brain for regions containing a neural code consistent with our predictions. This approach involves stepping through each voxel in the brain, and in each case running a representational similarity analysis on the cluster of voxels surrounding that central voxel (for all analyses, we used a spherical searchlight with 10-mm radius). We used a variation of this approach, where the value at each voxel was the average value of all searchlight analyses that included that voxel. This information-averaging approach more accurately reflects the multivariate nature of the analysis, and results in a smoother image (45). For computational efficiency, we restricted our analysis to a whole-brain gray-matter mask, created by averaging the normalized, segmented gray-matter images, and applying a threshold of 0.5. This searchlight approach was applied to the analysis described above, using a Fisher-transformed Pearson correlation to assess the mapping between neural overlap and false-memory likelihood in each searchlight. This approach was repeated for all subjects, and statistical significance at each voxel was assessed at the group level using a nonparametric permutation approach (46). This procedure provides a means of applying strict family-wise error correction for multiple comparisons without any parametric assumptions. Discussion Arndt J, Hirshman E (1998) True and false recognition in MINERVA2: Explanations from a global matching perspective. J Mem Lang 39(3):371–391. y g y 12. Boggio PS, et al. (2009) Temporal lobe cortical electrical stimulation during the en- coding and retrieval phase reduces false memories. PLoS One 4(3):e4959. 38. Chadwick MJ, Bonnici HM, Maguire EA (2014) CA3 size predicts the precision of memory recall. Proc Natl Acad Sci USA 111(29):10720–10725. 39. 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Weiskopf N, Hutton C, Josephs O, Turner R, Deichmann R (2007) Optimized EPI for fMRI studies of the orbitofrontal cortex: Compensation of susceptibility-induced gradients in the readout direction. MAGMA 20(1):39–49. false recognition in short-term memory. Neuroimage 56(3):1726–1734 16. Warren DE, Jones SH, Duff MC, Tranel D (2014) False recall is reduced by damage to the ventromedial prefrontal cortex: Implications for understanding the neural cor- relates of schematic memory. J Neurosci 34(22):7677–7682. 42. Kay KN, Rokem A, Winawer J, Dougherty RF, Wandell BA (2013) GLMdenoise: A fast, automated technique for denoising task-based fMRI data. Front Neurosci 7:247. y 17. Patterson K, Nestor PJ, Rogers TT (2007) Where do you know what you know? The rep- resentation of semantic knowledge in the human brain. Nat Rev Neurosci 8(12):976–987. 43. 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Roediger HL, McDermott KB (1995) Creating false memories: Remembering words not presented in lists. J Exp Psychol Learn Mem Cogn 21(4):803–814. 31. van Kesteren MTR, Ruiter DJ, Fernández G, Henson RN (2012) How schema and novelty augment memory formation. Trends Neurosci 35(4):211–219. 6. Roediger HL, III, Balota DA, Watson JM (2001) Spreading activation and arousal of false memories. The Nature of Remembering: Essays in Honor of Robert G. Crowder, eds Roediger HL, III, Nairne JS, Neath I, Surprenant AM (APA, Washington, DC), pp 95–115. 32. Mitchell TM, et al. (2008) Predicting human brain activity associated with the meanings of nouns. Science 320(5880):1191–1195. 7. Gallo DA (2010) False memories and fantastic beliefs: 15 years of the DRM illusion. Mem Cognit 38(7):833–848. 33. Ramirez S, et al. (2013) Creating a false memory in the hippocampus. Science 341(6144): 387–391. 8. 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Davis T, Xue G, Love BC, Preston AR, Poldrack RA (2014) Global neural pattern similarity as a common basis for categorization and recognition memory. J Neurosci 34(22):7472–7484. 11. Kim H, Cabeza R (2007) Differential contributions of prefrontal, medial temporal, and sen- sory-perceptual regions to true and false memory formation. Cereb Cortex 17(9):2143–2150. 37. Discussion Additionally, all three variables were controlled for in each analysis reported in Results. that result in a false memory, and 0 elsewhere. For each subject, we calculated the Spearman correlation between their TP neural overlap and false-memory data (within-subject correlation). We then calculated the Spearman correlation between that subject’s neural overlap and each other subject’s false-memory data, and averaged across these correlation values to provide a summary between-subject correlation. This procedure resulted in a within-subject and between-subject correlation for every subject. To assess whether there was significantly greater within-subject predictive information in the neural data, we compared the within- and between-subject correlations with a Wilcoxon sign rank test. A one-tailed test was used a result of our one-sided hypothesis that the correlation should be greater within- than between-subject. True-Memory Individuation Analysis. To be consistent with the false-memory analysis, we defined a “true memory” as a high-confidence old response to a previously presented DRM list item. The true-memory vector was created by calculating for each DRM list the proportion of words that were judged to be old with high confidence. This resulted in a true-memory vector of length 40 for each individual subject. The true-memory individuation analysis was otherwise identical to the false-memory individuation analysis described above. Control Analyses. To ensure that the results were driven by neural similarity that was specific to the false-memory strength and not additional extraneous factors, we conducted three control analyses. First, we established that word frequency ACKNOWLEDGMENTS. H.J.S. is funded by the James S. McDonnell Foundation and The Wellcome Trust; D.L.S. is supported by National Institute of Mental Health Grant MH060941. 26. Stadler MA, Roediger HL, 3rd, McDermott KB (1999) Norms for word lists that create false memories. Mem Cognit 27(3):494–500. 27. Roediger HL, 3rd, Watson JM, McDermott KB, Gallo DA (2001) Factors that determine false recall: A multiple regression analysis. Psychon Bull Rev 8(3):385–407. 28. Kriegeskorte N, Goebel R, Bandettini P (2006) Information-based functional brain mapping. Proc Natl Acad Sci USA 103(10):3863–3868. 3. Reyna VF, Corbin JC, Weldon RB, Brainerd CJ (2016) How fuzzy-trace theory predicts true and false memories for words, sentences, and narratives. J Appl Res Mem Cogn 5(1):1–9. 4. Deese J (1959) On the prediction of occurrence of particular verbal intrusions in im- mediate recall. J Exp Psychol 58(1):17–22. 29. Kriegeskorte N, Simmons WK, Bellgowan PSF, Baker CI (2009) Circular analysis in systems neuroscience: The dangers of double dipping. Discussion We used the Levenshtein edit distance to assess the similarity between each pair of words, as this has been shown to be a good predictor of various lexical effects (49) and is therefore appropriate for assessing low-level word similarity. For each DRM set, the visual similarity was defined as the average edit distance between the lure and list items. Finally, we explored whether the incidental task performed in the scanner could be driving the neural similarity results. To investigate this possibility, for each of the 40 DRM lists we quantified the number of list words where the subject had indicated the same category as the related lure concept for that list. The stronger the degree of correspondence, the stronger any task category representation should be for that particular DRM list. Each of these three control variables was correlated with the neural overlap data to determine whether each significantly contributed to the neural data. Additionally, all three variables were controlled for in each analysis reported in Results. (48) was not contributing to the neural data. Given that the neural overlap was based on the similarity between each lure and the respective list items, the ab- solute word frequency of the critical lure could not by itself explain this measure. Instead, we calculated the average difference in word frequency between each DRM lure and the four DRM list items. Second, we investigated whether visual similarity between the words could be contributing to the effects. We used the Levenshtein edit distance to assess the similarity between each pair of words, as this has been shown to be a good predictor of various lexical effects (49) and is therefore appropriate for assessing low-level word similarity. For each DRM set, the visual similarity was defined as the average edit distance between the lure and list items. Finally, we explored whether the incidental task performed in the scanner could be driving the neural similarity results. To investigate this possibility, for each of the 40 DRM lists we quantified the number of list words where the subject had indicated the same category as the related lure concept for that list. The stronger the degree of correspondence, the stronger any task category representation should be for that particular DRM list. Each of these three control variables was correlated with the neural overlap data to determine whether each significantly contributed to the neural data. 2. Qin S, Hermans EJ, van Marle HJF, Fernández G (2012) Understanding low reliability of memories for neutral information encoded under stress: Alterations in memory- related activation in the hippocampus and midbrain. J Neurosci 32(12):4032–4041. Discussion These were converted to t-statistics by dividing the parameter estimate by the estimate of the SE, thereby normalizing the re- sponses of each voxel (43). The resulting t-statistic images were left unsmoothed to preserve any fine-grained spatial information (44). False Memory Individuation Analysis. To test for the presence of unique TP neural information that predicts subject-specific false memories, we used an individuation analysis (30). The logic here is that if an individual has a unique set of TP neural representations that meaningfully influences cognition, then that individual’s neural overlap data should predict their own pattern of false- memory errors better than any other subject’s false-memory errors. To assess this possibility, we created a false-memory vector for each individual subject based on their specific pattern of false-memory errors in the behavioral DRM session. Given that we were specifically interested in genuine false memories rather than mistakes driven by uncertainty, we defined a false memory as a high-confidence old response to a related concept lure. The false-memory vector was in each case a binary vector of 40 values for the 40 DRM lists, with a 1 indicating the DRM lists Searchlight Analysis. We used a searchlight representational similarity analysis (22, 28) to search for brain regions containing the predicted neural code. Representational similarity analysis uses the neural pattern similarity between pairs of stimuli to infer the representational similarity. This approach is there- fore highly appropriate for assessing neural overlap between semantic repre- sentations, as the degree of overlap should be directly reflected in the representational similarity. We first assessed the neural overlap between each DRM list and its related lure concept by measuring the Pearson correlation between the pair of voxel patterns. In each case, we normalized the similarity Chadwick et al. 10184 \| www.pnas.org/cgi/doi/10.1073/pnas.1610686113 (48) was not contributing to the neural data. Given that the neural overlap was based on the similarity between each lure and the respective list items, the ab- solute word frequency of the critical lure could not by itself explain this measure. Instead, we calculated the average difference in word frequency between each DRM lure and the four DRM list items. Second, we investigated whether visual similarity between the words could be contributing to the effects. 1. Schacter DL, Guerin SA, St Jacques PL (2011) Memory distortion: An adaptive per- spective. Trends Cogn Sci 15(10):467–474. Chadwick et al. Discussion Skipper-Kallal LM, Mirman D, Olson IR (2015) Converging evidence from fMRI and aphasia that the left temporoparietal cortex has an essential role in representing abstract semantic knowledge. Cortex 69:104–120. 45. Björnsdotter M, Rylander K, Wessberg J (2011) A Monte Carlo method for locally multivariate brain mapping. Neuroimage 56(2):508–516. 20. Wang J, Conder JA, Blitzer DN, Shinkareva SV (2010) Neural representation of abstract and concrete concepts: A meta-analysis of neuroimaging studies. Hum Brain Mapp 31(10):1459–1468. 46. Nichols TE, Holmes AP (2002) Nonparametric permutation tests for functional neu- roimaging: A primer with examples. Hum Brain Mapp 15(1):1–25. 21. Clarke A, Tyler LK (2015) Understanding what we see: How we derive meaning from vision. Trends Cogn Sci 19(11):677–687. 47. Desikan RS, et al. (2006) An automated labeling system for subdividing the human cere- bral cortex on MRI scans into gyral based regions of interest. Neuroimage 31(3):968–980. 22. Kriegeskorte N, Kievit RA (2013) Representational geometry: Integrating cognition, computation, and the brain. Trends Cogn Sci 17(8):401–412. 48. Brysbaert M, New B (2009) Moving beyond Kucera and Francis: A critical evaluation of current word frequency norms and the introduction of a new and improved word frequency measure for American English. Behav Res Methods 41(4):977–990. 23. Bruffaerts R, et al. (2013) Similarity of fMRI activity patterns in left perirhinal cortex reflects semantic similarity between words. J Neurosci 33(47):18597–18607. NEUROSCIENCE 49. Yarkoni T, Balota D, Yap M (2008) Moving beyond Coltheart’s N: A new measure of orthographic similarity. Psychon Bull Rev 15(5):971–979. reflects semantic similarity between words. J Neurosci 33(47):18597–18 24. Devereux BJ, Clarke A, Marouchos A, Tyler LK (2013) Representational similarity analysis reveals commonalities and differences in the semantic processing of words and objects. J Neurosci 33(48):18906–18916. 50. Xia M, Wang J, He Y (2013) BrainNet Viewer: A network visualization tool for human brain connectomics. PLoS One 8(7):e68910. 25. Carlson TA, Simmons RA, Kriegeskorte N, Slevc LR (2014) The emergence of semantic meaning in the ventral temporal pathway. J Cogn Neurosci 26(1):120–131. 51. Morey R (2008) Confidence intervals from normalized data: A correction to Cousineau (2005). Tutor Quant Methods Psychol 4(2):61–64. PNAS \| September 6, 2016 \| vol. 113 \| no. 36 \| 10185 Chadwick et al.
https://openalex.org/W3106641936	https://eprints.qut.edu.au/206952/1/73260551.pdf	English	null	The human axial length and choroidal thickness responses to continuous and alternating episodes of myopic and hyperopic blur	PloS one	2,020	cc-by	10,065	This may be the author’s version of a work that was submitted/accepted for publication in the following source: Delshad, Samaneh, Collins, Michael, Read, Scott, & Vincent, Stephen (2020) The human axial length and choroidal thickness responses to continuous and alternating episodes of myopic and hyperopic blur. PLoS One, 15(12), Article number: e0243076. Delshad, Samaneh, Collins, Michael, Read, Scott, & Vincent, Stephen (2020) ( ) The human axial length and choroidal thickness responses to continuous and alternating episodes of myopic and hyperopic blur. PLoS One, 15(12), Article number: e0243076. This ﬁle was downloaded from: https://eprints.qut.edu.au/206952/ © The Author(s) This work is covered by copyright. 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If there is any doubt, please refer to the published source. https://doi.org/10.1371/journal.pone.0243076 https://doi.org/10.1371/journal.pone.0243076 PLOS ONE PLOS ONE RESEARCH ARTICLE Samaneh DelshadID, Michael John Collins, Scott Andrew Read, Stephen James VincentID Samaneh DelshadID, Michael John Collins, Scott Andrew Read, Stephen James VincentID Samaneh DelshadID, Michael John Collins, Scott Andrew Read, Stephen James VincentID Queensland University of Technology (QUT), Centre for Vision and Eye Research, School of Optometry and Vision Science, Institute of Health and Biomedical Innovation, Kelvin Grove, Queensland, Australia samaneh.delshad@connect.qut.edu.au a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Methods The right eye of sixteen young adults was exposed to 60 minute episodes of either continu- ous or alternating myopic and hyperopic defocus (+3 DS & -3 DS) over six separate days, with the left eye optimally corrected for distance. During alternating defocus conditions, the eye was exposed to either 30 or 15 minute cycles of myopic and hyperopic defocus, with the order of defocus reversed in separate sessions. The AxL and ChT of the right eye were measured before, during and after each defocus condition. Editor: Der-Chong Tsai, National Yang-Ming University Hospital, TAIWAN Received: May 5, 2020 Accepted: November 14, 2020 Published: December 2, 2020 Copyright: © 2020 Delshad 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. Editor: Der-Chong Tsai, National Yang-Ming University Hospital, TAIWAN Received: May 5, 2020 Accepted: November 14, 2020 Published: December 2, 2020 Copyright: © 2020 Delshad 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. OPEN ACCESS Citation: Delshad S, Collins MJ, Read SA, Vincent SJ (2020) The human axial length and choroidal thickness responses to continuous and alternating episodes of myopic and hyperopic blur. PLoS ONE 15(12): e0243076. https://doi.org/10.1371/journal. pone.0243076 Purpose To investigate the change in axial length (AxL) and choroidal thickness (ChT) in response to continuous and alternating episodes of monocular myopic and hyperopic defocus. * samaneh.delshad@connect.qut.edu.au Introduction Optical defocus can lead to predictable changes in choroidal thickness and eye growth in vari- ous animal species [1–5]. Rapid choroidal thickening in response to imposed myopic defocus [6, 7] precedes a slowing of longer-term eye growth [8–11], while rapid choroidal thinning in response to imposed hyperopic defocus [6, 7] precedes accelerated eye growth [8–11]. These compensatory changes result in the retinal photoreceptors moving closer to the defocused image plane. In humans, the quality of visual experience can also influence ocular growth. In eyes experiencing chronic deprivation of form vision through ocular conditions such as ptosis [12, 13], congenital cataract [14], corneal opacity [15], and vitreous haemorrhage [16], abnor- mal ocular growth is often observed. Similarly, manipulation of retinal image focus through optical treatment strategies such as bifocal or multifocal spectacles [17, 18], soft multifocal con- tact lenses [19, 20], or orthokeratology [21–24] have been linked with reduced progression of axial myopia in children. Investigations in human eyes have also shown a bi-directional response to short-term imposed continuous myopic and hyperopic defocus in children [25] and adults [26–31], with a small magnitude axial length reduction associated with rapid cho- roidal thickening in response to myopic defocus and axial elongation associated with rapid choroidal thinning in response to hyperopic defocus. In the absence of ocular pathology leading to a disruption in form vision, the visual control of eye growth could be associated with the dynamics of visual experience and the type and magnitude of defocus to which the eyes are exposed on a daily basis. Natural visual scenes typi- cally comprise objects at varying distances that produce myopic and hyperopic defocus, and well-focused retinal images, depending on where the eyes are fixating and focused within the environment [32–34]. The temporal integration of these defocus signals may provide input to the regulation of eye growth [6, 32, 33, 35]. Studies using various animal models have shown that over a period of days or weeks, the eyes use a complex method for integrating defocus sig- nals over time rather than a simple linear summation of the blur that it experiences [6, 35–44]. These studies suggest that the temporal integration of defocus signals is dependent upon the sign and power of the defocus experienced [6, 36–43], and the frequency and duration of indi- vidual episodes of blur [37, 44]. Introduction In humans, while studies have shown that the eye is responsive to short-term continuous myopic and hyperopic defocus by changing its axial length and choroidal thickness [25–31], it is not known how the eye responds to more complex temporally varying patterns of defocus, as might be encountered in real-world visual scenes. In this study, we tested the hypothesis that the short-term response of axial length and choroidal thickness to alternating episodes of myopic and hyperopic defocus in the human eye will reflect a simple temporal summation of the blur signals. Conclusions The human eye appears capable of temporal averaging of visual cues from alternating myo- pic and hyperopic defocus. In the short term, this integration appears to be a cancellation of the effects of the preceding defocus condition of opposite sign. Results Significant changes in AxL were observed over time, dependent upon the defocus condition (p < 0.0001). In general, AxL exhibited a greater magnitude of change during continuous than alternating defocus conditions. The maximum AxL elongation was +7 ± 7 μm (p = 0.010) in response to continuous hyperopic defocus and the maximum AxL reduction was -8 ± 10 μm of (p = 0.046) in response to continuous myopic defocus. During both 30 and 15 minute cycles of alternating myopic and hyperopic defocus of equal duration, the effect of opposing blur sessions cancelled each other and the AxL was near baseline levels following the final defocus session (mean change from baseline across all alternating defocus condi- tions was +2 ± 10 μm, p > 0.05). Similar, but smaller magnitude, changes were observed for ChT. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur Materials and methods Sixteen young adults (11 females, 5 males) aged between 19 and 34 years (mean ± SD, 24.8 ± 4.1 years) were recruited for the study. This sample size provided 80% power to detect 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur an intrasession axial length change of 10 μm at the 5% level, assuming a standard deviation of 10 μm [26]. All subjects were free from any systemic or ocular diseases and had no prior his- tory of eye injury or surgery. Before the study, each subject underwent an initial screening to ensure good ocular health and normal binocular vision, and to determine their refractive sta- tus. The spherical equivalent refraction across subjects ranged between +0.50 DS and -2.75 DS with a mean ± SD of -0.57 ± 1.12 DS. All subjects had anisometropia of less than 0.50 DS, astig- matism of -0.75 DC and exhibited visual acuity of at least 0.00 logMAR (20/20 Snellen acu- ity) in both eyes. No contact lens wearers were included in the study. Ethics approval was obtained from the Queensland University of Technology human research ethics committee prior to commencement of the study. Written informed consent was obtained from all sub- jects, who were treated in accordance with the Declaration of Helsinki. This study involved the measurement of axial length before, during and after six different defocus conditions. Subfoveal choroidal thickness was also measured before and after exposure to defocus. Each defocus condition was conducted on a separate day in order to allow any potential effects to dissipate from the prior defocus session (a minimum 24-hour washout period between the defocus conditions). The order of the defocus conditions was randomized between subjects to ensure no order-related bias in the data. To minimize the potential for a confounding interaction between the diurnal variations in axial length and subfoveal choroidal thickness [45, 46] and the eye’s response to defocus [47], all measurement sessions were con- ducted at a similar time of day (between 8:00 am and 2:00 pm with an average daily time differ- ence of 62 ± 23 minutes between sessions within each subject), and at least two hours after the subjects’ reported time of waking. None of the subjects enrolled in this study were taking any medications which could influence their ocular biometry measurements. PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 Materials and methods Since smoking may affect the measurement of choroidal thickness, smokers were also not included in this study [48]. Due to the influence of caffeine intake and dynamic exercise on ocular biometry, all par- ticipants were asked to abstain from consumption of caffeine or performing vigorous exercise for at least 1 hour prior to the start of each measurement session [49, 50]. Since prior visual tasks (e.g. intense near-work) could potentially affect measurements of axial length and cho- roidal thickness, each subject completed a 20 minute “washout period” during which they watched a movie of their choosing at a 6 m distance with their optimal distance refractive cor- rection before each measurement session. Following completion of the “washout period”, baseline measurements of axial length and subfoveal choroidal thickness were obtained from the right eye, and then, a 60 minute monocular “defocus period” was conducted. During the “defocus period”, each subjects’ right eye was exposed to six different defocus conditions (monocular defocus over the right eye’s optimal distance refraction), while watch- ing movies on a TV at 6 m, with the left eye optimally corrected for distance viewing to main- tain relaxed accommodation. This experimental paradigm has been used previously and allows control of the accommodation response with the non-tested (left) eye, while simulta- neously producing defocus conditions in the tested (right) eye [25–30]. Over six separate ses- sions, the right eye was exposed to 60 minute episodes of either continuous or alternating myopic and hyperopic defocus (+3 DS and -3 DS). During the alternating defocus conditions, the eye was exposed to either 30 minutes or 15 minutes of alternating cycles of myopic and hyperopic defocus, with the defocus order reversed in separate sessions (Fig 1). Five repeated measures of axial length (measured from the anterior corneal surface to the retinal pigment epithelium [RPE]) were obtained at baseline, and then every 15 minutes dur- ing both continuous and alternating defocus conditions, using the Lenstar optical biometer (LS 900, Haag Streit AG, Koeniz, Switzerland). This is a non-contact biometry device that works on the principle of optical low-coherence reflectometry (OLCR), using a broad-band light source (range of 20–30 nm), with a central wavelength of 820 nm. The Lenstar optical PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 3 / 16 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur Fig 1. Materials and methods Illustration of the type of defocus imposed on the right eye for each measurement day. On six separate measurement days, the right eye was exposed to one of the following conditions (in a randomized order): (A) continuous hyperopic (-3 D) defocus (1 x 60 minutes), (B) continuous myopic (+3 D) defocus (1 x 60 minutes), (C) alternating low frequency cycles of hyperopic then myopic defocus (2 x 30 minutes), (D) alternating low frequency cycles of myopic then hyperopic defocus (2 x 30 minutes), (E) alternating high frequency cycles of hyperopic then myopic defocus (4 x 15 minutes), and (F) alternating high frequency cycles of myopic then hyperopic defocus (4 x 15 minutes). https://doi.org/10.1371/journal.pone.0243076.g001 Fig 1. Illustration of the type of defocus imposed on the right eye for each measurement day. On six separate measurement days, the right eye was exposed to one of the following conditions (in a randomized order): (A) continuous hyperopic (-3 D) defocus (1 x 60 minutes), (B) continuous myopic (+3 D) defocus (1 x 60 minutes), (C) alternating low frequency cycles of hyperopic then myopic defocus (2 x 30 minutes), (D) alternating low frequency cycles of myopic then hyperopic defocus (2 x 30 minutes), (E) alternating high frequency cycles of hyperopic then myopic defocus (4 x 15 minutes), and (F) alternating high frequency cycles of myopic then hyperopic defocus (4 x 15 minutes). htt //d i /10 1371/j l 0243076 001 https://doi.org/10.1371/journal.pone.0243076.g001 https://doi.org/10.1371/journal.pone.0243076.g001 biometer has a display resolution of 10 μm. To improve the accuracy of the measurements of axial length, only five consecutive measurements with a cumulative standard deviation (as dis- played on the Lenstar) of 7 μm were included. If any of the five consecutive measurements yielded a cumulative SD of > 7 μm, that measurement was deleted and then immediately repeated with a new measurement. To control for accommodation and to provide continuous exposure to defocus during biometry, a binocular beam splitter periscope system was used to allow fixation of an external target (a high contrast Maltese cross) at a 6 m distance during measurement acquisition. PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 Materials and methods In order to provide the periscopic view of the Maltese cross, the sys- tem was adjusted and once the centre of the Maltese cross was aligned with the internal fixa- tion target of the biometer (the red fixation target of the Lenstar), the subject was asked to fixate the centre of the target at a 6 m distance. When using the periscope system, the subject’s sphero-cylinder distance refraction was mounted in a trial frame in front of each eye and the additional defocus lens was positioned in front of the right eye. A schematic diagram of this experimental set-up is shown in Fig 2. Fig 2. Schematic representation of the experimental set-up for axial length measures. A binocular periscope system was used to allow fixation of the distant target (Maltese cross) while the Lenstar biometer measured axial length. https://doi.org/10.1371/journal.pone.0243076.g002 Fig 2. Schematic representation of the experimental set-up for axial length measures. A binocular periscope system was used to allow fixation of the distant target (Maltese cross) while the Lenstar biometer measured axial length. https://doi.org/10.1371/journal.pone.0243076.g002 4 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur To assess the intrasession repeatability of axial length measurements, the within-session standard deviation (SD) and within-session range of five consecutive measurements of axial length across all subjects and all defocus conditions were calculated [51]. The intraclass correla- tion coefficient (ICC) was also calculated. The within-session SD was 5 μm, with a within-ses- sion range of 11 μm, and ICC of 0.999, indicating highly precise measurements of axial length. The subfoveal choroidal thickness of the right eye (defocused eye) was also measured using spectral domain optical coherence tomography (SD-OCT) (Copernicus SOCT-HR; Optopol Technology SA, Zawiercie, Poland). This device provides high resolution, cross-sectional images of the posterior eye, using a peak wavelength of 850 nm and has axial and transverse resolutions of 3 μm, and 5 μm (in tissue), respectively. The measurements of choroidal thick- ness were obtained at baseline, and then following removal of the defocus lens at the end of the 60 minute defocus period (immediately following the final measurement of axial length). The OCT images were obtained with a 5 mm horizontal foveal line scan, consisting of 40 B-scans, each with 1500 A-scans, with an acquisition time of 1.52 seconds. Materials and methods Following data collection, the five repeated measures of axial length at each measurement time point were averaged for each defocus condition, and across subjects. For the measure- ments of subfoveal choroidal thickness, the raw OCT images were extracted from the instru- ment and then analysed using custom written software [52]. For each OCT image, the software aligned and averaged the 40 individual B-scans, to generate a high-quality average B- scan image with reduced speckle noise and increased visibility of the posterior segment struc- tures. The resolution of the final OCT images was 2.26 microns per pixel. Each averaged OCT image for each subject at each measurement time point was then manually segmented by an experienced observer who was masked to the time of the measurement and type of defocus condition, for all of the scans. To provide an assessment of the repeatability and reliability of the subfoveal choroidal thick- ness segmentation, the masked observer manually segmented the baseline subfoveal choroidal thickness of all subjects for two randomly selected defocus conditions, twice. The coefficient of repeatability and 95% confidence interval of the coefficient of repeatability (derived from both defocus conditions) [51], were 6 μm and 3–9 μm, respectively. A paired sample t-test revealed no significant difference between the two measurements (p > 0.05). Bland-Altman analysis [53] revealed excellent agreement between the two measures of the subfoveal choroidal thick- ness, with a negligible mean difference of -0.5 μm and 95% limits of agreement of -9 to +7 μm. The intraobserver reliability was assessed using the ICC (two-way mixed model, absolute agree- ment) and was excellent at 0.999, with a 95% confidence interval of 0.998–1.00. The Shapiro-Wilk test of normality revealed that the axial length and subfoveal choroidal thickness data did not significantly depart from a normal distribution (p > 0.05). In order to assess the effects of defocus condition for each variable, the data were analysed using a repeated measures ANOVA with two within-subjects factors (time and type of defocus). Following the repeated measures ANOVA, for any variables with significant main effects and interactions (p < 0.05), pairwise comparisons with Bonferroni corrections were conducted. To assess the effects of defocus order and defocus frequency on each variable, a multifactorial linear mixed model analysis was used. PLOS ONE PLOS ONE Table 1. Mean change in axial length from baseline across different measurement times for continuous and alternating defocus conditions in all subjects. 15-minute 30-minute 45-minute 60-minute Defocus condition Mean change ± SD (μm) P value Mean change ± SD (μm) P value Mean change ± SD (μm) P value Mean change ± SD (μm) P value Condition A +5 ± 6 0.038 +6 ± 4 0.0001 +7 ± 7 0.010 +7 ± 7 0.027 Condition B -4 ± 5 0.040 -6 ± 7 0.043 -7 ± 7 0.025 -8 ± 10 0.046 Condition C +5 ± 8 0.219 +7 ± 9 0.045 +1 ± 12 1.000 0 ± 11 1.000 Condition D -4 ± 7 0.345 -6 ± 9 0.115 0 ± 11 1.000 -2 ± 12 1.000 Condition E +5 ± 8 0.330 -1 ± 9 1.000 -2 ± 9 1.000 -4 ± 7 0.251 Condition F -3 ± 6 0.307 -2 ± 7 1.000 -4 ± 6 0.265 -3 ± 8 1.000 Asterisk indicates a significant change (p < 0.05) in axial length from the baseline measurement. https://doi.org/10.1371/journal.pone.0243076.t001 Asterisk indicates a significant change (p < 0.05) in axial length from the baseline measurement. Asterisk indicates a significant change (p < 0.05) in axial length from the baseline measurement. Asterisk indicates a significant change (p < 0.05) in axial length from the baseline measurement Continuous defocus. Pairwise comparisons with Bonferroni corrections revealed that 15 minutes after the introduction of hyperopic defocus (Condition A), there was a significant axial elongation of +5 ± 6 μm from baseline (p = 0.038). Following this initial response, the axial length remained relatively stable until the end of the defocus period and was significantly different from baseline at all subsequent measurement times (all p < 0.05). The maximum axial elongation occurred at 45 minutes with the eye being +7 ± 7 μm longer than the baseline measurement (p = 0.010). The axial length at the end of 60 minutes was +7 ± 7 μm longer than the baseline measurement (p = 0.027) (Table 1) (Fig 3). The introduction of myopic defocus (Condition B) produced a significant axial length reduction of -4 ± 5 μm from baseline after 15 minutes (p = 0.040). Axial length Repeated measures ANOVA revealed a highly significant interaction between the type of defocus and time of measurement for axial length measures across the six defocus conditions (F (20,300) = 4.60, p < 0.0001). 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 The human eye’s response to alternating episodes of myopic and hyperopic blur PLOS ONE The mean change in axial length from baseline (0 minute time point) during 30 minute alternating cycles of hyperopic then myopic defocus (Condition C), and myopic then hyperopic defocus (Condition D) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from the baseline axial length (p < 0.05). https://doi org/10 1371/journal pone 0243076 g004 Fig 4. The mean change in axial length from baseline (0 minute time point) during 30 minute alternating cycles of hyperopic then myopic defocus (Condition C), and myopic then hyperopic defocus (Condition D) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from the baseline axial length (p < 0.05). https://doi.org/10.1371/journal.pone.0243076.g004 observed when the right eye was exposed to 30 minute alternating cycles of myopic then hyperopic defocus (Condition D). After the first 30 minutes of myopic defocus, the axial length was shorter than the baseline by -6 ± 9 μm (p > 0.05). This reduction in axial length then grad- ually cancelled during the next 30 minutes of hyperopic defocus of equal power, and by the end of 60 minutes, the eye was almost at the baseline level (mean difference of -2 ± 12 μm, p > 0.05) (Table 1) (Fig 4). Alternating defocus (15 minute cycles). When the eye was exposed to 15 minute alter- nating cycles of hyperopic and myopic defocus, a cancellation effect of opposing blur sessions did not occur as the axial length was observed to shorten slowly over time. During 60 minute alternating cycles of hyperopic then myopic defocus (Condition E), after the first 15 minutes of uninterrupted hyperopic defocus, axial length changed by +5 ± 8 μm from baseline (p > 0.05). This increase in axial length was then cancelled during the next 15 minutes of unin- terrupted myopic defocus, as the eye returned to below baseline level at 30 minutes. From 30 to 60 minutes, axial length continued to shorten slowly and reached a -4 ± 7 μm reduction from baseline following the final defocus session (p > 0.05) (Table 1) (Fig 5). PLOS ONE The axial length then continued to shorten gradually over the next 45 minutes, reaching a significant maximum axial length reduction of -8 ± 10 μm at the end of the 60 minutes (p = 0.046) (Table 1) (Fig 3). Alternating defocus (30 minute cycles). When the right eye was exposed to 30 minute alternating cycles of hyperopic then myopic defocus (Condition C), after the first 30 minutes of hyperopic defocus, the axial length was significantly longer than baseline by +7 ± 9 μm (p = 0.045). The introduction of myopic defocus then gradually cancelled the previous axial elongation effects of hyperopic defocus and over the next 30 minutes the eye approached the baseline axial length level (mean difference of 0 ± 11 μm from baseline at the end of the 60 minutes, p > 0.05) (Table 1) (Fig 4). A similar cancellation effect of opposing blur was Fig 3. The mean change in axial length from baseline (0 minute time point), during 60 minutes of continuous hyperopic defocus (Condition A) and continuous myopic defocus (Condition B) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from baseline axial length (p < 0.05). https://doi.org/10.1371/journal.pone.0243076.g003 Fig 3. The mean change in axial length from baseline (0 minute time point), during 60 minutes of continuous hyperopic defocus (Condition A) and continuous myopic defocus (Condition B) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from baseline axial length (p < 0.05). https://doi.org/10.1371/journal.pone.0243076.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 6 / 16 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur Fig 4. The mean change in axial length from baseline (0 minute time point) during 30 minute alternating cycles of hyperopic then myopic defocus (Condition C), and myopic then hyperopic defocus (Condition D) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from the baseline axial length (p < 0.05). https://doi.org/10.1371/journal.pone.0243076.g004 Fig 4. PLOS ONE A similar pattern of response was also observed for axial length during 15 minute alternating cycles of myopic then hyperopic defocus (Condition F), as it shortened gradually over time. Following comple- tion of the two cycles of myopic then hyperopic defocus, the axial length was -3 ± 8 μm less than the baseline measurement at 60 minutes (p > 0.05) (Table 1) (Fig 5). Effects of defocus order and defocus frequency. Assessment of the effect of defocus order revealed that regardless of the frequency of the cycles, there was no significant difference in the final axial length response to defocus observed when the eye was exposed to myopic then hyperopic defocus, or hyperopic then myopic defocus (p > 0.05). Similarly, there was no significant difference in the final axial length response between the 15 minute and 30 minute cycling frequencies for each order of defocus, indicating no significant effects of defocus fre- quency (p > 0.05). Axial length changes: Continuous vs alternating defocus After the first 30 minutes of uninterrupted hyperopic defocus, baseline axial length increased by a similar magnitude during both the continuous hyperopic defocus (Condition A) and the PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 7 / 16 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur Fig 5. The mean change in axial length from baseline (0 minute time point) during 15 minute alternating cycles of hyperopic then myopic defocus (Condition E), and myopic then hyperopic defocus (Condition F) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from the baseline axial length (p < 0.05). https://doi.org/10.1371/journal.pone.0243076.g005 Fig 5. The mean change in axial length from baseline (0 minute time point) during 15 minute alternating cycles of hyperopic then myopic defocus (Condition E), and myopic then hyperopic defocus (Condition F) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from the baseline axial length (p < 0.05). https://doi org/10 1371/journal pone 0243076 g005 Fig 5. The mean change in axial length from baseline (0 minute time point) during 15 minute alternating cycles of hyperopic then myopic defocus (Condition E), and myopic then hyperopic defocus (Condition F) for all subjects. Vertical error bars represent the standard error of the mean difference in axial length, and horizontal error bars represent the standard error in measurement time. Asterisks indicate a significant mean difference from the baseline axial length (p < 0.05). https://doi.org/10.1371/journal.pone.0243076.g005 https://doi.org/10.1371/journal.pone.0243076.g005 alternating hyperopic then myopic defocus (Condition C) conditions (+6 ± 4 μm vs +7 ± 9 μm, p > 0.05, Figs 3 and 4 respectively). From 30 minutes to 60 minutes, axial length remained stable during continuous hyperopic defocus (+1 ± 6 μm increase, p > 0.05, Fig 3), while it reduced significantly by -7 ± 10 μm during alternating hyperopic then myopic defocus (p = 0.022, Fig 4). The magnitude of change in axial length from 30 minutes to 60 minutes between the continuous hyperopic defocus and the alternating hyperopic then myopic defocus conditions was significantly different (p = 0.033). PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 Discussion This study has shown for the first time that the human eye is capable of temporal averaging of visual cues from alternating myopic and hyperopic defocus. Whilst exposure to continuous defocus led to significant bi-directional changes in axial length, the change in axial length was minimal when the eye was exposed to alternating periods of myopic and hyperopic defocus. During a 30 minute cycling frequency, alternating episodes of defocus of opposite power largely cancelled each other, and the eye remained at near baseline levels after 60 minutes. During a 15 minute cycling frequency, the eye’s response to myopic defocus appeared to be greater than the response to hyperopic defocus, as the eye was slightly shorter than the baseline measurement at 60 minutes, however this change was not statistically significant. Similar but smaller magnitude changes were also observed for subfoveal choroidal thickness. The findings from the continuous defocus conditions are consistent with those from previ- ous studies of human [26, 29–31] and other animal eyes [6, 7, 36] where small but significant changes in axial length and choroidal thickness have been reported after short term exposure to continuous myopic and hyperopic defocus. A recent investigation in human eyes has shown a significant change in axial length, approximately 2 minutes after exposure to imposed defo- cus [26]. Similarly, significant bi-directional changes in subfoveal choroidal thickness after 10 to 35 minutes have been reported [30]. An investigation in school children found no signifi- cant change in axial length after 60 minutes exposure to defocus [25], however, as the measure- ments were taken following the instillation of 1% cyclopentolate (an antimuscarinic agent that is known to affect the thickness of the choroid and axial length) [54, 55], these findings may have been influenced by the effects of the drug. Further, in a recent investigation, a 30 minute exposure to myopic defocus with full field and multifocal contact lenses yielded no significant change in choroidal thickness [56]. This difference in outcomes may be due to the shorter duration of exposure to defocus or to the influence of the contact lens correction. It is evident from our findings during the continuous defocus conditions that the human eye is able to respond to the sign of blur rapidly (within 15 minutes) and make distinct bi-directional changes in its axial length. Axial length changes: Continuous vs alternating defocus Condition A Condition B Condition C Condition D Condition E Condition F Mean change ± SD (μm) -4 ± 11 +8 ± 11 +7 ± 14 +2 ± 15 +5 ± 17 +5 ± 17 P value 0.208 0.028 0.087 0.685 0.278 0.273 Asterisk indicates a significant change (p < 0.05) in baseline subfoveal choroidal thickness after 60 minutes of defocus exposure. https://doi org/10 1371/journal pone 0243076 t002 Table 2. Mean change in subfoveal choroidal thickness from baseline across different defocus conditions in all subjects. Subfoveal choroidal thickness Imposing defocus resulted in some significant changes in subfoveal choroid thickness over time. At 60 minutes, the subfoveal choroid was -4 ± 11 μm thinner than baseline after continu- ous hyperopic defocus (Condition A) (p > 0.05), while it was significantly thicker by +8 ± 11 μm following continuous myopic defocus (Condition B) (p = 0.030) (Table 2). When exposed to 30 minute or 15 minute alternating cycles of defocus, subfoveal choroidal thickness did not differ significantly from baseline after hyperopic then myopic defocus (Con- dition C & Condition E), or after myopic then hyperopic defocus (Condition D & Condition F) (p > 0.05) (Table 2). There were no significant effects of defocus order and defocus fre- quency for any of the analyses of the subfoveal choroidal thickness measures (all p > 0.05). Axial length changes: Continuous vs alternating defocus After the first 30 minutes of uninterrupted myopic defocus, axial length decreased by -6 ± 7 μm during continuous myopic defocus (Condition B) and by -6 ± 9 μm during the alter- nating myopic then hyperopic defocus condition (Condition D) (p > 0.05, Figs 3 and 4). From 30 minutes to 60 minutes, axial length changed by -2 ± 6 μm during continuous myopic defo- cus (p > 0.05, Fig 3) and by +4 ± 12 μm during the alternating myopic then hyperopic defocus condition (p > 0.05, Fig 4). There was a significant difference in the magnitude of change in axial length from 30 minutes to 60 minutes between the continuous myopic defocus and the alternating myopic then hyperopic defocus conditions (p = 0.044). After the first 15 minutes of uninterrupted hyperopic defocus, baseline axial length increased significantly and by a similar magnitude during both the continuous hyperopic defo- cus (Condition A) and the alternating hyperopic then myopic defocus (Condition E) condi- tions (+5 ± 6 μm vs +5 ± 8 μm, p > 0.05, Figs 3 and 5 respectively). From 15 minutes to 60 minutes, axial length remained relatively stable during continuous hyperopic defocus (mean change of +2 ± 7 μm p > 0.05, Fig 3) while it shortened significantly by -9 ± 8 μm (p < 0.0001, Fig 5) during the alternating hyperopic then myopic defocus condition. After the first 15 minutes of uninterrupted myopic defocus, axial length changed by -4 ± 5 μm during continuous myopic defocus (Condition B) and by -3 ± 6 μm during alternat- ing myopic then hyperopic defocus condition (Condition F) (p > 0.05, Figs 3 and 5). From 15 minutes to 60 minutes, axial length changed by -4 ± 8 μm during continuous myopic defocus (p = 0.05, Fig 3) while it remained stable during alternating myopic then hyperopic defocus condition (+1 ± 9 μm, p > 0.05, Fig 5). 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 The human eye’s response to alternating episodes of myopic and hyperopic blur PLOS ONE Table 2. Mean change in subfoveal choroidal thickness from baseline across different defocus conditions in all subjects. PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 Discussion Although the underlying mechanisms of this response are not fully understood; a variety of potential mechanisms such as the role of contrast cues from contrast PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 9 / 16 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur adaptation (changes in contrast sensitivity at different spatial frequencies) [57–60], colour cues from chromatic aberration [61–64], or optical vergence cues from image defocus [65] have all been suggested as potential ways in which the human eye decodes the sign of blur. The maximum mean axial length change observed during continuous hyperopic defocus was +7 ± 7 μm of elongation and during continuous myopic defocus was a -8 ± 10 μm reduc- tion. These findings correspond closely with published data in human eyes where changes of similar magnitude in axial length following continuous short term exposure to myopic and hyperopic defocus have been reported [26, 29]. However, when the pattern of defocus was alternated at a 30 minute frequency, the eye underwent a significantly different temporal pat- tern of change than it did during continuous defocus. The axial length response to each type of defocus after 30 minutes was almost cancelled during the succeeding 30 minute exposure to defocus of opposite power, and the axial length was near baseline levels after 60 minutes. This finding occurred irrespective of the order of defocus, indicating that the cancelling effects of myopic and hyperopic defocus for this particular duration and alternating frequency were of similar strength. When the frequency of alternating defocus cycles was 15 minutes, the myopic and hyper- opic defocus did not appear to completely cancel the preceding effects of each other; rather, a slight non-significant trend towards a more dominant response to myopic defocus was observed, and axial length was observed to reduce slightly. In animal models of blur integra- tion, a greater potency of myopic defocus in arresting the ocular elongation effect of hyperopic defocus has been reported. For instance, when myopic and hyperopic defocus were presented sequentially or simultaneously in chicks [6, 36–39], tree shrews [40, 41], monkeys [42], and marmosets [43], the eye responded preferentially to myopic defocus and developed less myo- pia/more hyperopia. Even when episodes of myopic and hyperopic defocus had equal duration when imposed alternatively over the chick eye, the axial growth still reduced, suggesting that myopic defocus provides a stronger growth signal [37]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 Discussion Whilst this amount of change is above the axial resolution of the OCT device (3 μm in tissue), it is within the range of the coefficient of repeatability and its 95% confidence interval for the measurements of choroidal thickness in this study (6 μm and 3–9 μm, respectively). Therefore, this limitation should be noted when considering the choroidal thickness changes observed in this study. Previous studies suggest two potential models in which defocus signals could be integrated over time; a simple linear model, and a more complex non-linear model [6, 35, 37, 66, 67]. Based on a simple linear model, the effects of equal powers of defocus of opposite sign would be added linearly so that the resulting compensation would be relative to the average exposure of defocus that the retina experienced, summed over a period of time (e.g. +3 D for 30 minutes followed by -3 D for 30 minutes = 0 change). However, based on a non-linear model, more complex outcomes of blur integration are expected where the final compensation could be multifactorial, depending on the sign and power of the defocus experienced (myopic or hyper- opic blur), and the frequency and duration of individual episodes of blur. Whilst our findings from the alternating defocus conditions suggest a simple summation of the effects of defocus of opposing sign over time, the findings from our continuous defocus conditions do not reflect a simple linear model. We observed that the rate of axial elongation and axial length reduction resulting from exposure to equal amounts of hyperopic and myopic defocus was not linearly proportional to the time exposed to blur. During continuous defocus exposure, almost 70% of the final axial elongation observed in response to hyperopic defocus (+7 ± 7 μm) occurred after 15 minutes of exposure to defocus, and 50% of the final axial length reduction (-8 ± 10 μm) in response to myopic defocus occurred after 15 minutes of exposure to defocus, indicating that the time course of the eye’s response to blur may be non-linear and may vary depending on the sign of defocus. Whilst experiments with animals have shown characteristics which strongly suggest a complex, non-linear model of temporal blur integration [6, 36–44] the mixed findings from our investigations do not allow us to confidently propose the model of temporal blur integration in human eyes. Further studies in this field seem warranted. Discussion Since the slight bias towards the domi- nating effects of myopic defocus observed in our study was not statistically significant, we can- not conclude if such properties also exist in the human eye. Future investigations involving longer durations of exposure to defocus or utilizing different magnitudes of defocus may pro- vide additional insights into the relative influence of myopic versus hyperopic blur. Defocus-mediated changes in axial length are expected to occur through modulations in the thickness of the choroid posterior to the retina, and thus affects the measurements of the axial length to the overlying RPE [26–30]. We found the bi-directional changes in the thickness of the subfoveal choroid to be consistent with the direction of the observed changes in axial length at the end of each defocus condition. In general, subfoveal choroidal thickening occurred along with axial length reduction, and subfoveal choroidal thinning occurred along with axial elongation. However, for the continuous defocus conditions, subfoveal choroidal thickening accounted for 87% of the observed mean axial length reduction during myopic defocus, and only accounted for 57% of the observed mean axial elongation during hyperopic defocus. A similar pattern of response was also observed for axial length and subfoveal choroi- dal thickness during alternating defocus. The discrepancies in the magnitude of axial length and choroidal thickness changes could have arisen in two possible ways. First, the final mea- surement of the subfoveal choroidal thickness was obtained approximately one minute after the removal of the defocus lens, therefore, during this brief period of clear viewing during the OCT imaging process, some decay in the effects of defocus could have occurred. Alternately, other factors such as expansion or contraction of the sclera might have also contributed to the observed axial length elongation and reduction with defocus. Since current imaging technol- ogy does not allow visualization of the thickness of the posterior sclera in most eyes, future PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 10 / 16 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur studies to understand the potential role of the sclera in mediating the human eye’s response to short-term defocus seem warranted. The changes in choroidal thickness with defocus (thickening or thinning) were found to range between 4 to 8 μm across the different defocus conditions investigated in this study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 Author Contributions Author Contributions Conceptualization: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Data curation: Samaneh Delshad. Formal analysis: Samaneh Delshad. Investigation: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Methodology: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Project administration: Samaneh Delshad. Resources: Michael John Collins. Validation: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Visualization: Samaneh Delshad. Writing – original draft: Samaneh Delshad. Writing – review & editing: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Ste phen James Vincent. Conceptualization: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Conceptualization: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Data curation: Samaneh Delshad. Formal analysis: Samaneh Delshad. Investigation: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Methodology: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Project administration: Samaneh Delshad. Resources: Michael John Collins. Validation: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Stephen James Vincent. Visualization: Samaneh Delshad. Visualization: Samaneh Delshad. Writing – original draft: Samaneh Delshad. Writing – review & editing: Samaneh Delshad, Michael John Collins, Scott Andrew Read, Ste- phen James Vincent. Discussion It has been proposed that the transient exposure to hyperopic defocus associated with near activities (e.g. due to lag of accommodation) [68–71], ocular aberrations [72, 73] or peripheral defocus [74] might predispose the eye to myopia. We found that exposure to myopic defocus is able to quickly counterbalance the axial elongation and subfoveal choroidal thinning effects of hyperopic defocus. If short-term ocular changes in response to defocus are associated with longer term refractive error development in the human eye, then optical methods of introduc- ing myopic blur for at least a similar duration to that of exposure to hyperopic blur, may coun- teract the myopigenic stimulus. However, it must be noted that only one level of myopic and hyperopic defocus and limited alternating defocus frequencies were tested in this study. The inter-subject variability in the axial length and choroidal thickness response to blur observed in this study could have been due to individual differences in retinal sensitivity to blur (e.g. an equal level of defocus degrades visual acuity by varying amounts between individ- uals). Also, while we attempted to control many of the known factors that influence choroidal thickness and axial length (diurnal rhythms, near work, medications, smoking, caffeine intake, and dynamic exercise) there are many other systemic factors that could potentially vary from day to day and affect the highly vascularized and sympathetically innervated choroid. In conclusion we have shown for the first time that the human eye is capable of temporal integration of myopic and hyperopic defocus signals. Over 60 minutes of blur exposure, this PLOS ONE \| https://doi.org/10.1371/journal.pone.0243076 December 2, 2020 11 / 16 PLOS ONE The human eye’s response to alternating episodes of myopic and hyperopic blur integration was effectively a simple summation (cancellation) of the effects of the preceding opposite sign blur condition. integration was effectively a simple summation (cancellation) of the effects of the preceding opposite sign blur condition. S1 Dataset. 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W2512517824.txt	https://emergency.zaslavsky.com.ua/index.php/journal/article/download/576/605	uk		Стан систолічної та діастолічної функції міокарда при сепсисі в дітей: клінічні та прогностичні аспекти	Medicina neotložnyh sostoânij	2,022	cc-by	5,951	Îðèãèíàëüíûå èññëåäîâàíèÿ Original Researches УДК 616.831.9-002-053.2/6 ÃÅÎÐÃ²ßÍÖ Ì.À., ÊÎÐÑÓÍÎÂ Â.À., ÑÒÎËßÐÎÂ Ê.ª. Õàðê³âñüêà ìåäè÷íà àêàäåì³ÿ ï³ñëÿäèïëîìíî¿ îñâ³òè ÑÒÀÍ ÑÈÑÒÎË²×ÍÎ¯ ÒÀ Ä²ÀÑÒÎË²×ÍÎ¯ ÔÓÍÊÖ²¯ Ì²ÎÊÀÐÄÀ ÏÐÈ ÑÅÏÑÈÑ² Â Ä²ÒÅÉ: êë³í³÷í³ òà ïðîãíîñòè÷í³ àñïåêòè Резюме. У статті обговорюється проблема гемодинамічних розладів при сепсисі та септичному шоку в дітей. Наведено результати досліджень серцевого викиду, систолічної, діастолічної функції лівого шлуночка серця при сепсисі та септичному шоку в дітей. Продемонстровано розвиток різних типів діастолічної дисфункції та зниження серцевого викиду при септичному шоку в дітей. Надано докази негативного впливу діастолічної дисфункції на перебіг та прогноз при сепсисі й синдромі поліорганної недостатності в дітей. Ключові слова: діти, сепсис, діастолічна дисфункція, гемодинаміка. Сепсис та його ускладнення є однією з актуальних проблем інтенсивної терапії в дітей. Частота захворюваності на тяжкий сепсис (ТС) у дітей у США становить 0,56 на 1000 населення, а серед дітей першого року життя — 5,2 на 1000. Госпітальна летальність сягає 10,3 %, що при загальній щорічній кількості захворілих понад 42 000 осіб визначає 4400 летальних кінців у дітей від тяжкого сепсису [1]. За сучасними даними, смертність від сепсису в розвинутих країнах становить 5,9 на 100 000 немовлят, а в країнах СНД смертність від сепсису становить 12–15 % [2]. Одним з головних факторів, що визначає градації тяжкості сепсису, є серцево-судинна дисфункція, яка свідчить про наявність у пацієнта ТС або септичного шоку (СШ). Роль дисфункції міокарда та її прогностичне значення у хворих на ТС значно менше вивчені, ніж у хворих на СШ. Варто відзначити, що в доступній літературі містяться результати досліджень переважно систолічної функції лівого шлуночка (ЛШ), у той час як діастолічна функція при септичних станах досліджена значно менше. Між тим існують теоретичні передумови, що дають можливість прогнозувати наявність не лише систолічної, але й діастолічної дисфункції (ДД) ЛШ. Певна кількість зарубіжних досліджень підтверджують наявність ДД міокарда в дорослих, хворих на сепсис [3–5]. У той же час ДД при сепсисі в дітей не вивчалася. Вважається, що ДД розвивається раніше за систолічну, що, імовірно, може бути використано для прогнозування наслідків гемодинамічних розладів та терапевтичної корекції [6]. Серед можливих чинників ДД відзначаються: ішемія міокарда, скорочення часу діастоли через тахікардію, електроліт¹ 1 (72) • 2016 ні розлади та синдром «капілярного витоку», розлади споживання міокардом ацильних фрагментів через ушкодження «карнітинового човника» та деякі інші фактори [7–10]. Також наочним є той факт, що тривалість діастоли та кінцево-діастолічний тиск є факторами, які лімітують кровопостачання ЛШ, що, у свою чергу, забезпечує кисневий баланс та ефективність його роботи в умовах підвищеного гемодинамічного попиту. Отже, метою нашого дослідження було вивчення інтегральних показників гемодинаміки, стану систолічної та діастолічної функції ЛШ, показників структурного ушкодження міокарда, вазоактивних медіаторів і зв’язків між ними та визначення прогностичної значущості діастолічної дисфункції міокарда при С, ТС та СШ у дітей. Ìàòåð³àëè ³ ìåòîäè Дослідження проведено у 2006–2010 рр. на кафедрі дитячої анестезіології та інтенсивної терапії Харківської медичної академії післядипломної освіти, на базі відділення анестезіології та інтенсивної терапії (ВАІТ) КЗОЗ «Харківська обласна дитяча інфекційна клінічна лікарня». У роботі проаналізовано й узагальнено результати обстеження й лікування 170 хворих, середній вік 32,1 ± 3,7 міс., із Адреса для листування з авторами: Георгіянц Маріне Акопівна Е-mail: eniram@bigmir.net © Георгіянц М.А., Корсунов В.А., Столяров К.Є., 2016 © «Медицина невідкладних станів», 2016 © Заславський О.Ю., 2016 www.mif-ua.com 103 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches сепсисом, ТС та СШ, діагноз яких визначався за критеріями консенсусу SCCM/ESICM/ACCP/ATS/ SIS International Sepsis Definitions Conference, 2002. Хлопчиків було 95 (55,8 %), дівчаток — 75 (44,2 %). Контрольну групу становили 44 практично здорові дитини, середній вік 40,7 ± 8,5 міс. Хлопчиків було 20 (45,0 ± 8,0 %), дівчаток — 24 (55,0 ± 8,0 %). Моніторниг включав електрокардіографію, визначення частоти серцевих скорочень (ЧСС), пульсоксиметрію (SpO2), вимірювання систолічного (АТс), діастолічного (АТд) та середнього артеріального тиску (САТ) осцилометричним методом, контроль центрального венозного тиску (ЦВТ) (монітори UM-300, Україна). При надходженні до ВАІТ усім хворим проводили ехокардіоскопію у М-режимі та імпульсно-хвильове допплерівське сканування трансмітрального кровотоку за допомогою ультразвукових сканерів Ultima PA (Україна) та Sono-Sayt Titan (США). Вимірювали кінцево-діастолічний (КДР) та кінцево-систолічний (КСР) розміри ЛШ з наступним розрахунком за L. Teichholz et al. (1976) індексів кінцево-систолічного (ІКСО) та кінцево-діастолічного об’ємів (ІКДО) ЛШ, ударного індексу (УІ), фракції викиду (ФВ). Серцевий індекс розраховували за формулою: СІ = УІ • ЧСС. Також вимірювали параметри трансмітрального кровотоку: максимальні швидкості хвиль раннього діастолічного наповнення (Е) (Vmax E) та передсердної систоли (А) (Vmax A), мінімальні швидкості Е (Vmin E) та А (Vmin A), час уповільнення DT (deceleration time) хвилі Е, час ізоволемічної релаксації IVRT (isovolumic relaxation time), час ізоволемічного скорочення IVCT (isovolumic contraction time) ЛШ та ПШ; Em — пікову швидкість раннього діастолічного зсуву трикуспідального фіброзного кільця на боці латеральної стінки правого шлуночка. На підставі отриманих даних розраховували середні швидкості хвилі Е (Vmean E) та А (Vmean A). Тиск заклинювання легеневих капілярів (ТЗЛК) визначали за формулою: ТЗЛК = 1,24 • Vmax E/Em + 1,9, де Vmax Е — максимальна швидкість транстрикуспідального діастолічного потоку (Nagueh S., 1998). Розраховували відношення максимальної швидкості хвилі раннього діастолічного наповнення до максимальної швидкості хвилі передсердної систоли Е/А ЛШ = Vmax E ЛШ/Vmax А ЛШ. Розраховували показник процентної частки систоли лівого передсердя в наповненні лівого шлуночка (ФП ЛШ) за формулою: ФП ЛШ = Vmax A/(Vmax A + Vmax E) • 100 (Bejamin E. et al., 1992). Кінцево-діастоліч- ний тиск ЛШ (КДТЛШ) розраховували за формулою: КДТЛШ = 1,06 + 15,15 • VTI peak А/VTI Peak Е (Stork T.V., 1989). Податливість ЛШ (P/V) визначали за відношенням КДТЛШ/КДОЛШ (Marino P., 2010). Коронарний перфузійний тиск (КПТ) розраховували за формулою: КПТ = КДТЛШ – – АТд. Час систоли (Tsist) визначали за формулою: Tsist = ET A + IVCT MK, час діастоли (Tdiast) за формулою: Tdiаst = ET Peak Ea + ET Peak Aa + IVRT MK. Індекс «напруга — час» (ІНЧ) — за формулою: ІНЧ = АТс • Tsist. Індекс «діастолічний тиск — час» (ІДТЧ) — за формулою: ІДТЧ = КПТ • Tdiast. На підставі цих показників визначали індекс життєздатності міокарда: ІЖМ = ІДТЧ/ІНЧ (Morgan G., Mikhail M., 2010). Оцінку вірогідності відмінностей величин між групами проводили на підставі критерію t (Стьюдента). Ðåçóëüòàòè òà ¿õ îáãîâîðåííÿ За критеріями SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference (2002) усіх хворих було розподілено на три групи: група сепсису (група С) — 71 дитина (41,8 %), група тяжкого сепсису (ТС) — 64 дитини (38,2 %) та група септичного шоку (група СШ) — 34 дитини (20,0 %). За антропометричними показниками та віком хворі, які увійшли до груп С, ТС та СШ, вірогідно не відрізнялися (p > 0,05) (табл. 1). Аналіз показників тяжкості органних розладів свідчить, що в групі С порівняно з групою ТС та СШ була вірогідно меншою кількість хворих, які потребували респіраторної підтримки (р < 0,001). Кількість хворих, які потребували респіраторної підтримки, у групах ТС та СШ вірогідно не відрізнялась (p > 0,05). Загальна кількість хворих, які отримували симпатоміметики, дорівнювала 67 (39,4 ± 3,7 %). У групі С вони використовувалися вірогідно рідше, ніж у групах ТС та СШ (р < 0,001). У свою чергу, застосування симпатоміметиків у групі СШ порівняно із групою ТС також було вірогідно частішим (р < 0,001). Тяжкість органних розладів за SOFA була вірогідно меншою у групі С порівняно із групами ТС та СШ, а у групі ТС, у свою чергу, порівняно з групою СШ (р < 0,001) (табл. 2). Вивчення показників центральної гемодинаміки свідчить, що одним з найбільш поширених гемодинамічних симптомів С, ТС або СШ є тахікардія, адже ЧСС у всіх досліджуваних групах була вірогідно вищою порівняно з контрольною (p < 0,001). САТ також був вірогідно нижчим порівняно з гру- Таблиця 1. Гендерна та антропометрична характеристика обстежених хворих (х ± Sх) Показники Вік, міс. Стать, хлопчики/дівчатка Маса, кг Довжина тіла, см 2 Площа поверхні тіла, м 104 Група С Група ТС Група СШ 34,5 ± 5,6 32,3 ± 6,5 28,1 ± 8,0 42/29 36/29 17/17 13,9 ± 1,4 13,2 ± 1,7 10,8 ± 1,9 93,8 ± 3,7 89,9 ± 4,7 83,5 ± 5,7 0,59 ± 0,04 0,54 ± 0,05 0,52 ± 0,06 Ìåäèöèíà íåîòëîæíûõ ñîñòîÿíèé, ISSN 2224-0586 ¹ 1 (72) • 2016 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches пою контролю у всіх досліджуваних групах, проте в групі СШ він був вірогідно нижчим, ніж у групі С та ТС (p < 0,001) (табл. 3). Стан переднавантаження оцінювався за показниками тиску (ЦВТ, ТЗЛК) та об’єму ЛШ (КДР, КДО, ІКДО), а післянавантаження — за показником ІЗПСО. Аналіз отриманих даних свідчить, що ЦВТ у досліджуваних групах хворих не мав відмінностей від групи контролю, але він був вірогідно нижчим у групі С порівняно із групами СШ та ТС (p < 0,05 та p < 0,001 відповідно). Також ми не знайшли вірогідних відмінностей ТЗЛК між досліджуваними групами та порівняно із контролем (p > 0,05). У всіх обстежених групах КДР та ІКДО були вірогідно меншими, ніж у контролі (p < 0,01 та p < 0,001). Між досліджуваними групами вірогідних відмінностей цих показників ми не спостерігали (p > 0,05). Показник КДО був вірогідно меншим порівняно з контролем у групі ТС (p < 0,05) і не мав вірогідних відмінностей між досліджуваними групами та контролем (p > 0,05). Показники КСР, КСО та ІЗПСО у досліджуваних групах та порівняно із контролем не мали вірогідних відмінностей (p > 0,05) (табл. 4). Отже, зважаючи на вірогідне зменшення об’ємних показників переднавантаження ЛШ та відсутність вірогідних відмінностей показників тиску заповнення камер серця, мусимо дійти попередньої гіпотези щодо імовірної ролі зміни діастолічної функції серця при септичних станах у дітей. Серцевий викид оцінювався за показниками УО, УІ, ФВ та СІ. Аналіз отриманих даних свід- чить, що показник УО у групах С, ТС та СШ був вірогідно меншим, ніж у групі контролю (p < 0,05, p < 0,01 та p < 0,001 відповідно). У групі СШ він також був вірогідно нижчим порівняно із групою С (p < 0,05). УІ у групах С, ТС та СШ також був вірогідно меншим, ніж у групі контролю (p < 0,001). Доцільно звернути увагу на те, що в групі СШ він був також вірогідно нижчим, ніж у групах С та ТС (p < 0,001). У групах С та ТС СІ не зазнавав вірогідних змін порівняно із контролем та не мав вірогідних відмінностей між групами (p > 0,05). Натомість у групі СШ СІ був вірогідно меншим, ніж у контролі та групах С і ТС (p < 0,05 та p < 0,01 відповідно). ФВ ЛШ у групах С, ТС та СШ була вірогідно меншою порівняно із контролем (p < 0,01, p < 0,05 та p < 0,001 відповідно). У групі СШ вона також була вірогідно меншою, ніж у групах С та ТС (p < 0,05). При цьому необхідно відзначити, що середні значення ФВ не сягали загрозливо критичного рівня (менше від 45 %), але у групі С вона була нижче за 45 % у 1 (1 ± 1 %), у групі ТС — у 3 (5 ± 3 %), а у групі СШ — у 6 (18 ± 7 %) хворих. Отже, у групі СШ кількість хворих із критично зниженою ФВ ЛШ була вірогідно більшою, ніж у групі С, і не відрізнялася вірогідно від групи ТС (p < 0,05 та p > 0,05 відповідно) (табл. 5). Отже, зниження серцевого викиду відбувається паралельно з прогресуванням септичного стану й стає загрозливим при СШ. Провідну роль у розвитку СШ у дітей відіграє саме погіршення переднавантаження. Таблиця 2. Частота розвитку та показники тяжкості органних розладів (х ± Sх) Показники Кількість хворих на ШВЛ Кількість хворих, що отримували симпатоміметики Група С Група ТС Група СШ Загалом 14* (19,7 ± 5,0 %) 55 (84,6 ± 4,0 %) 25^^^ (73,5 ± 8,0 %) 94 (55,3 ± 4,8 %) 2* (2,8 ± 2,0 %) 37### (58,0 ± 6,0 %) 31^^^ (91,2 ± 5,0 %) 70 (41,2 ± 3,7 %) 9,2 ± 0,6^^^ 4,8 ± 0,3 Оцінка за шкалою SOFA, бали 2,2 ± 0,2*** 5,7 ± 0,3### Примітка: тут і в табл. 3–5, 7, 8: вірогідність відмінностей між групами С та ТС: * — p < 0,05; — p < 0,01; * — p < 0,001; ТС та СШ: # — p < 0,05; ## — p < 0,01; ### — p < 0,001; С та СШ: ^ — p < 0,05; ^^ — p < 0,01; ^^^ — p < 0,001; усі групи та контроль:  — p < 0,05;  — p < 0,01;  — p < 0,001. Таблиця 3. Показники гемодинаміки у великому колі кровообігу (х ± Sх) Показники Група С ЧСС, уд/хв САТ, мм рт.ст. Група ТС Група СШ Група контролю 133,8 ± 3,3 142,4 ± 3,7 146,4 ± 5,2 101,4 ± 3,9 69,6 ± 1,3 69,0 ± 1,8###,  57,9 ± 2,4^^^,  73,9 ± 0,4 Таблиця 4. Показники переднавантаження та післянавантаження (х ± Sх) Показники ЦВТ, мм рт.ст. Група С Група ТС Група СШ Група контролю 2,0 ± 0,3* 3,9 ± 0,4 3,8 ± 0,7^ 4,0 ± 2,0 ТЗЛК, мм рт.ст. 5,47 ± 0,61 5,74 ± 0,59 4,54 ± 0,38 5,22 ± 0,09 КДР, см 2,76 ± 0,09 2,57 ± 0,10 2,50 ± 0,15 3,04 ± 0,10 КСР, см 1,80 ± 0,07 1,67 ± 0,08 1,77 ± 0,14 1,88 ± 0,07 КДО, мл 31,7 ± 2,6 28,3 ± 3,1† 27,94 ± 4,7 37,2 ± 2,8 11,5 ± 1,2 10,7 ± 1,7 13,2 ± 2,9 11,3 ± 1,0 КСО, мл 2 52,5 ± 1,5 49,1 ± 2,1 45,9 ± 2,7 65,0 ± 2,4 ІЗПСО, дин•с•см–5•м2 1322,5 ± 55,2 1318,4 ± 70,3 1270,8 ± 101,7 1371,8 ± 68,4 ІКДО, мл/м ¹ 1 (72) • 2016 www.mif-ua.com 105 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches Діастолічну функцію ЛШ було вивчено у 92 хворих середнім віком 43,6 ± 5,7 міс., з масою тіла 15,7 ± 1,4 кг, довжиною тіла 97,8 ± 3,4 см. З них хлопчиків було 52 (57,0 ± 5,0 %), дівчаток — 40 (43,0 ± 5,0 %). Хворих на С було 37 (40,0 ± 5,0 %), ТС — 36 (39,0 ± 5,0 %) та СШ — 19 (21,0 ± 5,0 %). Дослідження проводилось лише за умов чіткого диференціювання хвиль Е та А трансмітрального кровотоку, адже при тахікардії це не завжди можливо. Відомо, що існує перший тип ДД (порушення релаксації), який характеризується зниженням показника E/A < 1,0–1,3 та пролонгацією часу DT, другий тип ДД (псевдонормальний) — E/A у нормі (1,3–2,0), але DT менше за норму, та третій тип (рестриктивний) — E/A > 2,0 і DT менше за норму. Оскільки в контрольній групі хворих було отримано значення Е/А (Х ± ) — 1,66 ± 0,13, що загалом відповідає літературним даним, усіх обстежених хворих із С, ТС та СШ за показником Е/А було розподілено саме на такі підгрупи: E/A < 1,3, Е/А = 1,3–2,0 та E/A > 2,0 та проаналізовано головні показники діастолічної функції ЛШ відповідно до змін співвідношення Е/А. Перш за все привертає увагу віковий розподіл хворих, адже середній вік хворих у групі E/A < 1,3 був вірогідно нижчим, ніж у групах Е/А = 1,3–2,0 та E/A > 2,0 (p < 0,05 та p < 0,01 відповідно) (табл. 6). Vmax Peak E не мала вірогідних відмінностей між групами E/A < 1,3 та Е/А = 1,3–2,0 (p > 0,05). Але у групі E/A > 2,0 Vmax Peak E була вірогідно вищою, ніж у групах E/A < 1,3 та Е/А = 1,3–2,0 (p < 0,001). Vmax Peak A у групі E/A < 1,3 була вірогідно більшою, ніж у інших групах, а в групі Е/А = 1,3–2,0 — вірогідно більшою, ніж у групі E/A > 2,0 (p < 0,001). ET Peak E у групі E/A < 1,3 не відрізнявся вірогідно від групи Е/А = 1,3–2,0 та був вірогідно коротшим, ніж у групі E/A > 2,0 (p > 0,05 та p < 0,001 відповідно). У групі Е/А = 1,3–2,0, у свою чергу, ET Peak E був вірогідно меншим, ніж у групі E/A > 2,0 (p < 0,001). ET Peak A був вірогідно меншим порівняно із іншими групами лише в групі E/A < 1,3 (p < 0,05). Показники Е/А та ФПН ЛШ у групі E/A < 1,3 були вірогідно меншими, ніж у групах Е/А = 1,3–2,0 та E/A > 2,0, а в групі Е/А = 1,3–2,0 меншими, ніж у групі E/A > 2,0 (p < 0,001). DT MK у групі E/A < 1,3 був вірогідно меншим, ніж у групі Е/А = 1,3–2,0 та E/A > 2,0 (p < 0,05 та p < 0,001 відповідно). У свою чергу, у групі E/A > 2,0 DT MK був вірогідно більшим, ніж у групі Е/А = 1,3–2,0 (p < 0,001). IVCT MK та IVRT МK не мали вірогідних відмінностей між групами (p > 0,05). Комплайєнс ЛШ Р/V у групі E/A < 1,3 був вірогідно меншим, ніж у інших групах, а у групі Е/А = 1,3–2,0 — вірогідно меншим, ніж у групі E/A > 2,0 (p < 0,001) (табл. 6). Вивчення показників діастолічної функції ЛШ при розподілі за групами С, ТС та СШ дало підстави констатувати, що Vmax Peak E у групі С була вірогідно вищою, ніж у контролі (p < 0,01). У групах ТС та СШ цей показник від контролю вірогідно не відрізнявся (p > 0,05). У групі СШ Vmax Peak E була вірогідно меншою, ніж у групі С (p < 0,01). Vmax Peak A у групах С, ТС та СШ була вірогідно Таблиця 5. Показники систолічної функції та серцевого викиду (х ± Sх) Показники Група С, n = 71 Група ТС, n = 65 Група СШ, n = 34 Група контролю, n = 44 20,2 ± 1,6 17,6 ± 1,7 14,2 ± 2,0  25,8 ± 1,9 УІ, мл/м2 33,9 ± 1,0 31,7 ± 1,1###,  26,0 ± 1,1^^^,  43,7 ± 2,3 СІ, л/хв/м2 4,47 ± 0,15 4,47 ± 0,19## 3,76 ± 0,17^^,  4,38 ± 0,22 66 ± 1 66 ± 1  60 ± 2^,  70 ± 1 УО, мл ФВ, % #, ^, Таблиця 6. Характеристика основних показників діастолічної функції лівого шлуночка залежно від типу співвідношення Е/А (х ± Sх) Показники E/A < 1,3, n = 49 # Е/А = 1,3–2,0, n = 26 E/A > 2,0, n = 17 Вік, міс. 24,9 ± 5,9 51,7 ± 11,2 85,0 ± 17,2 С/ТС/СШ 12/23/14 15/9/2 10/4/3 ### Vmax Peak E, см/с 104,7 ± 4,9 Vmax Peak A, см/с 99,9 ± 4,1### ET Peak E, мс ET Peak A, мс ### 106,0 ± 13,2 87,9 ± 11,8# ### 113,7 ± 5,0 133,3 ± 5,7 72,5 ± 3,0* 52,3 ± 3,9 132,6 ± 10,3 196,0 ± 14,9 97,6 ± 7,9 122,0 ± 9,9 E/A 1,05 ± 0,03 1,58 ± 0,04* 2,76 ± 0,23 ФПН ЛШ, % 49,2 ± 0,7### 39,1 ± 0,6* 27,9 ± 1,2 ### 78,0 ± 6,6* 113,4 ± 11,1 IVCT MK, мс 41,9 ± 3,0 47,7 ± 3,1 49,3 ± 8,0 IVRT МK, мс 50,8 ± 3,1 53,4 ± 3,5 50,7 ± 3,8 DT MK, мс 52,7 ± 5,5 3,32 ± 0,51*** 8,8 ± 1,4 Р/V, мм рт.ст./мл 1,16 ± 0,14### Примітка: вірогідність відмінностей між групами: E/A < 1,3 і Е/А = 1,3–2,0: * — р < 0,05, — p < 0,01, * — p < 0,001; E/A < 1,3 і E/A > 2,0: # — р < 0,05, ## — p < 0,01, ### — p < 0,001; Е/А = 1,3–2,0 і E/A > 2,0:  — р < 0,05,  — p < 0,01,  — p < 0,001. 106 Ìåäèöèíà íåîòëîæíûõ ñîñòîÿíèé, ISSN 2224-0586 ¹ 1 (72) • 2016 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches вищою, ніж у групі контролю (p < 0,01, p < 0,001 та p < 0,05 відповідно). Vmin Peak E у групах С та ТС не мала вірогідних відмінностей від контролю, у групі СШ була вірогідно меншою, ніж у контрольній групі (відповідно p > 0,05 та p < 0,05). У групах С та ТС цей показник був вірогідно більший, ніж у групі СШ (p < 0,05). Vmin Peak A у всіх досліджуваних групах був вірогідно вищим, ніж у групі контролю (p < 0,001). Міжгрупових відмінностей цього показника не спостерігалося (p > 0,05). Показник ET Peak E у групах С та ТС не відрізнявся вірогідно ні між групами, ні з групою контролю (p > 0,05). Натомість у групі СШ він був вірогідно меншим, ніж у групі С та у групі контролю (p < 0,01 та p < 0,001 відповідно). ET Peak A у досліджуваних групах вірогідно не відрізнявся, а також не відрізнявся із контролем у групах С та ТС (p > 0,05). Але в групі СШ він був вірогідно меншим порівняно із контролем (p < 0,01). Співвідношення Е/А у групі С не відрізнялося від контролю (p > 0,05), а у групах ТС та СШ було вірогідно меншим, ніж у контролі (p < 0,01). Також у цих групах Е/А було вірогідно нижчим, ніж у групі С (p < 0,05). Аналогічних змін зазнав ФПН ЛШ. У групі С він не мав вірогідних відмінностей від норми, але був вірогідно меншим, ніж у групах ТС та СШ (p > 0,05 та p < 0,01 відповідно). У свою чергу, у групах ТС та СШ цей показник був вірогідно меншим, ніж у групі контролю (p < 0,001). Схожих змін зазнавав DT MK (табл. 7). У групі С він не мав вірогідних відмінностей від норми, але був вірогідно меншим, ніж у групах ТС та СШ (p > 0,05 та p < 0,01 і p < 0,001 відповідно). У групах ТС та СШ цей показник був вірогідно меншим, ніж у групі контролю (p < 0,001). Показник часу ізоволемічного скорочення IVCT MK не мав вірогідних відмінностей ні між групами С, ТС та СШ, ні між ними та групою контролю (p > 0,05). Натомість показник IVRT МK у групах С, ТС та СШ був вірогідно меншим, ніж у групі контролю (p < 0,05, p < 0,001 та p < 0,01 відповідно). Показник Р/V у групах С та СШ вірогідно від норми не відрізнявся (p > 0,05). У групі ТС він був вірогідно меншим, ніж у групі С та у групі контролю (p < 0,05 та p < 0,01 відповідно) (табл. 7). Отже, при септичних станах у дітей найбільш часто зустрічається тип ДД, що характеризується зменшенням співвідношення Е/А, скороченням часу DT та IVRT, зниженням комплайєнсу ЛШ. Цей тип ДД асоційований із тяжкістю стану та раннім віком дитини. Його наявність негативно впливає на ефективність венозного повернення під час діастоли та може знижувати серцевий викид. Імовірним наслідком цього типу ДД може бути зниження ефективності інфузійної терапії, розвиток капілярного витоку та рідинно-рефрактерного шоку. Приклад допплерограми трансмітрального кровотоку при ТС продемонстровано на рис. 1. Ретроспективний аналіз показав, що у групі з Е/А < 1,3 симпатоміметики отримували 27 з 49 (54,0 ± 7,0 %) хворих, у групі з Е/А = 1,3–2,0 — 6 з 26 (23,0 ± 8,0 %), а в групі з Е/А > 2,0 — 5 з 17 (29,0 ± 11,0 %) хворих. При порівнянні частоти призначення симпатоміметиків з’ясувалось, що у групі з Е/А < 1,3 вони призначались вірогідно частіше, ніж у групах з Е/А = 1,3–2,0 та Е/А > 2,0 (p < 0,001). У доросліших хворих з меншою тяжкістю септичного стану превалюють псевдонормальний тип ДД та третій тип ДД. Дані типи ДД при септичних станах у дітей можна вважати більш сприятливими Рисунок 1. Виражений перший тип діастолічної дисфункції при тяжкому сепсисі в дитини 5 міс. Таблиця 7. Показники діастолічної функції ЛШ залежно від тяжкості септичного стану (х ± Sх) Група С Група ТС Група СШ Група контролю Vmax Peak E, см/с Показники 123,0 ± 4,1 109,7 ± 5,8 98,6 ± 7,7^^ 107,5 ± 3,8 Vmax Peak A, см/с 79,2 ± 4,7 89,2 ± 5,2 81,1 ± 6,8 64,8 ± 2,1 Vmin Peak E, см/с 19,9 ± 2,2 16,9 ± 1,3 13,0 ± 1,2  16,8 ± 1,3 Vmin Peak A, см/с 43,7 ± 4,6 50,8 ± 5,0 52,2 ± 6,5 25,9 ± 1,8 # ^,  ^^, 146,5 ± 10,9 125,9 ± 19,2 99,6 ± 7,8 103,0 ± 16,5 80,0 ± 6,5 101,5 ± 4,1 1,40 ± 0,12 1,31 ± 0,13  1,66 ± 0,03 E/A ФПН ЛШ, % DT MK, мс 1,78 ± 0,15* 38,65 ± 1,39 44,71 ± 1,55 101,6 ± 9,4 148,9 ± 7,2 ET Peak E, мс ET Peak A, мс ^, ^^, 45,44 ± 2,09 ^^^,  36,67 ± 0,44 92,9 ± 5,1 90,6 ± 8,7 59,9 ± 4,2 IVCT MK, мс 42,8 ± 3,4 47,0 ± 3,4 43,5 ± 4,9 43,3 ± 2,7 IVRT МK, мс 56,4 ± 3,0 49,3 ± 3,8 47,5 ± 4,2 87,1 ± 2,5 4,47 ± 0,79* 2,31 ± 0,53 2,70 ± 1,04 4,3 ± 0,2 Р/V, мм рт.ст./мл ¹ 1 (72) • 2016 www.mif-ua.com 50,5 ± 6,9  107 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches фузії та кисневого забезпечення міокарда. Tsist у досліджуваних групах не мав вірогідної відмінності від групи контролю (p > 0,05). Але в групі С він був вірогідно тривалішим, ніж у групі ТС (p < 0,01). ІНЧ у групах С, ТС та СШ також не мав вірогідних відмінностей із контролем (p > 0,05). При цьому у групах ТС та СШ він був вірогідно меншим, ніж у групі С (p < 0,05 та p < 0,001 відповідно). У групах С, ТС та СШ Тdiаst був вірогідно меншим, ніж у контролі (p < 0,05, p < 0,001 та p < 0,001 відповідно). У групах ТС та СШ він також був коротшим, ніж у групі С (p < 0,05). ІДТЧ у групах С, ТС та СШ також був вірогідно меншим, ніж у групі контролю (p < 0,05, p < 0,001 та p < 0,001 відповідно). У групі СШ ІДТЧ також був вірогідно меншим, ніж у групах С та ТС (p < 0,05 та p < 0,001 відповідно). ІЖМ у нормі має становити від 0,7 до 1,0 [12]. За нашими спостереженнями, у здорових дітей він дорівнював 0,72 ± 0,06 ум.од. ІЖМ у групах С, ТС та СШ був вірогідно меншим, ніж у групі контролю (p < 0,001, p < 0,01 та p < 0,001 відповідно). У групі СШ цей показник був також вірогідно меншим, ніж у групах ТС та С (p < 0,05 та p < 0,01 відповідно). КДТЛШ у групах С, ТС та СШ був вірогідно підвищеним порівняно із групою контролю (p < 0,05, p < 0,001 порівняно з першим типом, адже вони супроводжуються меншими розладами серцевого викиду та вірогідно рідше потребують призначення симпатоміметичної підтримки. При цих типах ДД спостерігається більш сприятливий прогноз для життя. У групі з Е/А = 1,3–2,0 померло 2 з 26 (8,0 ± 5,0 %) хворих, у групі з Е/А > 2,0 — 1 з 17 (6,0 ± 6,0 %) хворих, натомість у групі з Е/А < 1,3 — 7 з 49 (14,0 ± 5,0 %). Показники летальності між групами з Е/А = 1,3–2,0 та Е/А < 1,3 та Е/А > 2,0 і Е/А < 1,3 вірогідно відрізнялись (p < 0,05 та p < 0,01 відповідно). Стан діастолічної функції ЛШ становить інтерес не лише через вплив на серцевий викид та систолічну функцію, а й тому, що, як відомо, кровопостачання ЛШ здійснюється лише під час діастоли, у напрямку від ендокарду до епікарду, а отже, скорочення діастоли та збільшення діастолічного тиску в ЛШ можуть викликати ішемію міокарда, яка, у свою чергу, погіршує енергозалежну релаксацію ЛШ. Зважаючи на існуючі дані щодо наявності структурного ушкодження міокарда ЛШ при септичних станах (підвищення рівня тропоніну та креатинфосфокінази), викликає інтерес стан коронарного кровообігу [11]. Отже, було проведено дослідження показників, що відбивають стан коронарної пер- Таблиця 8. Показники кисневого забезпечення міокарда ЛШ (х ± Sх) Показник Група С Група ТС Група СШ Контроль 277,1 ± 8,8** 243,1 ± 8,9 253,1 ± 11,7 255,4 ± 15,1 27803,4 ± 1290,8* 23677,8 ± 1263,7 20966,7 ± 1223,5^^^ 26487,3 ± 1778,3 289,8 ± 21,2 * 229,8 ± 9,1 231,7 ± 13,6  356,4 ± 15,6 13994,1 ± 1348,0 10647,9 ± 1638,5  ІЖМ, ум.од. 0,49 ± 0,04 0,46 ± 0,07  КДТЛШ, мм рт.ст. 12,0 ± 1,0**  КПТ, мм рт.ст. 45,2 ± 2,1*  Tsist, мс ІНЧ, ум.од. , Тdiаst, мс ІДТЧ, ум.од. , , #, #, 20,7 ± 2,2 ##, 37,4 ± 2,8  ^, ^^^, 5504,2 ± 1196,8 17453,9 ± 1092,4  0,72 ± 0,06  9,8 ± 0,5 ^^^,  50,0 ± 1,0 0,25 ± 0,05 23,0 ± 3,6 22,0 ± 3,7  ^^, ^^, Таблиця 9. Деякі показники центральної гемодинаміки та діастолічної функції ЛШ залежно від результату септичного стану (х ± Sх) Показники Сприятливий результат, n = 144 Несприятливий результат, n = 26 р 27,7 ± 3,9 23,4 ± 7,0 > 0,05 Маса, кг 12,1 ± 0,9 10,8 ± 1,8 > 0,05 Довжина тіла, см 87,5 ± 2,7 84,0 ± 5,1 > 0,05 139,6 ± 2,2 150,5 ± 7,2 > 0,05 66,1 ± 1,1 69,0 ± 3,5 > 0,05 ІКДО, мл/м 50,8 ± 1,3 42,5 ± 2,8 < 0,05 УІ, мл/м2 32,1 ± 0,7 27,3 ± 1,7 < 0,05 5,4 ± 0,2 4,1 ± 0,4 < 0,01 Вік, міс. ЧСС, уд/хв САТ, мм рт.ст. 2 2 СІ, л/хв/м ФВ, % 65 ± 1 66 ± 2 > 0,05 Vmax Peak A, см/с 83,9 ± 3,4 85,7 ± 10,0 > 0,05 Vmax Peak Е, см/с 115,9 ± 3,5 94,2 ± 9,9 < 0,05 E/A 1,55 ± 0,09 1,15 ± 0,11 < 0,01 DT MK, мс 72,6 ± 5,0 50,7 ± 4,8 < 0,01 Р/V, мм рт.ст./мл 3,19 ± 0,47 1,53 ± 0,51 < 0,05 КДТЛШ, мм рт.ст. 16,04 ± 1,17 28,11 ± 5,04 < 0,05 108 Ìåäèöèíà íåîòëîæíûõ ñîñòîÿíèé, ISSN 2224-0586 ¹ 1 (72) • 2016 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches та p < 0,001 відповідно). У групі СШ та ТС він також був вірогідно більшим, ніж у групі С (p < 0,01). У свою чергу, КПТ порівняно із групою контролю також вірогідно знижувався у всіх досліджуваних групах хворих — С, ТС та СШ (p < 0,05, p < 0,001 та p < 0,001 відповідно). У групі СШ він був вірогідно нижчим, ніж у групах С та ТС (p < 0,01 та p < 0,001 відповідно). А у групі ТС — вірогідно нижчим, ніж у групі С (p < 0,05) (табл. 8). Отже, ДД при септичних станах у дітей безпосередньо впливає на коронарну перфузію та кисневе забезпечення міокарда. Погіршення кисневого забезпечення міокарда прогресує паралельно ступеню тяжкості септичного стану: С — ТС — СШ. Імовірно, що гемодинамічний фактор є важливим чинником структурного ушкодження міокарда ЛШ і сприяє погіршенню як енергозалежних діастолічних процесів, так і систолічної функції ЛШ. Як зазначалося, ДД ЛШ має несприятливий вплив на перебіг СШ у дорослих, проте її вплив на перебіг септичних станів у дітей не вивчено. З метою з’ясування можливого впливу ДД на результат при септичних станах у дітей було вивчено стан її показників у пацієнтів, які вижили, та в померлих. Серед обстежених хворих сприятливий результат (одужання) спостерігався у 144 дітей, несприятливий — у 26 хворих. Аналіз отриманих даних дає підстави вважати, що за віком та антропометричними характеристиками хворі, які одужали, та померлі хворі вірогідно не відрізнялись (р > 0,05). У групі хворих із несприятливим результатом захворювання спостерігалися вірогідно нижчі показники ІКДО ЛШ, УІ ЛШ та СІ ЛШ (р < 0,05 та р < 0,01 відповідно). Показники САТ, ЧСС, ФВ та Vmax Peak A не мали вірогідних відмінностей (р > 0,05). Натомість Vmax Peak Е у групі хворих із несприятливим результатом захворювання був вірогідно меншим, ніж у групі із сприятливим результатом захворювання (р < 0,05). Внаслідок зменшення швидкості раннього діастолічного току крові в групі хворих із несприятливим наслідком септичного стану спостерігались вірогідно нижчі співвідношення Е/А (р < 0,01) та Р/V (р < 0,05) та вірогідно більший КДТЛШ (р < 0,05). DT Е MK у групі хворих із несприятливим наслідком септичного стану також був вірогідно коротшим, ніж у групі із сприятливим результатом септичного стану (р < 0,01) (табл. 9). Отже, отримані дані свідчать, що ДД першого типу при септичних станах у дітей може вважатися прогностично несприятливим типом розладу трансмітрального кровотоку. Наявність даного варіанту ДД супроводжується високим ризиком розвитку синдрому поліорганної недостатності, високою летальністю, негативно впливає на показники серцевого викиду при септичних станах у дітей і потребує своєчасної діагностики та корекції. Âèñíîâêè 1. Центральна гемодинаміка у великому колі кровообігу при септичних станах характеризується збільшенням ЧСС, що відбувається на тлі погіршен¹ 1 (72) • 2016 ня переднавантаження, яке прогресує відповідно до ступеня тяжкості септичного стану (С — ТС — СШ). 2. Зниження показників артеріального тиску за відсутності вірогідних змін ІЗПСО обумовлені переважно зменшенням серцевого викиду, що відбувається як за рахунок обмеженого переднавантаження, так і за рахунок погіршення систолічної функції лівого шлуночка, що спостерігається переважно у хворих на СШ. 3. При септичних станах у дітей відбувається розвиток діастолічної дисфункції лівого шлуночка, що обумовлена як розладами релаксації, так і рестриктивними порушеннями. Найбільш несприятливим типом діастолічної дисфункції є перший її тип із зменшенням співвідношення Е/А < 1,3. 4. Погіршення комплайєнсу лівого шлуночка при септичних станах поряд із гіповолемією може бути важливим чинником обмеження переднавантаження та формування рідинно-рефрактерного шоку. 5. Низький комплайєнс лівого шлуночка та скорочення часу діастоли сприяють збільшенню кінцево-діастолічного тиску в ньому та призводять до погіршення коронарної перфузії під час діастоли, що може бути чинником розвитку структурного ушкодження міокарда. Ñïèñîê ë³òåðàòóðè 1. Watson R.S. The Epidemiology of Severe Sepsis in Children in the United States / R.S. Watsons // Am. J. of Resp. and Critical Care Med. — 2003. — Vol. 167. — P. 695-701. 2. Исаков Ю.Ф. Сепсис у детей / Ю.Ф. Исаков, Н.В. Белобородова — М.: Издатель Мокеев, 2001. — 369 с. 3. Diastolic dysfunction and mortality in severe sepsis and septic shock / G. Landesberg, D. Gilpn, Meroz Y. [et al]. // Europen Heart Journal. — 2012. — Vol. 33. — P. 895-903. 4. Diastolic dysfunction and mortality in early severe sepsis and septic shock: a prospective, observational echocardiography study / S.M. Brown, J.E. Pittman, E.L. Hirshberg [et al.] // Clinical Ultrasound Journal. — 2012. — Vol. 4. — P.2-9. 5. Left ventricular diastolic dysfunction in the intensive care unit: trends and perspectives. Review Article / L. Elisen, P. Davlouros, D. Karakitsos [et al.] // Critical Care Research and Practice. — 2012. — Vol. 3. — 34-39. doi:10.1155/2012/964158 6. Пыков М.И. Детская ультразвуковая диагностика / М.И. Пыков, К.В. Ватолин. — М.: Видар-М, 2001. — 680 с. 7. Management of Myocardial Dysfunction in Severe Sepsis / M. Jozwiak, R. Persichini, X. Monnet [et al.] // Respir. Crit. Care Med. — 2011. — Vol. 32(2). — P. 206-214. 8. Huntr J.D. Sepsis end heart. Review article / J.D. Huntr, M. Doddi // Br. J. Anaesth. — 2010. — Vol. 104. — P. 3-11. 9. Pathophysiology of Sepsis-Related Cardiac Dysfunction: Driven by Inflammation, Energy Mismanagement, or Both? / D. Konstantinos, A. Lymperopoulos, P. Johannes Kennel [et al.] // Curr. Heart Fail. Rep. — 2015 Apr. — 12(2). — 130-40. doi 10.1007/ s11897-014-0247-z. 10. Sepsis-induced Cardiomyopathy / J. Francisco Romero-Bermejo, M. Ruiz-Bailen, J. Gil-Cebrian [et al.] // Current Cardiology Reviews. — 2011. — Vol. 7. — P. 163-183. 11. Troponin Elevation in Severe Sepsis and Septic Shock: The Role of Left Ventricular Diastolic Dysfunction and Right Ventricular Dilatation / G. Landesberg, A. Jaffe, D. Gilon [et al.] // Crit. Care Med. — 2014. — Vol. 42. — P. 790-800. 12. Морган-мл. Дж.E. Клиническая анестезиология. Кн 2. — Пер. с англ. / Дж.E. Морган-мл., М.С. Михайл. — М.: Издательство БИНОМ; СПб.: Невский Диалект, 2010. — 366 с. www.mif-ua.com Отримано 12.11.15 109 Îðèãèíàëüíûå èññëåäîâàíèÿ / Original Researches Ãåîðãèÿíö Ì.À., Êîðñóíîâ Â.À., Ñòîëÿðîâ Ê.Å. Õàðüêîâñêàÿ ìåäèöèíñêàÿ àêàäåìèÿ ïîñëåäèïëîìíîãî îáðàçîâàíèÿ Heorhiiants M.A., Korsunov V.A., Stoliarov K.Ye. Kharkiv Medical Academy of Postgraduate Education, Kharkiv, Ukraine ÑÎÑÒÎßÍÈÅ ÑÈÑÒÎËÈ×ÅÑÊÎÉ È ÄÈÀÑÒÎËÈ×ÅÑÊÎÉ ÔÓÍÊÖÈÈ ÌÈÎÊÀÐÄÀ ÏÐÈ ÑÅÏÑÈÑÅ Ó ÄÅÒÅÉ: êëèíè÷åñêèå è ïðîãíîñòè÷åñêèå àñïåêòû STATE OF SYSTOLIC AND DIASTOLIC MYOCARDIAL FUNCTION IN SEPSIS IN CHILDREN: Clinical and Prognostic Aspects Резюме. В статье обсуждается проблема гемодинамических нарушений при сепсисе и септическом шоке у детей. Приводятся результаты исследования сердечного выброса, систолической и диастолической функции левого желудочка сердца при сепсисе и септическом шоке у детей. Продемонстрировано развитие разных типов диастолической дисфункции и снижение сердечного выброса при септическом шоке у детей. Представлены доказательства негативного влияния диастолической дисфункции на течение и прогноз сепсиса и полиорганной недостаточности у детей. Ключевые слова: дети, сепсис, диастолическая дисфункция, гемодинамика. Summary. The article discusses the problem of hemodynamic disorders in sepsis and septic shock in children. The results of studies of cardiac output, systolic, diastolic left ventricular function in sepsis and septic shock in children are presented. The development of different types of diastolic dysfunction and reduced cardiac output in septic shock in children are shown. The paper provides evidence of diastolic dysfunction negative impact on the course and prognosis in sepsis and multiple organ failure syndrome in children. Key words: children, sepsis, diastolic dysfunction, hemodynamics. 110 Ìåäèöèíà íåîòëîæíûõ ñîñòîÿíèé, ISSN 2224-0586 ¹ 1 (72) • 2016
https://openalex.org/W4361922079	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S3_from_Targeted_Exome_Sequencing_of_Krebs_Cycle_Genes_Reveals_Candidate_Cancer_Predisposing_Mutations_in_Pheochromocytomas_and_Paragangliomas/22463499/1/files/39914793.pdf	Malagasy	null	Supplementary Figure S4 from Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer–Predisposing Mutations in Pheochromocytomas and Paragangliomas	null	2,023	cc-by	64	Supplementary Figure 3 Supplementary Figure 3 EVWT-GOT2c.223T>G-GOT2 0.0 0.1 0.2 0.3 0.4 Aspartate/glutamate ratio (RU) c.357A>T-GOT2 B A n.s. 0 0.1 0.2 0.3 0.4 0.5 0.6 Succinate/fumarate ratio (RU) n.s. EVWT-OGDHLc.750G>T-OGDHL p = 0.022 EVWT-GOT2c.223T>G-GOT2 0.0 0.1 0.2 0.3 0.4 Aspartate/glutamate ratio (RU) c.357A>T-GOT2 A n.s. p = 0.022 B 0 0.1 0.2 0.3 0.4 0.5 0.6 Succinate/fumarate ratio (RU) n.s. EVWT-OGDHLc.750G>T-OGDHL B A
https://openalex.org/W3037663256	https://www.mdpi.com/2077-0383/9/6/1978/pdf?version=1593003639	English	null	Disturbances of Continuous Sleep and Circadian Rhythms Account for Behavioral Difficulties in Children with Autism Spectrum Disorder	Journal of clinical medicine	2,020	cc-by	12,859	Disturbances of Continuous Sleep and Circadian Rhythms Account for Behavioral Diﬃculties in Children with Autism Spectrum Disorder Schroder 1,8,9 1 Centre National de la Recherche Scientiﬁque CNRS UPR 3212, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 8 allée du Général Rouvillois, 67000 Strasbourg, France; enise.yavuz@etu.unsitra.fr (E.Y.-K.); pbourgin@unistra.fr (P.B.); schroderc@unistra.fr (C.M.S.) 1 Centre National de la Recherche Scientiﬁque CNRS UPR 3212, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 8 allée du Général Rouvillois, 67000 Strasbourg, France; enise.yavuz@etu.unsitra.fr (E.Y.-K.); pbourgin@unistra.fr (P.B.); schroderc@unistra.fr (C.M.S.) 2 y p g 2 Department of Child and Adolescent Neurodevelopmental Psychiatry, Le Vinatier Hospital, 95 Boulevard Pinel, 69678 Bron CEDEX, France; Marie-Maude.GEOFFRAY@ch-le-vinatier.fr 95 Boulevard Pinel, 69678 Bron CEDEX, France; Marie-Maude.GEOFFRAY@ch-le-vinatier.fr 3 Health Services and Performance Research (HESPER), Claude Bernard University Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbane CEDEX, France , , ; 3 Health Services and Performance Research (HESPER), Claude Bernard University Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbane CEDEX, France 4 Department of Neurology and Clinical Neurophysiology, University Hospital Bretonneau, 2 Boulevard Tonnellé, 37044 Tours, France; N.LIMOUSIN-CHAMPFAILLY@chu-tours.fr 5 Pediatric Sleep, Hôpital Femme-Mère-Enfant, Hospices civils of Lyon, 59 Boulevard Pinel, 4 Department of Neurology and Clinical Neurophysiology, University Hospital Bretonneau, 2 Boulevard Tonnellé, 37044 Tours, France; N.LIMOUSIN-CHAMPFAILLY@chu-tours.fr 5 Pediatric Sleep, Hôpital Femme-Mère-Enfant, Hospices civils of Lyon, 59 Boulevard Pinel, 69500 Bron, France; patricia.franco@chu-lyon.fr 5 Pediatric Sleep, Hôpital Femme-Mère-Enfant, Hospices civils of Lyon, 59 Boulevard Pinel, 69500 Bron, France; patricia.franco@chu-lyon.fr p y 6 Lyon Neuroscience Research Center U1028/UMR5292, Claude Bernard University Lyon 1, 6 Lyon Neuroscience Research Center U1028/UMR5292, Claude Bernard University Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbane CEDEX, France Lyon Neuroscience Research Center U1028/UMR5292, Claude Bernard Un 43 Boulevard du 11 Novembre 1918, 69622 Villeurbane CEDEX, France 7 UMR 1253, iBrain, Université de Tours, Inserm, CHRU de Tours, Centre Universitaire de Pédopsychiatrie, 2 Boulevard Tonnellé, 37044 Tours, France; frederique.brilhault@univ-tours.fr q 8 Sleep Disorders Center, International Research Center for ChronoSomnology, Strasbourg University Hospitals, 1 place de l’Hôpital, 67000 Strasbourg, France 8 Sleep Disorders Center, International Research Center for ChronoSomnology, Strasbourg University Hospitals, 1 place de l’Hôpital, 67000 Strasbourg, France p , gy, Strasbourg University Hospitals, 1 place de l’Hôpital, 67000 Strasbourg, France g y p p p g 9 Department of Child and Adolescent Psychiatry, Strasbourg University Hospitals & University of Strasbo Medical School, 67000 Strasbourg, France * Correspondence: eve.reynaud@chru-strasbourg.fr Received: 27 May 2020; Accepted: 21 June 2020; Published: 24 June 2020 Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Disturbances of Continuous Sleep and Circadian Rhythms Account for Behavioral Diﬃculties in Children with Autism Spectrum Disorder Enise Yavuz-Kodat 1, Eve Reynaud 1,* , Marie-Maude Geoﬀray 2,3, Nadège Limousin 4, Patricia Franco 5,6, Frédérique Bonnet-Brilhault 7 , Patrice Bourgin 1,8 and Carmen M. Schroder 1,8,9 Enise Yavuz-Kodat 1, Eve Reynaud 1,* , Marie-Maude Geoﬀray 2,3, Nadège Limousin 4, Patricia Franco 5,6, Frédérique Bonnet-Brilhault 7 , Patrice Bourgin 1,8 and Carmen M. Keywords: autism; ASD; children; sleep; circadian rhythm; pediatrics 1. Introduction Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by persistent impairments in social communication and social interaction and the presence of restricted, repetitive patterns of behavior, interests or activities [1]. Its worldwide reported prevalence approaches 1% of the population, and comorbidities are highly prevalent [2–4]. Indeed, over 70% of individuals diagnosed with ASD have concurrent somatic or psychiatric conditions such as behavioral disorders, anxiety, gastrointestinal problems, seizures and sleep disorders [5–7]. Sleep disorders are among the most common associated disorders in this population, with prevalence rates ranging from approximately 50% to 80% [8,9]. The most common parent-reported sleep problems in children with ASD are related to bedtime resistance, sleep initiation, long periods of nighttime awakening and shortened sleep time [8,10,11]. Studies using objective measures of sleep have observed similar results with increased sleep onset latency, decreased sleep eﬃciency and increased number and duration of night waking in this population compared to typically developing children [12–16]. Objective sleep was measured either with the gold standard polysomnography or with actigraphy (ACT). The latter is being more and more used to assess sleep in children with ASD, as it is more feasible and allows ecological recordings over longer periods of time, and was recently validated against polysomnography [17]. Alongside studies on sleep itself, a growing number of studies have reported abnormalities of the circadian organization of sleep–wake rhythms in children with ASD. The circadian timing system orchestrates numerous rhythms such as core body temperature, the neuroendocrine system, cardiac function as well as the timing of the sleep–wake cycle. Endogenous melatonin, which is released by the pineal gland at night, plays a crucial role in the regulation of the sleep–wake cycle, acting as a time cue to stabilize and synchronize various circadian rhythms. In a recent systematic review, two main circadian rhythm disorders were reported in children with ASD: a phase delay of sleep periods and an irregular sleep–wake pattern [18]. These circadian rhythm abnormalities in ASD may be linked to mutations in the expression of clock genes [19,20]. Most importantly, a dysregulation in the melatonin rhythm with a decrease in melatonin secretion over the night and globally over the 24-h cycle has been suggested as the main underlying pathophysiological mechanism of circadian rhythm disorders in individuals with ASD [21–24]. Increasingly, studies monitor the 24-h rest–activity pattern using a wrist-worn actigraphy as a proxy of the circadian sleep–wake rhythm. Received: 27 May 2020; Accepted: 21 June 2020; Published: 24 June 2020 Abstract: Sleep disorders are among the most common comorbidities in children with Autism Spectrum Disorder (ASD), and subjectively deﬁned sleep disturbances have been related to ASD symptom severity. However, no study has investigated the diﬀerential impact of objectively measured sleep and circadian rhythm disturbances on behavioral diﬃculties in this population. Fifty-two children with ASD aged 3–10 years underwent assessments of sleep and circadian rest–activity rhythms objectively with actigraphy and subjectively with the Children’s Sleep Habits Questionnaire. Behavioral diﬃculties were assessed using the ABC-C. Group comparison analyses were used to compare sleep and circadian rhythm parameters of children with higher and lower behavioral diﬃculties and dominance analysis to rank predictors and address multicollinearity. Children with high irritability had a shorter continuous sleep period compared to those with lower irritability (−60 min, p = 0.04), as well as those with high stereotypic behaviors compared to children with less stereotypies (−75 min, p = 0.006). Objective circadian and sleep disturbances accounted together for, respectively, 17%, 18% and 36% of the variance in social withdrawal, irritability and stereotypic behaviors. The identiﬁcation of both sleep and circadian rhythm disturbances as explanatory factors for behavioral diﬃculties warrants their inclusion in the existing behavioral management strategies for children with ASD. Keywords: autism; ASD; children; sleep; circadian rhythm; pediatrics Keywords: autism; ASD; children; sleep; circadian rhythm; pediatrics J. Clin. Med. 2020, 9, 1978; doi:10.3390/jcm9061978 www.mdpi.com/journal/jcm www.mdpi.com/journal/jcm J. Clin. Med. 2020, 9, 1978 2 of 19 1. Introduction Sleep–wake rhythms over long periods in children with ASD have rarely been assessed objectively with ACT but were rather carried out in adults with ASD [25–27]. In typically developing children, numerous studies have evidenced the association of sleep disturbances with behavioral outcomes (for review, see [28,29]). The majority of these studies have assessed sleep subjectively and only a few studies have investigated circadian rhythms in relation to daytime functioning. In children with ASD, most studies agree that subjective sleep problems are linked to behavioral diﬃculties [14,16,30–32]. However, results in the literature diﬀer with respect to speciﬁc subjective sleep problems and their relation to speciﬁc behavioral diﬃculties. Aathira et al. (2017) [33] found an association between poor sleep and overall behavioral diﬃculties in their study which included 71 children with ASD aged 3–10 years. The diﬀerences were particularly important for the symptomatology of being “withdrawn”. Allik et al. (2006) [32] found that parent-reported insomnia was related to hyperactivity in 32 children with Asperger’s Syndrome/High-Functioning Autism with a mean age of 10.8 years. In a sample of 1784 children aged 3–18 years, Goldman et al. (2011) [34] observed contradictory results regarding the association between sleep and hyperactivity, which diﬀered according to the type of behavioral questionnaire used. Subjective sleep disturbances have also been related to the severity of ASD symptoms such as the presence of restricted and repetitive behaviors and diﬃculties in reciprocal social interactions, as well as irritability, aﬀective disturbances and disruptive behaviors [14,30,31,35,36]. J. Clin. Med. 2020, 9, 1978 3 of 19 Only a few studies have investigated objectively measured sleep in relation to behavioral diﬃculties. In a recent study, Bangerter et al. (2020) [37] showed an association between actigraphy-derived sleep eﬃciency, number of awakenings and hyperactivity and anxiety in children with ASD. However, Anders et al. (2012) [38] found no association between actigraphy-deﬁned sleep problems and behavioral diﬃculties, including internalizing and externalizing behaviors in young children with ASD. Furthermore, studies using polysomnography in relation to behavioral diﬃculties are scarce and are usually conducted in children over 10 years on average [12,15,39]. Only one study has investigated the aforementioned relation in children aged 4–10 years [14]. The authors showed a positive correlation between sleep latency and several behavioral problems such as aﬀective problems, anxious/depressed problems and aggressive behaviors. 1. Introduction The literature on the impact of circadian rhythm disorders on daytime functioning in children with ASD is even more scarce and discrepancies remain [22,23,40]. Indeed, Tordjman et al. [22,23] showed a negative correlation between nocturnal urinary melatonin and autism severity, with lower levels of melatonin secretion being associated with lower overall level of verbal language, less imitative social play and more repetitive use of objects; however, other authors did not conﬁrm these ﬁndings [40]. To the best of our knowledge, no study has yet objectively investigated the impact of both sleep and circadian rhythm disturbances on behavioral diﬃculties in children with ASD. Characterizing sleep and circadian rhythms diﬃculties separately is essential as they have diﬀerent pathophysiological pathways, as they may have a diﬀerential impact on daytime functioning and as they may lead to diﬀerent treatments. The main objective of this study was to investigate, for the ﬁrst time, the relationship between subjective and objective measures of sleep and circadian rest–activity rhythm to problem behaviors, and to determine the relative contribution of each in explaining problem behaviors in children with ASD. 2.1. Participants Fifty-two children aged 3–10 years were recruited as part of a longitudinal French multicenter clinical research program (University Hospitals of Strasbourg, Lyon and Tours) (Clinical Trial NCT02878499) examining the role of sleep and circadian rhythms disorders in children with ASD. The study complied with the standards of good clinical practices and the Declaration of Helsinki of 1975, as revised in 2008. All procedures contributing to this work have been approved by the regional French Institutional Review Board (Comité de Protection des Personnes “Est IV”, 4 November 2012, Strasbourg, France). Written, signed and informed consent was obtained prior to participation from the parents of participants and assent was obtained from the child when possible. Inclusion criteria were to be a child aged 3–10 years, with a diagnosis of ASD using the Diagnostic and Statistical Manual of Mental Disorders (DSM IV-TR or DSM-5 criteria) [1]. All participants underwent a clinical diagnostic evaluation by certiﬁed practitioners and met criteria for ASD using the Autism Diagnostic Observation Scale cutoﬀ(ADOS [41]), as well as the criteria on all domains of the Autism Diagnostic Interview-Revised (ADI-R [42]). Children were included independently of their degree of developmental delay or intellectual disability associated with ASD. They had to be on no medication or on stable medication 2 months before the inclusion and during the assessment periods. Exclusion criteria were ASD associated with known neurogenetic disorders (e.g., associated with fragile X syndrome, Rett syndrome, Down syndrome, Smith–Magenis syndrome, Bourneville tuberous sclerosis, Von Recklinghausen’s disease, cytomegalovirus encephalitis, congenital rubella syndrome and phenylketonuria), epilepsy, comorbid severe physical disability or severe allergy. Finally, participants were not allowed to have had transmeridian travels over two time zones or more, 3 months before the assessments. 4 of 19 J. Clin. Med. 2020, 9, 1978 2.2. Measures 2.2. Measures 2.2.1. Objective Sleep and Circadian Measures, Using Actigraphy Actigraphy is a convenient objective and noninvasive method for measuring continuously, in the home environment, both sleep and circadian rest–activity rhythms over long periods of time. Each child was asked to wear an activity monitor with luxmeter (MotionWatch 8®, CamNtech Ltd., Fenstanton, Cambridgeshire, UK) for 15 days, on their non-dominant wrist, or on the left wrist by default if the child was not yet lateralized. The actigraph is an electronic device containing a piezo-electric accelerometer that measures the intensity, amount and duration of physical movement in all directions. Sleep Analysis Actigraphy data were scored automatically for sleep/wake using the Actiwatch Activity and Sleep Analysis 7® software algorithm, version 7.23. As our previous actigraphy validation study in children with ASD indicated that the low sensitivity-threshold setting shows the best ﬁt when compared to polysomnography (PSG) [17], this setting is thus reported here. The low sensitivity-threshold is deﬁned as a threshold level of 80 counts, i.e., an activity score of 80 counts or more during an epoch of 1 min is necessary for that epoch to be scored as wake. Sleep parameters obtained by the sleep analysis were: Total Sleep Time (TST), Sleep Latency (SL), Wake After Sleep Onset (WASO) and Sleep Eﬃciency (SE). TST was deﬁned as the time between sleep onset and sleep oﬀset minus the time of WASO, and SL was deﬁned as the time between bedtime and sleep onset. WASO was deﬁned as the number of minutes scored as wake between sleep onset and sleep oﬀset. SE was deﬁned as the ratio of TST to the time in bed (i.e., time from bedtime to get up time). In addition to classic sleep parameters provided by sleep analysis software, we assessed the longest continuous sleep episode (LSE). LSE is deﬁned as the longest duration of uninterrupted sleep and has recently been identiﬁed as a clinically meaningful sleep parameter in a study population of children and adolescents with ASD [43–47]. Previous studies have measured LSE subjectively using sleep diaries, whereas it was objectively measured using actigraphy in the present study. As such, LSE was deﬁned here as the longest uninterrupted sleep episode or interrupted by less than ﬁve consecutive minutes of scored wake on ACT. This modiﬁcation was necessary due to the brief episodes of sleep that may be identiﬁed incorrectly as a wake episode [17]. The deﬁnition of LSE was based on a comparison of ACT and PSG in this sample (unpublished data), which identiﬁed a tolerance margin of 5 min as the most ﬁtting one, when compared to LSE measured by PSG. In parallel to the actigraphy recording, parents were requested to ﬁll a paper-based sleep diary and to indicate a set of information on their child’s sleep including bedtime (the time the child is in bed and ready to sleep) in order to calculate sleep latency. 2.1. Participants Actigraphic activity is measured in counts deﬁned as the amplitude of the signal produced by its accelerometer, with the number of counts being proportional to the intensity of the movement. The accelerometer samples movement amplitude 32 times per second. Its peak of intensity is deﬁned for each second as the highest amplitude across the 32 records, and peak intensity values were summed into an epoch of 1 min. Sleep Analysis Sleep diary data were used to reduce actigraphy artifacts, including the removal of the device (e.g., bathing, showering and swimming pool) and any reason for an atypical night of sleep (e.g., sickness, sleepover, late evening event, etc.). Circadian Measures Non-Parametric Circadian Rhythm Analyses (NPCRA; MotionWare 1.2.31, Cambridge Neurotechnology, Fenstanton, Cambridgeshire, UK) were performed using rest–activity data to assess three actigraphy-derived circadian rhythm parameters: interdaily stability (IS), intradaily variability (IV) and relative amplitude (RA). Algorithms that determine these variables were described by Van Someren et al. (1999) [48]. To perform NPCRA, at least 4 continuous 24-h days of monitoring 5 of 19 J. Clin. Med. 2020, 9, 1978 were required (whereas only nighttime recording of at least 4 days were required for sleep analysis). If the actigraph device was removed for more than half an hour and up to 3 h a day, the activity level of this missing period was replaced by the average of the mean activity level of the day before and the day after this missing period. If more than 3 h of recording were missing in a day, that day was not considered in the NPCRA. As parents kept a sleep diary during the period of monitoring, it was possible to handle missing data and/or artifacts with parents’ comments (e.g., child removed the actigraph device while in the swimming pool). The parameter of IS gives an indication on the day-to-day stability of the rest–activity rhythm. IS values range between 0, for no stability other than Gaussian noise, to 1, indicating that the rest–activity pattern is repeated perfectly every single day. A higher IS value indicates a more stable rhythm. IV provides information on the variability of the rhythm within a day and quantiﬁes the degree of fragmentation of the behavioral rhythm. In other words, IV indicates the frequency and extent of transitions between rest and activity. Its value ranges between 0, indicating that transitions between rest and activity within a day are tightly consolidated, to 2, which means that the fragmentation of transitions between rest and activity is random. y RA is an estimate of the strength or robustness of the circadian rhythm. It is deﬁned as the normalized diﬀerence between the most active 10-h period and least active 5-h period in a 24-h pattern. Higher values indicate greater amplitude with values ranging between 0 and 1. The midpoint of sleep (MSF), another actigraphy-derived circadian parameter, was also computed [49]. It is deﬁned as the midpoint between sleep onset and sleep end with the calculation as follow: time of sleep onset + TST/2. 2.2.2. Subjective Sleep Measures The Children’s Sleep Habits Questionnaire (CSHQ) Circadian Measures This measure is indicative of the circadian preference of a child, i.e., its chronotype. In our study sample of children aged 3–10 years at inclusion, we did not correct for sleep debt accumulated during work or school days (no signiﬁcant diﬀerences between sleep on school days and weekends). The Children’s Sleep Habits Questionnaire (CSHQ) The CSHQ is a parent-rated questionnaire comprised of 45 items, 33 of which contribute to the scoring, designed to screen for sleep problems in children aged 4–10 years [50]. Parents were asked to complete the questionnaire based on their child’s sleep habits over the last typical week. Each scored question is rated on a three-point scale as occurring “usually” (i.e., 5–7 times per week), “sometimes” (i.e., 2–4 times per week) or “rarely” (i.e., never or once a week). To date, the CSHQ has generally been used to assess the severity of sleep problems or to categorize children as “good sleepers” or “poor sleepers” based on the CSHQ total score, with a clinically sensitive cut-oﬀscore established to ≥41 for the identiﬁcation of probable sleep problems [50]. In their initial study, Owens and colleagues (2000) demonstrated a very poor to acceptable internal consistency of the eight subscales (Cronbach’s alphas: 0.36–0.70), although test–retest reliability was acceptable (correlations values: 0.62–0.79). This initial study did not assess the factor structure and, thereafter, only a few studies have investigated its psychometric properties in typically developing children, with none of them supporting the original eight subscales structure of the CSHQ [51]. The psychometric examination of the CSHQ factor structure allows to establish a proﬁle of speciﬁc types of sleep problems rather than assessing the overall severity of sleep problems. To date, despite its widespread use in children with ASD, only 3 studies have investigated the factor structure of the CSHQ in this population. Johnson and colleagues [52] proposed a revised ﬁve-factor structure while Katz and colleagues [53] suggested a four-factor structure, in children with ASD aged 2–10 years and 4–10 years, respectively. We based the scoring of the CSHQ on a recent study investigating the factor structure of the questionnaire in a population corresponding closely to our population of interest: ASD children aged 4–5 years [54]. In this ASD-adapted CSHQ, the questionnaire is comprised of 5 subscales: Bedtime Routine (BTR), Sleep Onset and Duration (SO&D), Night Wakening (NW), Sleep Disordered Breathing (SDB) and Morning Wakening (MoW). J. Clin. Med. 2020, 9, 1978 6 of 19 Aberrant Behavior Checklist-Community (ABC-C) The ABC-C is a 58-item parent-rated questionnaire that was initially developed for the assessment of a range of disruptive behaviors in people with intellectual and developmental disabilities [55]. The ABC-C was shown to have sound psychometric properties with high internal consistency across the subscales (mean alpha = 0.91), excellent test–retest reliability (mean r = 0.98) and acceptable inter-rater reliability (mean r = 0.63). Its use has been validated in children with ASD [56]. Each question is rated by parents/caregivers on a four-point scale from 0 (“not a problem”) to 3 (“the problem is severe in degree”). The ABC-C is comprised of ﬁve factors: ABC-I, Irritability, Agitation, Crying (15 items); ABC-II, Lethargy and social withdrawal (16 items); ABC-III, Stereotypic Behavior (7 items); ABC-IV, Hyperactivity, Noncompliance (16 items); and ABC-V, Inappropriate Speech (4 items). The latter was not considered in this study because many young children with ASD in our study had not acquired suﬃcient levels of speech. Higher scores indicate more severe behavioral diﬃculties. Since the number of items diﬀers between ABC-C factors, the scores were reported as a percentage to ease comparison (i.e., (raw score × 100/maximum score of each ABC-C factor)). Recent studies have reported nonlinear associations between sleep and daytime functioning [57,58]. Thus, ABC-C subscales were dichotomized individually according to their median value, to study the diﬀerence in sleep and circadian rest–activity rhythm between subjects with higher and lower behavioral diﬃculties, allowing for an easier clinical interpretation. When participants scored equal to or above the median on an ABC-C factor, they were referred as the group with “higher problem behavior” and as the group with “lower problem behavior” when participants scored under the median on that ABC-C factor. 2.2.4. Adaptive Behaviors To describe the level of adaptive functioning of the study sample, the Vineland Adaptive Behavior Scales (VABS [59]) was administered. The VABS is a semi-structured interview conducted with parents to assess a range of adaptive behaviors such as communication, socialization, daily living skills and motor skills. Equivalent ages for each domain were reported here. 2.3. Data Analysis and Statistics Two sets of analyses were conducted in the present study. First, we investigated the association between each of the sleep and circadian variables with the 4 ABC-C factors. To compare sleep and circadian measures between the group with higher and the group with lower problem behavior for each ABC-C factor, independent sample t tests, Wilcoxon Mann–Whitney or Welch’s t-tests were performed, according to data distribution and homogeneity of variance. Secondly, we carried out dominance analysis (DA) to determine, among objective sleep and circadian exposures, those most strongly associated with behavioral diﬃculties. Dominance analysis allows both addressing multicollinearity between predictors and ranking predictors according to the relative importance of their contribution in explaining the variance of the outcome. Addressing multicollinearity was essential as sleep and circadian measures are strongly correlated; thus, classic linear regression was not considered. DA consists in exhaustive series of pairwise comparisons of the predictors, considering all possible subset regression models with and without the other predictors. For example, when considering 3 predictors (X1, X2 and X3), 7 models are created: three models including one predictor alone, three including 2 predictors each (X1 and X2), (X1 and X3) and (X2 and X3)) and a last model including all three predictors. The additional contribution of a given predictor is measured by the increase in explained variance that results from adding that predictor to the regression model. Thus, the additional contributions of X1 are the increases in the proportion of variance accounted for when X1 is added to each subset of the remaining predictors (i.e., the null subset, X2, X3, X2 and X3). One predictor is said J. Clin. Med. 2020, 9, 1978 7 of 19 to completely dominate another if its additional contribution to each of the subset models is greater than that of the other predictor [60–62]. Three DA were tested: in the ﬁrst model, named the “sleep model”, all objective sleep predictors were entered (TST, WASO, SL and LSE) except sleep eﬃciency as its calculation is dependent on the other sleep predictors. In the second DA, named the “circadian model”, all circadian predictors were entered: IS, IV, RA and MSF. In the last DA, the “circadian and sleep model”, both objective sleep and circadian predictors were entered: TST, WASO, SL, LSE, IS, IV, RA and MSF. The coeﬃcient of determination, R2, for each model (R2 total) and for the dominant predictor within each model are reported. 2.3. Data Analysis and Statistics p Statistical analysis was performed using R version 1.2.5001. The calc.yhat package was used to perform dominance analysis [61]. Statistical signiﬁcance was set at p value < 0.05. 3.1. Study Participants The sample included 52 children comprised of 41 boys (79%) and 11 girls, with a mean age of 5.39 years ± 1.50 (SD) and an age range of 2.75–9.57 years. Descriptive characteristics of the study participants are set out in Table 1. As measures were not always interpretable (e.g., incomplete questionnaires, insuﬃcient number of days of ACT, etc.), the number of participants for which the measure was available is speciﬁed. The average developmental delay (i.e., the diﬀerence between chronological age and developmental age) in adaptive behaviors according to the Vineland Adaptive Behaviors Scale subdomains ranged 1.85–2.93 years and was especially important in the subdomains of communication and socialization. Table 1. Descriptive characteristics of the study participants. Mean (SD) Range VABS subdomains (n = 47) (developmental age) Communication (years) 2.7 (1.4) (0.8–5.6) Socialization (years) 2.4 (1.3) (0.9–6.5) Daily living skills (years) 3.1 (1.8) (0.9–11.1) Motor skills (years) 3.4 (1.2) (1.4–6.2) ABC-C subscales (n = 52) I: Irritability, agitation, crying 43.3 (24.0) (0.0–93.3) II: Lethargy, social withdrawal 27.4 (15.0) (4.2–72.9) III: Stereotypic behavior 27.9 (19.3) (0.0–76.0) IV: Hyperactivity, Non-compliance 50.5 (23.5) (2.0–98.0) CSHQ total score (n = 50) 51.6 (9.4) (34–78) ASD-adapted CSHQ (n = 50) Bedtime routine 6.2 (2.4) (4–12) Sleep onset and duration 10.4 (2.7) (6–16) Night waking 7.8 (1.9) (6–12) Sleep disordered breathing 3.5 (1.3) (3–9) Morning wakening 6.7 (2.1) (4–12) Actigraphy sleep measures (n = 40) TST (min)TST(hours) 503.9 (52.5) (372.0–603.1) 8.40 (52.5) (6.2–10.1) WASO (min) 51.8 (23.0) (21.9–126.1) SE (%) 82.6 (6.0) (65.9–91.0) SL (min) 36.8 (23.6) (4.0–94.1) LSE (min) 354.0 (90.0) (184.8–553.1) LSE (hours) 5.9 (1.5) (3.1–9.2) Table 1. Descriptive characteristics of the study participants. Table 1. Descriptive characteristics of the study participants. 8 of 19 J. Clin. Med. 2020, 9, 1978 Table 1. Cont. 3.2. Comparison of Sleep and Circadian Rhythm Measures between Behavioral Diﬃculty Groups Comparison of Sleep and Circadian Rhythm Measures between Behavioral Diﬃculty Groups Table 2 reports the diﬀerences in sleep and circadian measures between the groups with higher problem behaviors and the groups with lower problem behavior, separately for the four ABC-C factors. Within objective sleep measures, the longest sleep episode showed the highest diﬀerence between groups with low or high levels of behavioral disturbances. On average, the higher irritability group slept continuously 60 min less than the lower irritability group (p = 0.04). The group with higher stereotypic behaviors slept continuously 75 min less than the lower stereotypic behaviors group (p = 0.006) (see Figure 1). In other words, the less continuously the children slept, the more irritability and stereotypic behavior they displayed. No diﬀerences were observed regarding the objective circadian rest–activity rhythm measures. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 10 of 19 Figure 1. Longest continuous sleep episode as a function of problem behaviors in children with ASD. FI—irritability, Factor I, Irritability on the Aberrant behavior checklist–Community (ABC-C). FIII— stereotypic behavior, Factor III, stereotypic behavior on the Aberrant behavior checklist–Community. Low, group with an ABC-C score below the median, calculated separately by factor; High, group with an ABC-C score equal or above the median, calculated separately by factor. Figure 1. Longest continuous sleep episode as a function of problem behaviors in children with ASD. FI—irritability, Factor I, Irritability on the Aberrant behavior checklist–Community (ABC-C). FIII—stereotypic behavior, Factor III, stereotypic behavior on the Aberrant behavior checklist–Community. Low, group with an ABC-C score below the median, calculated separately by factor; High, group with an ABC-C score equal or above the median, calculated separately by factor. Figure 1. Longest continuous sleep episode as a function of problem behaviors in children with ASD. FI—irritability, Factor I, Irritability on the Aberrant behavior checklist–Community (ABC-C). FIII— stereotypic behavior, Factor III, stereotypic behavior on the Aberrant behavior checklist–Community. Low, group with an ABC-C score below the median, calculated separately by factor; High, group with an ABC-C score equal or above the median, calculated separately by factor. Figure 1. Longest continuous sleep episode as a function of problem behaviors in children with ASD. FI—irritability, Factor I, Irritability on the Aberrant behavior checklist–Community (ABC-C). FIII—stereotypic behavior, Factor III, stereotypic behavior on the Aberrant behavior checklist–Community. 3.1. Study Participants Mean (SD) Range Actigraphy circadian measures (n = 39) IS 0.59 (0.11) (0.33–0.78) IV 0.65 (0.09) (0.44–0.88) RA 0.93 (0.05) (0.79–0.98) MSF (hh:mm) 02:07 (01:06) (23:46–04:19) VABS, Vineland Adaptive Behavior Scales; ABC-C, Aberrant behavior checklist-community; ABC-I, Factor I Irritability; ABC-II, Factor II social withdrawal; ABC-III, Factor III stereotypic behavior; ABC-IV, Factor IV Hyperactivity; ABC-total, Total score on the ABC-C; CSHQ, Children’s sleep habits questionnaire; BR, bedtime resistance; SOD, sleep onset delay; SD, sleep duration; NW, night waking; Para, parasomnia; SDB, sleep disordered breathing; DS, daytime sleepiness; CSHQ total; Total score on the CSHQ; ASD-adapted CSHQ, ASD-adapted Children’s sleep habits questionnaire; BTR, Bedtime Routine; SO&D, Sleep Onset and Duration; NW, Night Wakening; SDB, Sleep Disordered Breathing; MoW, Morning Wakening; TST, Total Sleep Time; WASO, Wake After Sleep Onset; SE, Sleep Eﬃciency; SL, Sleep Latency; LSE, longest Sleep Episode; IS, Interdaily Stability; IV, Intradaily Variability; RA, Relative Amplitude; MSF, Midpoint of Sleep. Mean (SD) 3.2. Comparison of Sleep and Circadian Rhythm Measures between Behavioral Diﬃculty Groups Low, group with an ABC-C score below the median, calculated separately by factor; High, group with an ABC-C score equal or above the median, calculated separately by factor. When comparing subjective sleep measures between the groups with higher problem behaviors to the groups with lower problem behaviors, a significant difference was found on several ASD- adapted CSHQ subscales, as well as on the total CSHQ, scored according to the original scoring. The group with higher irritability differed significantly from the group with lower irritability on bedtime routine (p = 0.033). A difference between groups was also observed for social withdrawal on sleep onset and duration (p = 0.034). The group with higher hyperactivity did not differ from the group with lower hyperactivity but a tendency was found for bedtime routine (p = 0.069). The group with higher social withdrawal significantly differed from the group with lower social withdrawal on the CSHQ total score (p = 0 048) When comparing subjective sleep measures between the groups with higher problem behaviors to the groups with lower problem behaviors, a signiﬁcant diﬀerence was found on several ASD-adapted CSHQ subscales, as well as on the total CSHQ, scored according to the original scoring. The group with higher irritability diﬀered signiﬁcantly from the group with lower irritability on bedtime routine (p = 0.033). A diﬀerence between groups was also observed for social withdrawal on sleep onset and duration (p = 0.034). The group with higher hyperactivity did not diﬀer from the group with lower hyperactivity but a tendency was found for bedtime routine (p = 0.069). The group with higher social withdrawal signiﬁcantly diﬀered from the group with lower social withdrawal on the CSHQ total score (p = 0.048). J. Clin. Med. 2020, 9, 1978 9 of 19 Table 2. Sleep and circadian rhythm measures by ABC-C factor group. Table 2. Sleep and circadian rhythm measures by ABC-C factor group. 3.2. Comparison of Sleep and Circadian Rhythm Measures between Behavioral Diﬃculty Groups I-Irritability Mean (SD) II-Social Withdrawal Mean (SD) III-Stereotypy Mean (SD) IV-Hyperactivity Mean (SD) Low High Low High Low High Low High Objective sleep (ACT) TST (min) 509.6 (63.3) 498.2 (39.8) 506.8 (59.5) 501.2 (46.7) 512.2 (54.5) 495.6 (50.5) 503.4 (62.4) 504.6 (34.6) SE (%) 82.8 (7.2) 82.3 (4.8) 82.4(6.8) 82.7(5.4) 84.0 (5.5) 81.1(6.3) 82.2(7.1) 83.1 (4.1) SL (min) 38.3 (23.4) 35.3 (24.4) 39.4 (28.1) 34.4 (19.1) 36.2(25.6) 37.4 (22.2) 36.9 (23.0) 36.7 (25.3) WASO (min) 48.4 (26.1) 55.2 (19.3) 50.0 (21.23) 53.5 (24.8) 56.3 (26.8) 84.0 (5.6) 52.3 (28.0) 51.1 (13.2) LSE (min) 383.1 (101.6) * 324.8 (67.1) * 370.3 (101.9) 339.1 (77.2) 391.7 (90.5) 316.2 (73.7) 354.1 (100.3) 353.7 (75.0) Circadian rhythm (ACT) IS 0.62 (0.09) 0.58 (0.12) 0.61 (0.11) 0.58 (0.10) 0.62 (0.10) 0.57 (0.11) 0.59 (0.11) 0.60 (0.10) IV 0.65 (0.08) 0.65 (0.10) 0.64 (0.09) 0.66 (0.10) 0.65 (0.08) 0.65 (0.11) 0.65 (0.09) 0.65 (0.10) RA 0.93 (0.05) 0.92 (0.04) 0.93 (0.04) 0.92 (0.05) 0.93 (0.04) 0.92 (0.05) 0.93 (0.05) 0.92 (0.05) MSF (hh:mm) 01:57 (01:15) 02:17 (00:54) 01:59 (01:13) 02:14 (00:59) 02:18 (00:57) 01:56 (01:13) 02:07 (01:05) 02:08 (01:09) Subjective sleep (ASD-adapted CSHQ) BTR 5.4 (2.0) * 7.0 (2.6) * 5.7 (2.5) 6.8 (2.2) 6.0 (2.7) 6.4 (2.1) 5.6 (2.0) 6.9 (2.7) SO&D 9.8 (2.4) 11.1 (2.8) 9.7 (2.9) * 11.2 (2.2) * 10.1 (2.9) 10.8 (2.4) 10.2 (2.6) 10.8 (2.8) NW 7.6 (1.8) 8.0 (2.0) 7.3 (1.6) 8.3 (2.1) 7.4 (1.6) 8.2 (2.1) 7.9 (1.9) 7.7 (1.9) SDB 3.6 (1.5) 3.4 (1.1) 3.5 (1.4) 3.6 (1.3) 3.6 (1.5) 3.4 (1.1) 3.8 (1.7) 3.2 (0.7) MoW 6.8 (2.1) 6.6 (2.3) 6.4 (2.1) 6.9 (2.2) 6.6 (2.2) 6.7 (2.1) 6.7 (2.2) 6.6 (2.2) CSHQ total 49.3 (7.6) 53.4 (10.4) 48.9 (8.8) * 54.2 (9.2) * 50.2 (9.4) 52.8 (9.2) 50.8 (9.3) 52.2 (9.4) * p < 0.05, ** p < 0.01. * p < 0.05, ** p < 0.01. Low, group with an ABC-C score below the median, calculated separately by factor; High, group with an ABC-C score equal or above the median, calculated separately by factor; ABC-C, Aberrant behavior checklist-community ABC-I, Factor I Irritability; ABC-II, Factor II social withdrawal; ABC-III, Factor III stereotypic behavior; ABC-IV, Factor IV Hyperactivity; ABC-total, Total score on the ABC-C; ACT, actigraphy; TST, Total Sleep Time; SE, Sleep Eﬃciency; SL, Sleep Latency; WASO, Wake After Sleep Onset; LSE, longest Sleep Episode; IS, Interdaily Stability; IV, Intradaily Variability; RA, Relative Amplitude; MSF, Midpoint of Sleep (average and standard deviation are ported in hh:mm). ASD-adapted Children’s sleep habits questionnaire subdomains: BTR, Bedtime Routine; SO&D, Sleep Onset and Duration; NW, Night Wakening; SDB, Sleep Disordered Breathing; MoW, Morning Wakening; CSHQ total, Children’s sleep habits questionnaire total score. 3.3. Dominance Analyses The dominance analyses (DA) enabled to determine separately for the four ABC-C factors, the importance of the contribution of objective sleep and circadian predictors and to identify which predictor is most instrumental. The results are reported in Table 3, with the ﬁrst analysis including as predictors the objective sleep measures (TST, WASO, LSE and SL), the second including the objective circadian measures (IS, IV, RA and MSF) and the third including both. Total R2 indicates the coeﬃcient of determination of the model (i.e., the proportion of the variance of ABC-C factors explained by the predictors combined), a dominant predictor is reported with its unique variance when fulﬁlling complete dominance requirement (cf. Methods Section, paragraph 2.3. Data Analysis and Statistics). Table 3. Dominance analysis of sleep, circadian predictors and disruptive behaviors. I-Irritability II-Social Withdrawal III-Stereotypy IV-Hyperactivity Sleep R2 total 7.0% 6.0% 7.0% 0.8% Dominant predictor (R2) LSE (2.5%) TST (4.1%) LSE (4.3%) SL (0,3%) Circadian R2 total 4.5% 9.8% 18.4% 0.3% Dominant predictor (R2) IS (1.7%) MSF (5.2%) MSF (15.8%) RA (0.3%) Sleep and Circadian R2 total 17.7% 16.7% 35.9% 0.7% Dominant predictor (R2) LSE (6.1%) MSF (4.7%) LSE & MSF * RA (0.4%) * Complete dominance between LSE and MSF cannot be established. MSF accounted uniquely for 18.2% and LSE for 14.3% for the stereotypic behaviors factor. IV, intradaily variability; RA, relative amplitude; MSF, mid-point of sleep; ABC-I, Factor I Irritability; ABC-II, Factor II social withdrawal; ABC-III, Factor III stereotypic behavior; ABC-IV, Factor IV Hyperactivity; TST, total sleep time; LSE, longest sleep episode. Table 3. Dominance analysis of sleep, circadian predictors and disruptive behaviors. * Complete dominance between LSE and MSF cannot be established. MSF accounted uniquely for 18.2% and LSE for 14.3% for the stereotypic behaviors factor. IV, intradaily variability; RA, relative amplitude; MSF, mid-point of sleep; ABC-I, Factor I Irritability; ABC-II, Factor II social withdrawal; ABC-III, Factor III stereotypic behavior; ABC-IV, Factor IV Hyperactivity; TST, total sleep time; LSE, longest sleep episode. In the sleep and circadian model, the eight objective predictors accounted for 17.7% of the variance in irritability, and the longest sleep episode (LSE) contributed 6.1% of unique variance (see Figure 2). The same model accounted for 16.7% of the variance in social withdrawal, with the midpoint of sleep (MSF) as a complete dominant factor with 4.7% of unique variance. 3.2. Comparison of Sleep and Circadian Rhythm Measures between Behavioral Diﬃculty Groups Low, group with an ABC-C score below the median, calculated separately by factor; High, group with an ABC-C score equal or above the median, calculated separately by factor; ABC-C, Aberrant behavior checklist-community ABC-I, Factor I Irritability; ABC-II, Factor II social withdrawal; ABC-III, Factor III stereotypic behavior; ABC-IV, Factor IV Hyperactivity; ABC-total, Total score on the ABC-C; ACT, actigraphy; TST, Total Sleep Time; SE, Sleep Eﬃciency; SL, Sleep Latency; WASO, Wake After Sleep Onset; LSE, longest Sleep Episode; IS, Interdaily Stability; IV, Intradaily Variability; RA, Relative Amplitude; MSF, Midpoint of Sleep (average and standard deviation are ported in hh:mm). ASD-adapted Children’s sleep habits questionnaire subdomains: BTR, Bedtime Routine; SO&D, Sleep Onset and Duration; NW, Night Wakening; SDB, Sleep Disordered Breathing; MoW, Morning Wakening; CSHQ total, Children’s sleep habits questionnaire total score. J. Clin. Med. 2020, 9, 1978 10 of 19 10 of 19 3.3. Dominance Analyses The variance in stereotypy was explained by the combination of sleep and circadian parameter accounting for 35% of the variance. Although no factor reached individual complete dominance, MSF and LSE dominated the other predictors. The MSF contributed approximately 18% of unique variance in stereotypic behavior and LSE accounted for 14.3% of unique variance in stereotypic behavior. For the last ABC-C factor, hyperactivity, the sleep and circadian model accounted for only 0.7% of the variance in hyperactivity and relative amplitude contributed 0.4% of unique variance. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 11 of 19 Figure 2). The same model accounted for 16.7% of the variance in social withdrawal, with the midpoint of sleep (MSF) as a complete dominant factor with 4.7% of unique variance. The variance in stereotypy was explained by the combination of sleep and circadian parameter accounting for 35% of the variance. Although no factor reached individual complete dominance, MSF and LSE dominated the other predictors. The MSF contributed approximately 18% of unique variance in stereotypic behavior and LSE accounted for 14.3% of unique variance in stereotypic behavior. For the last ABC-C factor, hyperactivity, the sleep and circadian model accounted for only 0.7% of the variance in hyperactivity and relative amplitude contributed 0.4% of unique variance. Figure 2. Differential effect of sleep and circadian rhythm parameters on problem behaviors in children with ASD. Percentage of explained variance of ABC-C factors across dominance analysis models Each column represents a model; the filled dots indicate which variables are included For Figure 2. Diﬀerential eﬀect of sleep and circadian rhythm parameters on problem behaviors in children Figure 2. Differential effect of sleep and circadian rhythm parameters on problem behaviors in children with ASD. Percentage of explained variance of ABC-C factors across dominance analysis Figure 2. Diﬀerential eﬀect of sleep and circadian rhythm parameters on problem behaviors in children 11 of 19 J. Clin. Med. 2020, 9, 1978 with ASD. Percentage of explained variance of ABC-C factors across dominance analysis models. Each column represents a model; the ﬁlled dots indicate which variables are included. For each factor, the four variables with the highest unique variance were retained, and the corresponding models included 1–4 variables. Models representing less than 1% are not shown. 4. Discussion This is the ﬁrst study to undertake an objective and subjective assessment of sleep and circadian rest–activity rhythm in association with behavioral diﬃculties in young children with ASD. Longer continuous sleep in children with ASD was associated with less behavioral diﬃculties during the day. Indeed, the duration of the longest uninterrupted sleep episode, as measured objectively by actigraphy, was 60 min shorter in the group with higher irritability than the group with lower irritability, and 75 min shorter in the group with higher stereotypy than the group with lower stereotypy (see Figure 1). A 60–75-min period is equivalent to at least one complete sleep cycle (REM sleep and non-REM sleep) in children (50–80 min, depending on age). Both REM sleep and non-REM sleep are related to child growth and memory consolidation [63–65], and there is a general consensus that consolidated sleep throughout a whole night is optimal for the plasticity changes needed for learning and memory consolidation in children with ASD [66]. This study thus corroborates the interest of the longest sleep episode as a marker of sleep quality related to daytime outcomes in children with ASD. In accordance with these results, a previous study found that a clinically meaningful improvement in the duration of the longest sleep episode, after a 13-week treatment of pediatric prolonged-release melatonin, led to a signiﬁcant improvement in daytime behavioral problems in a population of 125 ASD children aged 2–17.5 years [46]. Additionally, this study found that the longest sleep episode was also associated with improvement in quality of life in parents. Together, these ﬁndings support the importance of continuous nocturnal sleep for daytime behavioral functioning and highlight the importance of this sleep measure when assessing sleep in children with ASD, as well as the repercussion of sleep on overall quality of life of their families. Using dominance analyses, we observed that objective circadian and objective sleep disturbances accounted together for, respectively, 17%, 18% and 36% of the variance in social withdrawal, irritability and stereotypic behaviors. The longest sleep episode and the midpoint of sleep were the most dominant predictors to explain the variance of these problem behaviors (see Figure 2). Although it is essential to provide objective sleep and circadian measures, subjective data have their own value, as some sleep disorders diagnoses, in particular insomnia, are solely deﬁned through subjective criteria. 3.3. Dominance Analyses FI–irritability, Factor I, Irritability on the Aberrant Behavior Checklist–Community; FII–social withdrawal, Factor II, social withdrawal on the Aberrant Behavior Checklist–Community; FIII–stereotypic behavior, Factor III, stereotypic behavior on the Aberrant Behavior Checklist–Community. Sleep variables: LSE (longest sleep episode), TST (total sleep time) and SL (sleep latency). Circadian variables: RA (relative amplitude), IS (interdaily stability), IV (intradaily variability) and MSF (midpoint of sleep). 4. Discussion In the present study, children with higher irritability had more diﬃculties in the following subjective measures: bedtime routine, while children with higher social withdrawal showed insuﬃcient sleep duration and higher bedtime resistance. Moreover, the group with higher social withdrawal had signiﬁcantly more severe sleep problems, based on the CSHQ total score. The group with higher hyperactivity did not diﬀer from the group with lower hyperactivity on subjective measures, but a tendency was found for bedtime routine. Comparative literature is scarce, as the majority of studies using objective assessment of sleep have only focused on characterizing sleep in children with ASD. Only a few studies have investigated actigraphy-derived sleep measures in relation to behavioral diﬃculties. A recent study has shown an association between actigraphy-derived sleep eﬃciency, number of awakenings and hyperactivity and anxiety in 144 children with ASD aged 14.6 on average [37]. In contrast, apart from the longest sleep episode mentioned above, other objective sleep measures in our study were not signiﬁcantly associated to behavioral diﬃculties in our population of very young children with ASD. Our ﬁndings are in accordance with a previous study which has found no association between actigraphy-deﬁned 12 of 19 J. Clin. Med. 2020, 9, 1978 sleep problems and behavioral diﬃculties, including internalizing and externalizing behaviors, in 68 children with ASD with a mean age of 3.9 years [38]. Discrepancies among studies may thus be attributed to the age of the study population, with links between objective sleep measures and behavior being more diﬃcult to establish in very young children with ASD. Contradictory results may also be explained by diﬀerent actigraphy-related methods for the estimation of sleep parameters. Indeed, the algorithm that determines sleep or wake and the deﬁnition of sleep parameters diﬀer. For example, in the study of Bangerter et al. (2020) [37], sleep eﬃciency was deﬁned as the percentage of minutes of sleep within the interval between sleep start and sleep end, whereas it was calculated as the actual sleep time divided by the time in bed in the present study. This diﬀerence may bias toward a higher estimation of sleep eﬃciency in their study compared to ours. 4. Discussion Furthermore, we attempted to compare the results with other objective sleep measures such as polysomnography, but very few studies have investigated sleep in relation to behavioral diﬃculties and the majority have been conducted in children over 10 years on average and have rather focused on the relation to autism severity [12,39,67]. Only one study investigated the aforementioned relation in children aged 4–10 years [14]. The authors showed a positive correlation between sleep latency and several behavioral problems such as aﬀective problems, anxious/depressed problems and aggressive behaviors. Altogether, these ﬁndings suggest that further studies in children with ASD relating objective sleep measure to behavioral diﬃculties are required in order to clearly establish the precise association between these variables for each age group. As for subjective sleep, a growing literature has investigated its impact on behavioral diﬃculties. Globally, studies agree that sleep problems are linked to behavioral diﬃculties in children with ASD [14,16,30–32]. However, the results in the literature diﬀer with respect to speciﬁc sleep problems and their relation to speciﬁc behavioral diﬃculties. In accordance with our ﬁndings, Aathira et al. (2017) [33] also found an association between poor sleep, deﬁned with a cutoﬀscore on the CSHQ, and overall behavioral diﬃculties, measured by the total score on the Child Behavior Checklist (CBCL), in their study on 71 children with ASD aged 3–10 years. The diﬀerences were particularly important for the symptomatology of being “withdrawn”. Similarly, the association between sleep and irritability has been established in previous studies [31,68]. Indeed, Allik et al. (2006) [32] found that parent-reported insomnia was related to hyperactivity rated on the Strength and Diﬃculties Questionnaire (SDQ) in 32 children with Asperger’s Syndrome/High Functioning Autism with a mean age of 10.8 years. In another study, in a sample of 1784 children aged 3–18 years, Goldman et al. (2011) [34] established a signiﬁcant diﬀerence in hyperactivity between ASD-good sleepers and ASD-poor sleepers on the Parental Concern Questionnaire but not on the CBCL externalizing behaviors [34]. Unlike these studies, we did not ﬁnd any association between hyperactivity and subjective sleep [30,32,34,69]. The discrepancy between our results and the literature can be partly explained by the diﬀerence in age and the method used across studies in order to characterize either sleep or hyperactivity. Indeed, as can be seen in the study by Goldman et al. (2011), the type of questionnaire used to assess hyperactivity can explain diverging results. 4. Discussion Moreover, the aforementioned studies examined the relationship between sleep and hyperactivity in children who are older than those in our study sample, and the eﬀects of sleep problems are likely to diﬀer according to age. It is also possible that, in young children with ASD, hyperactivity may be exacerbated but that sleep does not play a preponderant role in this particular behavior in the studied age and that any added eﬀects of sleep may only be visible in studies with larger sample sizes. We tested this hypothesis by conducting separate analyses in preschool-aged children (<6 years, n = 33) and school-aged children (≥6 years, n = 19). In schooled-aged children, the group with higher hyperactivity had a signiﬁcantly lower sleep eﬃciency compared to the group with lower hyperactivity (p = 0.028), while this association was not found in preschool-aged children (p = 0.12). As the methods to assess and analyze subjective sleep are diﬀerent across studies, precise comparison is hindered. Indeed, the majority of studies used the original scoring of the CSHQ while the present study used the ASD-adapted scoring. Since changes in sleep diﬃculties occur over time, from preschoolers to middle childhood, a recent study included children with ASD aged 4–5 years old J. Clin. Med. 2020, 9, 1978 13 of 19 and provided a novel ﬁve-factor model [54]. In the present study, the mean age of the sample was 5.39 years old which is similar to that of Zaidman-Zait et al. (2020) [54]. Thus, this novel ﬁve-factor model was retained for our analyses. Results on actigraphy-derived circadian measures did not show any diﬀerence in children with higher behavioral diﬃculties compared to children with lower behavioral diﬃculties. While it is possible that there is no diﬀerence to be found, another explanation for these results may be that actigraphy-derived non-parametric circadian rhythm analyses (NCPRA) may be an inadequate proxy of circadian rhythms in our population of very young children with ASD and signiﬁcant sleep disturbances. As mentioned above, NCPRA give three actigraphy-derived circadian rhythm parameters: IS, IV and RA. These circadian rest–activity rhythm measures represent a proxy of the endogenous rhythm; they do not reﬂect it directly. While this approximation may be adequate in the population in which they were conceived, they are not necessarily ﬁtting in all population. 4. Discussion Indeed, they were initially investigated in adults with Alzheimer’s disease [48] and were later used in other populations such as in Parkinson’s disease, bipolar disorder or in healthy infants [70–72]. There are diﬀerent possible reasons for which the NPCRA might not correctly identify the circadian rhythm of young children. Children’s sleep–wake cycles depend on their parents rather than their internal rhythms, many still nap and thus display a biphasic circadian rhythm and they present more movement during sleep. While actigraphy-derived measures of sleep have been validated against its gold standard, polysomnography [17], this is yet to be done for NPCRA measures, which have not yet been validated against a gold standard measurement of circadian rhythms such as melatonin or core body temperature. Although between-group analyses did not show any diﬀerence in the midpoint of sleep, a proxy of circadian preference or chronotype [49], dominance analysis (DA) enabled to highlight the importance of these circadian measures in explaining behavioral diﬃculties when studied together with sleep measures. Indeed, the midpoint of sleep completely dominated all other predictors in explaining social withdrawal, accounting uniquely for around 5% of the variance. DA also emphasized the role of the chronotype measures studied together in explaining stereotypy and social withdrawal accounting, respectively, for 18.7% and 4.7% of their variance. These results can be paralleled to those of Sun et al. (2018) [73] who demonstrated, in 12-month-old typically developing infants, the implication of the circadian rest–activity rhythm in the development of social learning. Clinically signiﬁcant social withdrawal has been reported as a common behavior in ASD children in previous studies [74]. Altogether these ﬁndings suggest that circadian preference, or chronotype, may explain in part the social disability that is described in children with ASD, possibly linked to either genetic factors or to rhythm desynchronization. Furthermore, the relation of sleep and circadian abnormalities to stereotypic behaviors may be explained by a common physiopathological pathway. Indeed, serotonin, which is the precursor of the circadian neurohormone melatonin, has been found to be involved in various functions such as sleep, circadian rhythms, aﬀective regulation and stereotypic behaviors [75,76]. Increased whole-blood serotonin levels and decreased plasma melatonin have previously been reported in patients with ASD [20,77]. 4. Discussion The link between serotonin levels and stereotypic behaviors is further evidenced by the eﬃcacy of medications that inhibit serotonin transport [78] and an exacerbation of these behaviors after a pharmacological depletion of tryptophan which leads to reduced serotonin synthesis [79]. A particular strength of the present study is the objective assessment of both sleep and circadian rhythms with actigraphy, while the majority of studies addressing the same topic have assessed sleep mainly subjectively. Furthermore, only a small body of research has evaluated circadian rhythms objectively in relation to daytime behavior and none related to behavioral diﬃculties. Our study sample included ASD children aged 3–10 years across the spectrum, with and without associated intellectual disability. This allows generalizing our results to the overall population of ASD children and not only to high-functioning ASD children, which is often the case in the literature. Even though our study sample is larger than most studies assessing sleep objectively in relation to daytime behavior, it may not possess suﬃcient statistical power to show between-group diﬀerences. Indeed, a clinically 14 of 19 J. Clin. Med. 2020, 9, 1978 signiﬁcant mean diﬀerence of 28 min in WASO was observed between the groups with lower versus higher stereotypic behaviors, but statistical signiﬁcance was not reached. A further limitation of the present study is the use of a single measure to characterize children’s daytime behavior. Indeed, it would have been valuable to assess behaviors with questionnaires that are speciﬁc of ASD symptoms such as, the Social responsiveness Scale [80] or the Repetitive Behavior Scale [81], or using observational data. In the present study, we conducted a cross-sectional analysis, which does not allow establishing causality and addressing the issue of temporality. To do so, a longitudinal analysis is required. The present analyses were run on the baseline measures of an ongoing longitudinal research project. The longitudinal analyses will allow us to address temporality in the future. The objective evaluation of the circadian rest–activity rhythm is both a strength and a limitation. Indeed, actigraphy enables to noninvasively record circadian rest–activity rhythms, thereby providing an estimation of the child’s ecological sleep–wake rhythm over long periods of time, but it remains a proxy of the circadian timing system. Therefore, future studies should address this limitation and use gold standard phase markers of the circadian rhythm such as 24 h melatonin proﬁle or core body temperature. 4. Discussion One possible way to better characterize the circadian rhythm with actigraphy would be to validate the NPCRA against gold standard measures ﬁrst, and then to establish normative values across diﬀerent age groups in both typically developing children and in children with ASD. Only recently, a few studies have attempted to address this research gap [82–84]. However, only one study investigated the NPCRA, involving solely 24-h recordings, in children ages 4–11 years [83]. Further work on this topic using longer follow-up periods is therefore needed. As mentioned above, in the present study, we used actigraphy rather than polysomnography as actigraphy has recently been validated against polysomnography in children with ASD [17] and because actigraphy allows for a continuous record of sleep during a week or more, in the home environment, unlike polysomnography, which is often conducted in a sleep laboratory for 1–2 days. In addition to that, we performed dominance analyses, which allow addressing multicollinearity between predictors and ranking predictors according to the relative importance of their contribution in explaining the variance of the outcome unlike classic regression analysis. Author Contributions: Conceptualization, C.M.S.; methodology and formal analysis, E.Y.-K., E.R., and C.M.S.; investigation, C.M.S., E.Y.K., N.L., M.-M.G., P.F., and F.B.-B.; data curation, E.Y.-K.; writing—original draft preparation, E.Y.K.; writing—review and editing, E.Y.K., E.R., P.B., and C.M.S.; and funding acquisition, C.M.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Hospital Program for Clinical Research (PHRC National Autisme et Sommeil, HUS No. 5060, 2012). E.R. received a grant from the French Society for Sleep Research and Sleep Medicine (SFRMS). Acknowledgments: The authors would like to greatly thank the patients and their parents for their participation. We would like to thank everyone on the clinical and administrative teams for their assistance with this study. 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This would enable future development of a comprehensive intervention program in young children with ASD that may improve responses to currently available behavioral management programs. Author Contributions: Conceptualization, C.M.S.; methodology and formal analysis, E.Y.-K., E.R., and C.M.S.; investigation, C.M.S., E.Y.K., N.L., M.-M.G., P.F., and F.B.-B.; data curation, E.Y.-K.; writing—original draft preparation, E.Y.K.; writing—review and editing, E.Y.K., E.R., P.B., and C.M.S.; and funding acquisition, C.M.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Hospital Program for Clinical Research (PHRC National Autisme et Sommeil, HUS No. 5060, 2012). E.R. received a grant from the French Society for Sleep Research and Sleep Medicine (SFRMS). Acknowledgments: The authors would like to greatly thank the patients and their parents for their participation. 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https://openalex.org/W3046628386	http://www.ajnr.org/content/ajnr/41/9/1592.full.pdf	English	null	MRS as an Aid to Diagnose Malignant Transformation in Low-Grade Gliomas with Increasing Contrast Enhancement	American journal of neuroradiology	2,020	cc-by	5,353	ABSTRACT BACKGROUND AND PURPOSE: Increased contrast enhancement has been used as a marker of malignant transformation in low- grade gliomas. This marker has been found to have limited accuracy because many low-grade gliomas with increased contrast enhancement remain grade II. We aimed to investigate whether MR spectroscopy can contribute to the diagnosis of malignant transformation in low-grade gliomas with increased contrast enhancement. MATERIALS AND METHODS: Patients with low-grade gliomas who had contemporaneous MR spectroscopy and histopathology for tumor regions with increased contrast enhancement between 2004 and 2015 were retrospectively reviewed. Clinical data collected were sex and age, Karnofsky Performance Scale, histologic subtypes, isocitrate dehydrogenase 1 mutation status, disease duration, adjuvant therapy, and post-radiation therapy duration. Imaging data collected were contrast-enhancement size, whole-tumor size, MR spectroscopy metabolite ratios, and tumor grades of regions with increased contrast enhancement. Diagnostic values of these factors on malignant transformation of low-grade gliomas were statistically analyzed. RESULTS: A total of 86 patients with 96 MR spectroscopy studies were included. Tumor grades associated with increased contrast enhancement were grade II (n ¼ 42), grade III (n ¼ 27), and grade IV (n ¼ 27). On multivariate analysis, the NAA/Cho ratio was the only signiﬁcant factor (P, .001; OR, 7.1; 95% CI, 3.2–16.1) diagnostic of malignant transformation. With 0.222 as the cutoff value, the sensitivity, speciﬁcity, and accuracy of NAA/Cho for diagnosing malignant transformation were 94.4%, 83.3%, and 89.6%, respectively. CONCLUSIONS: MR spectroscopy complements conventional MR imaging in the diagnosis of malignant transformation in a sub- group of low-grade gliomas with increased contrast enhancement. enlargement of nonenhancing tumor and an increase of enhancement on postgadolinium T1-weighted images. At the time of imaging progression, the tumors may remain WHO grade II or may have progressed to WHO grade III or IV, ie, malignant transformation (MT). Definitive diagnosis of MT requires histopathologic examination, but due to potential surgical risk and costs, increased contrast enhancement is fre- quently used as a surrogate marker for MT in clinical prac- tice, research studies, and clinical trials of LGG. In the widely used Response Assessment in Neuro-Oncology criteria,2 an increase of enhancement is also regarded as MT, thus disease progression. L ow-grade gliomas (LGGs) are grade II World Health Organization (WHO) primary brain tumors, accounting for 14.6% of gliomas in population-based studies.1 LGGs may remain clinically and radiographically stable for years after initial diagno- sis and treatment. Received February 18, 2020; accepted after revision June 4. From the Departments of Medical Imaging and Intervention (C.H.T.), and Neurosurgery (K.-C.W., P.-Y.C.), Chang Gung Memorial Hospital at Linkou and Chang Gung University College of Medicine, Tao-Yuan, Taiwan; and Department of Radiology (M.C.), University of North Carolina School of Medicine, Chapel Hill, North Carolina. This work was supported, in part, by grants from the National Science Council Taiwan (NSC98-2314-B-182A-051-MY3 and NSC101-2314-B-182-084 to C.H.T.). Please address correspondence to Cheng Hong Toh, MD, PhD, Department of Medical Imaging and Intervention, Chang Gung Memorial Hospital at Linkou, No. 5, Fuxing St, Guishan District, Taoyuan City 333, Taiwan (R.O.C.); e-mail: eldomtoh@hotmail.com Indicates open access to non-subscribers at www.ajnr.org http://dx.doi.org/10.3174/ajnr.A6688 1592 Toh Sep 2020 www.ajnr.org Increasing Contrast Enhancement Transformation in Low-Grade Gliomas with MRS as an Aid to Diagnose Malignant C.H. Toh, M. Castillo, K.-C. Wei and P.-Y. Chen http://www.ajnr.org/content/41/9/1592 https://doi.org/10.3174/ajnr.A6688 doi: 2020, 41 (9) 1592-1598 AJNR Am J Neuroradiol of October 23, 2024. This information is current as ORIGINAL RESEARCH ADULT BRAIN ORIGINAL RESEARCH ADULT BRAIN MRS as an Aid to Diagnose Malignant Transformation in Low-Grade Gliomas with Increasing Contrast Enhancement C.H. Toh, M. Castillo, K.-C. Wei, and P.-Y. Chen C.H. Toh, M. Castillo, K.-C. Wei, and P.-Y. Chen ABSTRACT However, at an unpredictable time, some may show MR imaging features suggestive of disease progression, eg, L However, a recent study showed that up to 18% of LGGs with increased contrast enhancement remained WHO grade II.3 Despite having a sensitivity of 92%, increased contrast enhance- ment had a specificity of 57% for the detection of MT. Besides, increased contrast enhancement may also be due to treatment- related changes.4,5 In a consensus article recently published by Indicates open access to non-subscribers at www.ajnr.org http://dx.doi.org/10.3174/ajnr.A6688 1592 Toh Sep 2020 www.ajnr.org 1592 the Society for Neuro-Oncology and the European Association of Neuro-Oncology,6 the limited accuracy of increased contrast enhancement as an indicator of MT in LGGs has also been recog- nized. Therefore, using increased contrast enhancement as a marker of MT may result in overtreatment of patients whose tumors remain low-grade, errors in the results of research studies, and misinterpretation of clinical benefits of new therapies. In light of the limited implications of increased contrast enhance- ment, it is crucial to search for imaging markers that can diagnose MT accurately. operation. Patients with multiple operations for separate instan- ces of increased contrast enhancement were included as long as they had not already experienced MT and each event of increased contrast enhancement was treated independently. Histopathologic diagnosis was made by a board-certified neu- ropathologist according to the 2000 WHO classification of CNS tumors before 2007 and thereafter based on the 2007 WHO clas- sification. Patients younger than 18 years of age at initial diagno- sis or with a diagnosis of radiation necrosis due to increased contrast enhancement were excluded. Patients were also excluded if their MR spectroscopy showed poor spectral resolution or inclusion of normal-appearing brain or edema in the VOIs. Conventional MR imaging sequences such as T1WI, T2WI, FLAIR, and postcontrast T1WI provide only anatomic informa- tion. Patients This retrospective study was performed after institutional review board approval (Chang Gung Memorial Hospital). Patients were retrospectively selected from the brain tumor database of our institution if they met all the following criteria: 1) prior patho- logic diagnosis of LGG with follow-up MR imaging studies per- formed between 2004 and 2015 showing increased contrast enhancement; 2) having undergone surgery due to increased con- trast enhancement with a pathologic diagnosis of grade II, III, or IV glioma; 3) availability of MR spectroscopy from brain regions with increased contrast enhancement; and 4) confirmed removal of the brain regions with increased contrast enhancement on fol- low-up MR imaging. ABSTRACT In contrast, proton MR spectroscopy (MRS) offers unique metabolic information of brain tumor biology and enables quan- titative assessment of tissue metabolites such as Cho (a marker of cellular membrane turnover), Cr (a marker of energetic systems and intracellular metabolism), NAA (a marker of neuronal and axonal viability and density), lactate (a marker of anaerobic me- tabolism), and lipids (a marker of cellular breakdown or necro- sis).7 MR spectroscopy is used as an adjunct to conventional MR imaging because it helps in differentiating brain tumor from other lesions, identifies optimal biopsy sites in heterogeneous gliomas, monitors treatment response, and differentiates treat- ment-related changes and tumor recurrence.8 Clinical and Imaging Information Patient medical records were retrospectively reviewed to collect clinical information including sex, age and Karnofsky Performance Scale score at the time of increased contrast enhancement, histo- logic subtypes (diffuse astrocytoma, oligoastrocytoma, or oligoden- droglioma), isocitrate dehydrogenase 1 (IDH-1) mutation status, disease duration (time interval between first histopathologic diag- nosis of LGG and increased contrast enhancement), adjuvant ther- apy received before increased contrast enhancement, post-radiation therapy duration (time interval between end of radiation therapy and increased contrast enhancement), and tumor grades associated with increased contrast enhancement. Previous studies have demonstrated the utility of MR spec- troscopy in predicting or prognosticating MT and survival of those with LGGs.9-15 To the best of our knowledge, the role of MR spectroscopy in diagnosing MT among LGGs with increased contrast enhancement has not been investigated. In the present study, using histopathology as the criterion standard, we aimed to determine the value of MR spectroscopy to diagnose MT in a group of patients who had a prior diagnosis of LGG and an increase in contrast enhancement on follow-up MR imaging. The first follow-up MR imaging studies after the last opera- tion were reviewed for baseline residual tumor. MR spectroscopy metabolites measured in brain regions with increased contrast enhancement were recorded. The size of the contrast enhance- ment was measured by determining the product of the largest di- ameter and its perpendicular length on single postcontrast transverse T1-weighted or MPRAGE images. The size of whole tumor, which included both enhancing and nonenhancing com- ponents, was measured on transverse FLAIR or T2-weighted images. If multiple lesions were present, the largest 3 were selected and their products were summed. Follow-up studies, ei- ther intraoperative or postoperative MR imaging, were reviewed to confirm removal of brain regions with increased contrast enhancement. All imaging reviews were performed by 2 neurora- diologists. Differences were resolved by consensus. AJNR Am J Neuroradiol 41:1592–98 Sep 2020 www.ajnr.org Conventional MR Imaging and MR Spectroscopy Protocols Table 1: Clinical and imaging data of patients with low-grade gliomas demonstrating increased contrast enhancement thickness, 1 mm; matrix, 256 256; and FOV, 256 224 mm) with intravenous administration of 0.1 mmol per kilogram of body weight gadopentetate dimeglumine (Magnevist; Bayer HealthCare Pharmaceuticals). Table 1: Clinical and imaging data of patients with low-grade gliomas demonstrating increased contrast enhancement Clinical Information No. of Patients Sex Female 33 (34.4%) Male 63 (65.6%) Age range (yr) 20–39 35 (36.5%) 40–59 47 (49.0%) Older than 60 14 (14.5%) KPS $90 74 (77.1%) #80 22 (22.9%) Histologic subtype Diffuse astrocytoma 30 (31.3%) Oligoastrocytoma 27 (28.1%) Oligodendroglioma 39 (40.6%) IDH1 mutation Wild-type 7 (7.3%) Mutant 61 (63.5%) Not available 28 (29.2%) Baseline residual tumor Yes 81 (84.4%) No 15 (15.6%) Disease duration ,2 years 10 (10.4%) 2–5 years 35 (36.5%) 5–10 years 25 (26.0%) .10 years 26 (27.1%) Adjuvant therapy RT 75 (78.1%) Carmustine implant 9 (9.4%) Temozolomide 16 (16.7%) Post-RT duration ,6 months 2 (2.7%) 6–12 months 5 (6.7%) 12–24 months 9 (12%) .24 months 59 (78.6%) Whole tumor size ,4 cm2 17 (17.7%) 4–16 cm2 45 (46.9%) .16 cm2 34 (35.4%) Contrast enhancement size ,4 cm2 37 (38.5%) 4–16 cm2 36 (37.5%) .16 cm2 23 (24.0%) Tumor grade II 42 (43.8%) III 27 (28.1% IV 27 (28.1%) Note:—RT indicates radiation therapy; KPS, Karnofsky Performance Scale. To better guide VOI placement, we performed all MR spectros- copy studies after contrast medium administration. Postcontrast T1WI or MPRAGE in axial, coronal, and sagittal planes was used to define VOIs. The location of the VOIs was carefully chosen by an on-site neuroradiologist to include as much of the enhancing regions as possible and avoid inclusion of necrosis, cysts, hemor- rhage, edema, calcifications, and normal-appearing brain. Single- voxel MR spectroscopy studies were performed using a point- resolved spectroscopy sequence with automated shimming and water suppression. For MR spectroscopy performed on 3T machines, the acquisition parameters were the following: TR/TE, 2000/135ms; flip angle, 90°; voxel size, 15 15 15mm3; average, 128. For MR spectroscopy performed on a 1.5T machine, the ac- quisition parameters were the following: TR/TE, 1500/135ms; flip angle, 90°; voxel size, 16 18 20mm3; average, 192. The levels of metabolites in the VOIs were determined using the workstation software (Leonardo; Siemens). Conventional MR Imaging and MR Spectroscopy Protocols Peak integrals were obtained using a Levenberg-Marquardt fitting routine for principal peaks includ- ing lactate, NAA, Cr, and Cho at 1.33, 2.02, 3.02, and 3.22ppm, respectively. The peak-area metabolite ratios Cho/Cr, NAA/Cr, NAA/Cho, and lactate/Cr were calculated. Because NAA was not detectable in some patients, the Cho/NAA ratio was not calculated. Statistical Analysis y Clinical and imaging factors thought to be associated with MT were analyzed by univariate analysis. Categorical variables were tested with x 2 analysis, and continuous variables were tested with the Student t test. Selected variables with P values, .05 by univar- iate analysis were subjected to multivariate analysis using logistic regression with a forward stepwise selection procedure. Odds ratios and 95% CIs were calculated to demonstrate the relative risk of each significant factor for MT. Areas under the curve and cutoff values of statistically significant variables were determined by receiver operating characteristic curve analysis. Cutoff values with the highest sensitivity and lowest false-positive rates were chosen for the calculation of sensitivity, specificity, and accuracy of each significant variable. A commercially available statistical software package (SPSS 22; IBM) was used for analysis, and P val- ues , .05 were considered a statistically significant difference. Note:—RT indicates radiation therapy; KPS, Karnofsky Performance Scale. Conventional MR Imaging and MR Spectroscopy Protocols Preoperative MR imaging and MR spectroscopy studies were per- formed on a single occasion using 1.5T (Magnetom Espree; Siemens) or 3T (Magnetom Tim Trio; Siemens) clinical MR imaging scanners. Intraoperative MR imaging studies were per- formed with a 1.5T unit (Magnetom Espree). Typical pulse sequences used included transverse T1WI (TR/TE, 250/2.46 ms; section thickness, 4 mm; gap, 1 mm; matrix, 256 256; and FOV, 220 220mm), transverse T2WI (TR/TE, 4000/90 ms; section thickness, 4 mm; gap, 1; flip angle, 120°; echo-train length, 17; matrix, 512 358; and FOV, 220 220 mm), transverse and cor- onal FLAIR (TR/TE/TI, 8200/85/2500ms; section thickness, 4 mm; gap, 1 mm; matrix, 320 256; and FOV, 220 220 mm), postcontrast T1WI in transverse, coronal, and sagittal planes as well as 3D-MPRAGE (TR/TE/TI, 2530/4.03/1100 ms; section Increased contrast enhancement was defined as emergence of new contrast enhancement in previously nonenhancing tumors or regions thereof, new separate lesions with contrast enhance- ment, or at least a 25% increase in the size of enhancement for tumors with enhancement at baseline. Baseline referred to the first follow-up MR imaging after the last operation. Increased contrast enhancement was confirmed by neuroradiologists who compared the baseline MR imaging and the one before the next 1593 Table 1: Clinical and imaging data of patients with low-grade gliomas demonstrating increased contrast enhancement Clinical Information No. of Patients Sex Female 33 (34.4%) Male 63 (65.6%) Age range (yr) 20–39 35 (36.5%) 40–59 47 (49.0%) Older than 60 14 (14.5%) KPS $90 74 (77.1%) #80 22 (22.9%) Histologic subtype Diffuse astrocytoma 30 (31.3%) Oligoastrocytoma 27 (28.1%) Oligodendroglioma 39 (40.6%) IDH1 mutation Wild-type 7 (7.3%) Mutant 61 (63.5%) Not available 28 (29.2%) Baseline residual tumor Yes 81 (84.4%) No 15 (15.6%) Disease duration ,2 years 10 (10.4%) 2–5 years 35 (36.5%) 5–10 years 25 (26.0%) .10 years 26 (27.1%) Adjuvant therapy RT 75 (78.1%) Carmustine implant 9 (9.4%) Temozolomide 16 (16.7%) Post-RT duration ,6 months 2 (2.7%) 6–12 months 5 (6.7%) 12–24 months 9 (12%) .24 months 59 (78.6%) Whole tumor size ,4 cm2 17 (17.7%) 4–16 cm2 45 (46.9%) .16 cm2 34 (35.4%) Contrast enhancement size ,4 cm2 37 (38.5%) 4–16 cm2 36 (37.5%) .16 cm2 23 (24.0%) Tumor grade II 42 (43.8%) III 27 (28.1% IV 27 (28.1%) Note:—RT indicates radiation therapy; KPS, Karnofsky Performance Scale. RESULTS Between 2004 and 2015, a total of 222 patients with LGGs were regularly followed up in our institution after initial diagnosis and treatment. On follow-up MR imaging studies, 139 patients had 161 instances of increased contrast enhancement and proceeded to an operation. Six instances with a pathologic diagnosis of radi- ation necrosis were excluded. For the rest of the 155 instances, the pathologic diagnosis was grade II diffuse glioma (n = 59), grade III (n = 53), and grade IV (n = 43). MR spectroscopy was available in 47 of 57 grade II, 34 of 52 grade III, and 29 of 43 grade IV gliomas. Among the 110 MR spectroscopy studies, 14 were excluded due to poor spectral resolution (2 grade II, 5 grade III, and 2 grade IV), inclusion of normal brain in VOIs (1 grade II and 1 grade III), or brain regions with increased contrast enhancement that were not removed (2 grade II and 1 grade III). Successful removal of brain regions with increased contrast enhancement was confirmed in 33 instances with intraoperative MR imaging, in 30 with postoperative MR imaging performed within 1 week, and in 33 with postoperative MR imaging per- formed between 2 and 12 weeks. The final study population con- sisted of 86 patients with 96 MR spectroscopy studies (42 grade II, 27 grade III, and 27 grade IV). Seventy-five MR spectroscopy studies were performed on 3T MR imaging, and 21, on 1.5T. Clinical and imaging data are shown in Table 1. Among these 96 instances, 63 (65.6%) occurred in male patients. RESULTS The mean age Toh Sep 2020 www.ajnr.org 1594 Table 2: Univariate analysis of factors associated with malignant transformationa Table 2: Univariate analysis of factors associated with malignant transformationa Factors Malignant Transformation P Value OR 95% CI No Yes Sex .85 0.9 0.39–2.15 Male 28 35 Female 14 19 Age (yr) 49.1 6 12.3 44.2 6 12.6 .058 NA –0.17–10.1 KPS 88.1 6 11.9 90.7 6 8.2 .202 NA –6.73–1.44 Histologic subtype .604 NA NA Diffuse astrocytoma 12 18 Oligoastrocytoma 14 13 Oligodendroglioma 16 23 IDH1 mutation .525 1.7 0.31–9.67 Yes 25 36 No 2 5 Mean disease duration (yr) 6.9 6 5.1 7.1 6 5.4 .818 NA –2.42–1.92 Baseline residual tumor .044 3.7 0.97–14.15 Yes 39 42 No 3 12 RT .037 3.1 1.03–9.37 Yes 37 38 No 5 16 Post-RT duration (mo) 59.5 6 48.7 75.6 6 54.5 .182 NA –39.9–7.71 Chemotherapy .800 NA NA None 32 39 Carmustine implant 3 6 Temozolomide 7 9 Whole-tumor size (cm2) 10.8 6 11.1 17.1 6 11.1 .007 NA –10.8 to 1.7 Contrast-enhancement size (cm2) 6.1 6 6.6 12.3 6 11.1 .001 NA –10.7 to 2.8 Cho/Cr 3.31 6 4.26 5.60 6 4.61 .015 NA –4.1 to 0.5 NAA/Cho 0.41 6 0.28 0.10 6 0.07 ,.001 NA 0.22–0.38 NAA/Cr 1.02 6 1.19 0.58 6 0.78 .033 NA 0.04–0.84 Lac/Cr 0.43 6 1.41 0.77 6 1.62 .285 NA –0.96–0.28 Note:—NA indicates not applicable; Lac, lactate; RT, radiation therapy; KPS, Karnofsky Performance Scale. a Data are mean 6 SD for age, KPS, disease duration, post-RT duration, whole-tumor size, contrast enhancement size, and MR spectroscopy metabolite ratios. Note:—NA indicates not applicable; Lac, lactate; RT, radiation therapy; KPS, Karnofsky Performance Scale. a Data are mean 6 SD for age, KPS, disease duration, post-RT duration, whole-tumor size, contrast enhance Note:—NA indicates not applicable; Lac, lactate; RT, radiation therapy; KPS, Karnofsky Performance Scale. a Data are mean 6 SD for age, KPS, disease duration, post-RT duration, whole-tumor size, contrast enhancement size, and MR spectroscop among LGGs with increased contrast enhancement. On receiver operating characteristic curve analysis (Fig 3), the discriminative power of NAA/Cho measured with an area under the curve was 0.922 (P , .001; 95% CI, 0.86–0.98). With 0.222 as the cutoff value, the sensitivity, specificity, and accuracy of NAA/Cho in diagnosing MT were 94.4%, 83.3%, and 89.6%, respectively. RESULTS and Karnofsky Performance Scale scores at the time of increased contrast enhancement were 46.3years (range, 20–83) and 89.5 (range, 50–100), respectively. Initial histologic subtypes were dif- fuse astrocytoma (n¼ 30), oligoastrocytoma (n¼ 27), and oligo- dendroglioma (n¼ 39). Eighty-one instances had baseline residual tumors. IDH1 mutation status was available in 68 (70.8%) instan- ces with 61 (89.7%) being IDH1-mutant. Median disease duration was 5.2years for those remaining with grade II, and 5.1years for those with MT. Adjuvant therapy administered before an increase of contrast enhancement included radiation therapy (n¼ 75), Carmustine implants (n¼ 9), and temozolomide (n¼ 16). Median post-radiation therapy duration was 53.5months (range, 3.5–206 months). NAA was not detectable in 3 instances of MT (1 grade III and 2 grade IV). Lactate was present in 14 (33.3%) instances that remained grade II and 21 (38.9%) of MT. AJNR Am J Neuroradiol 41:1592–98 Sep 2020 www.ajnr.org DISCUSSION Our study showed that among patients with a prior diagnosis of LGG, increased contrast enhancement on MR imaging can be seen in LGGs that remained grade II or those that underwent MT. NAA/Cho ratios measured in regions with increased con- trast enhancement were significantly lower in LGGs with MT compared with those that remained grade II. Thus, the NAA/ Cho ratio may diagnose MT among LGGs with increased con- trast enhancement. Table 2 illustrates the results of univariate analysis in which baseline residual tumor (P =.044) and previous adjuvant radiation therapy (P¼ .037) were associated with MT. LGGs with MT had significantly larger whole-tumor size (P¼ .007), larger contrast- enhancement size (P ¼ .001), higher Cho/Cr ratio (P¼ .015), lower NAA/Cho ratio (P, .001), and lower NAA/Cr ratio (P¼ .033) compared with those without MT. Figures 1 and 2 illustrate meas- urements of metabolite levels in a LGG preserving grade II histol- ogy and an LGG with MT, respectively. The median time to MT in the present study was 5.1 years, which correlates well with previous reports in which it ranged from 2.7 to 5.4 years.16,17 Previously identified prognostic factors for MT include older age, male sex, multiple tumor locations, tu- mor size$5 cm, adjuvant temozolomide, presence of residual tu- mor, astrocytoma histology, and IDH wild-type.16-18 However, in our study, none of these factors were helpful in making the diag- nosis of MT. We speculate that this discrepancy could be due to different criteria for MT. In our study, MT was diagnosed with On multivariate analysis, the NAA/Cho ratio (P , .001; OR, 7.1, 95% CI, 3.2–16.1) was the only variable predictive of MT 1595 subsequent therapy. However, an operation may not always be possi- ble; therefore, noninvasive imaging markers play an important role in patient care. In our study, NAA/Cho was found to be diagnostic of MT and showed similar sensitivity (94.4%) but a higher specificity (83.3%). FIG 1. Measurement of metabolite levels using single-voxel MR spectroscopy in a low-grade gli- oma with increased contrast enhancement but preserved grade II histology. Transverse contrast- enhanced T1WI (left) shows an enhancing nodule in the right frontal lobe, posterior to the surgical cavity. A 15 15 15cm3 VOI was placed over the enhancing brain region. Spectrum (right) obtained with TE= 135 ms shows an increase in the Cho peak and a decrease in the NAA peak. DISCUSSION The NAA/Cho ratio is 0.41. Previous studies have tried to predict the outcome of LGG, particularly in the early course of the disease. Studies show that MR spectroscopy performed at baseline9,10 or at recurrence11,12 may predict progression-free survival and MT of LGG. MR spectroscopy may dif- ferentiate clinically stable LGGs from those that progress as a result of MT.14,15 The NAA/Cho ratio was found to be the best MR spectroscopy marker for LGG progression, with a sensitivity of 53.9%.13 The Cho/NAA ratio was shown to reliably differentiate recurrent glioma from post-radiation injury.4,5 Those studies demonstrated the poten- tial of MR spectroscopy in prognosticat- ing or predicting survival of those with LGG. However, their diagnostic and therapeutic impact on patient care was limited due to inconsistent MR spec- troscopy techniques and variable meth- ods of choosing VOI locations, and most important, many MT cases were not histologically proved. In our study, we investigated the diagnostic per- formance at the time of increased contrast enhancement. With histology as the criterion standard, we found that MR spectroscopy could diagnose MT with high diagnostic performance (area under the curve ¼ 0.924). FIG 1. Measurement of metabolite levels using single-voxel MR spectroscopy in a low-grade gli- oma with increased contrast enhancement but preserved grade II histology. Transverse contrast- enhanced T1WI (left) shows an enhancing nodule in the right frontal lobe, posterior to the surgical cavity. A 15 15 15cm3 VOI was placed over the enhancing brain region. Spectrum (right) obtained with TE= 135 ms shows an increase in the Cho peak and a decrease in the NAA peak. The NAA/Cho ratio is 0.41. FIG 2. Measurement of metabolite levels using single-voxel MR spectroscopy in a low-grade gli- oma with increased contrast enhancement and malignant transformation. Transverse contrast- enhanced T1WI (left) shows an enhancing nodule in the right medial frontal lobe, posterior to the surgical cavity. A 15 15 15cm3 VOI was placed over the enhancing brain region. Spectrum (right) obtained with TE= 135 ms shows an increase in the Cho peak and a decrease in the NAA peak. The NAA/Cho ratio is 0.20. FIG 2. Measurement of metabolite levels using single-voxel MR spectroscopy in a low-grade gli- oma with increased contrast enhancement and malignant transformation. Transverse contrast- enhanced T1WI (left) shows an enhancing nodule in the right medial frontal lobe, posterior to the surgical cavity. Toh Sep 2020 www.ajnr.org REFERENCES 1. Ostrom QT, Cioffi G, Gittleman H, et al. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2012-2016. Neuro Oncol 2019;21: v1–100 CrossRef Medline 2. van den Bent MJ, Wefel JS, Schiff D, et al. Response Assessment in Neuro-Oncology (a report of the RANO group): assessment of out- come in trials of diffuse low-grade gliomas. Lancet Oncol 2011;12:583– 93 CrossRef 3. Narang AK, Chaichana KL, Weingart JD, et al. Progressive low- grade glioma: assessment of prognostic importance of histologic reassessment and MRI findings. World Neurosurg 2017;99:751–57 CrossRef Medline FIG 3. Receiver operating characteristic curve analysis of the diagnos- tic performance of the NAA/Cho ratio in differentiating LGG pre- serving grade II histology and LGG with MT. 4. Fink JR, Carr RB, Matsusue E, et al. Comparison of 3 Tesla proton MR spectroscopy, MR perfusion and MR diffusion for distinguish- ing glioma recurrence from posttreatment effects. J Magn Reson Imaging 2012;35:56–63 CrossRef Medline the VOI location, and thus improves the reproducibility of metab- olite measurements. Finally, locations of VOIs were decided by an on-site neuroradiologist, possibly helping to achieve good spectral quality. 5. Matsusue E, Fink JR, Rockhill JK, et al. Distinction between glioma progression and post-radiation change by combined physiologic MR imaging. Neuroradiology 2010;52:297–306 CrossRef Medline 6. Schiff D, van den Bent M, Vogelbaum MA, et al. Recent develop- ments and future directions in adult lower-grade gliomas: Society for Neuro-Oncology (SNO) and European Association of Neuro-Oncology (EANO) Consensus. Neuro Oncol 2019;21:837– 53 CrossRef Medline quality. In this study, patients with LGGs with increased contrast enhancement who did not proceed to an operation were excluded. Initially, this exclusion may appear as selection bias, but given the low specificity of increased contrast enhancement, this seeming limitation is actually the strength of our study. LGGs that progressed with only enlargement of nonenhancing tumors were not included in this study because they need different methods for choosing VOI location, and the MT rate for this group was 23%, much lower than the 82% for those with increased contrast enhancement.3 Although we did not perform image-guided tissue sampling, we reviewed the intraoperative or postoperative MR imaging studies to confirm removal of brain regions with increased contrast enhancement. This step decreases the chances of tumor grade misclassification. DISCUSSION A 15 15 15cm3 VOI was placed over the enhancing brain region. Spectrum (right) obtained with TE= 135 ms shows an increase in the Cho peak and a decrease in the NAA peak. The NAA/Cho ratio is 0.20. We found that MR spectroscopy was useful for diagnosing MT. There were several reasons for this finding, including the use of a single-voxel tech- nique. Precise measurements of metab- olite levels in brain regions with increased contrast enhancement were essential for differentiation between LGGs with preserved grade II histology and those with MT. Therefore, spectral contami- nation by adjacent normal brain, edema, and fat should be mini- mized because it could introduce significant measurement errors. Compared with multivoxel MR spectroscopy, single-voxel MR spectroscopy has a shorter acquisition time, better shimming, a higher signal-to-noise ratio, and better spectral quality. Moreover, it is less susceptible to contaminations from adjacent tissues. In our study, MR spectroscopy was performed after administration of a gadolinium contrast medium because increased contrast enhance- ment better guides VOI placement, allows consistency in choosing tal lobe, posterior to the brain region. Spectrum a decrease in the NAA histopathology, while in other studies, a significant proportion of MT cases were diagnosed with only MR imaging. Increased contrast enhancement has been used as an indicator of MT in several clinical guidelines for LGG.2,19,20 However, our results do not support this observation. In the present study, the percentage of LGGs that remained grade II despite having increased contrast enhancement was 37% (59 of 161), higher than the previously reported 18%.3 In a recent study, the sensitiv- ity and specificity of increased contrast enhancement for diagnos- ing MT were 92% and 57%, respectively.3 Because of the low specificity, the authors suggested that histopathologic confirmation at the time of imaging progression should be attempted to guide Toh Sep 2020 www.ajnr.org 1596 FIG 3. Receiver operating characteristic curve analysis of the diagnos- tic performance of the NAA/Cho ratio in differentiating LGG pre- serving grade II histology and LGG with MT. CONCLUSIONS MR spectroscopy complements conventional MR imaging in the diagnosis of malignant transformation in a subgroup of low- grade gliomas with increased contrast enhancement. MR spec- troscopy may reduce the need for an operation and help in clini- cal decision-making for low-grade gliomas with increased contrast enhancement. Disclosures: Cheng Hong Toh—RELATED: Grant: National Science Council Taiwan, Comments: research grant.* Money paid to institution. Disclosures: Cheng Hong Toh—RELATED: Grant: National Science Council Taiwan, Comments: research grant. *Money paid to institution. REFERENCES A trend toward lower myo-inositol levels in high-grade gliomas compared with low-grade gliomas was reported.21 Further studies using short-TE MR spectroscopy are needed to determine the utility of myo-inositol in diag- nosing MT because our MR spectroscopy spectra obtained at a TE of 135 ms were not useful in demonstrating this metabo- lite. In the present study, NAA levels of LGGs with MT were lower than those that remained grade II. In some LGGs with MT, NAA was even undetectable. Therefore, NAA/Cho rather than Cho/NAA ratios were calculated. We speculate that a lower level or absence of NAA, which reflects more severe neuronal destruction, may be the principal metabolite change in MT. Further studies are needed to support our observations. 7. Bertholdo D, Watcharakorn A, Castillo M. Brain proton magnetic resonance spectroscopy: introduction and overview. Neuroimaging Clin N Am 2013;23:359–80 CrossRef Medline 8. Oz G, , Alger JR, Barker PB, et al; MRS Consensus Group. Clinical proton MR spectroscopy in central nervous system disorders. Radiology 2014;270:658–79 CrossRef Medline 9. Hattingen E, Raab P, Franz K, et al. Prognostic value of choline and creatine in WHO grade II gliomas. Neuroradiology 2008;50:759–67 CrossRef Medline 10. Bourdillon P, Hlaihel C, Guyotat J, et al. Prediction of anaplastic transformation in low-grade oligodendrogliomas based on mag- netic resonance spectroscopy and 1p/19q codeletion status. J Neurooncol 2015;122:529–37 CrossRef Medline 11. Jalbert LE, Neill E, Phillips JJ, et al. Magnetic resonance analysis of malig- nant transformation in recurrent glioma. Neuro Oncol 2016;18:1169–79 CrossRef Medline 12. Neill E, Luks T, Dayal M, et al. Quantitative multi-modal MR imaging as a non-invasive prognostic tool for patients with recur- rent low-grade glioma. J Neurooncol 2017;132:171–79 CrossRef Medline 13. Alimenti A, Delavelle J, Lazeyras F, et al. Monovoxel 1H magnetic resonance spectroscopy in the progression of gliomas. Eur Neurol 2007;58:198–209 CrossRef Medline 14. Tedeschi G, Lundbom N, Raman R, et al. Increased choline signal coinciding with malignant degeneration of cerebral gliomas: a se- rial proton magnetic resonance spectroscopy imaging study. J Neurosurg 1997;87:516–24 CrossRef Medline 15. Bobek-Billewicz B, Stasik-Pres G, Hebda A, et al. Anaplastic transformation of low-grade gliomas (WHO II) on magnetic AJNR Am J Neuroradiol 41:1592–98 Sep 2020 www.ajnr.org 159 19. Soffietti R, Baumert BG, Bello L, et al; European Federation of Neurological Societies. Guidelines on management of low-grade glio- mas: report of an EFNS-EANO Task Force. Eur J Neurol 2010;17:1124– 33 CrossRef Medline resonance imaging. 8 Toh Sep 2020 www.ajnr.org REFERENCES Folia Neuropathol 2014;52:128–40 CrossRef Medline resonance imaging. Folia Neuropathol 2014;52:128–40 CrossRef Medline 16. Murphy ES, Leyrer CM, Parsons M, et al. Risk factors for malignant transformation of low-grade glioma. Int J Radiat Oncol Biol Phys 2018;100:965–71 CrossRef Medline 20. Sepulveda-Sanchez JM, Muñoz Langa J, Arráez MA´, et al. SEOM clinical guideline of diagnosis and management of low-grade glioma. Clin Transl Oncol 2018 2017;20:3–15 CrossRef Medline 17. Tom MC, Park DYJ, Yang K, et al. Malignant transformation of molecularly classified adult low-grade glioma. Int J Radiat Oncol Biol Phys 2019;105:1106–12 CrossRef Medline y 18. Chaichana KL, McGirt MJ, Laterra J, et al. Recurrence and malig- nant degeneration after resection of adult hemispheric low-grade gliomas. J Neurosurg 2010;112:10–17 CrossRef Medline 21. Castillo M, Smith JK, Kwock L. Correlation of myo-inositol levels and grading of cerebral astrocytomas. AJNR Am J Neuroradiol 2000;21:1645–49 Toh Sep 2020 www.ajnr.org 1598
https://openalex.org/W2892232694	https://europepmc.org/articles/pmc6147945?pdf=render	English	null	Meconium microbiome associates with the development of neonatal jaundice	Clinical and translational gastroenterology	2,018	cc-by	6,490	© The Author(s) 2018 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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/. A R T I C L E O p e n A c c e s s Abstract Objective: Neonatal jaundice is a common disease that affects up to 60% of newborns. Gut microbiota mediated the excretion of bilirubin from the human body. However, the relationship between early gut microbiome and development of neonatal jaundice is not fully understood. Here we sought to characterize meconium microbiome of newborns and to clarify its association with risk of neonatal jaundice. Methods: We conducted a nested case–control study with 301 newborns providing meconium samples from 2014 to 2015. The main outcome was the development of neonatal jaundice at 42 day follow-up. 16S rRNA gene sequencing was performed to proﬁle the meconium microbiome. LEfSe was employed to identify different features between control and case groups. Logistic regression was used to estimate the risk effect of early gut microbiome on neonatal jaundice. Results: Logistic regression models suggested that higher ɑ-diversity was signiﬁcantly associated with lower risk of jaundice in cesarean infants (OR 0.72, 95% CI 0.52–0.98), but not in infants born naturally. Higher relative abundance of Biﬁdobacterium pseudolongum in newborn meconium was signiﬁcantly associated with lower risk of jaundice both in cesarean-born infants and in the total subjects (OR 0.24, 95% CI 0.07–0.68; OR 0.55, 95% CI 0.31–0.95, respectively). Spearman’s correlations showed that relative abundance of B. pseudolongum was signiﬁcantly correlated with ɑ-diversity (P < 0.01). Conclusion: Preventive and treatment methods implying early gut microbiome intervention could be promising for the management of neonatal jaundice. Meconium microbiome associates with the development of neonatal jaundice Tianyu Dong, PhD1,2, Ting Chen, PhD3, Richard Allen WhiteIII, PhD4, Xu Wang, PhD1,2, Weiyue Hu, PhD1,2, Yali Liang, MS5, Yuqing Zhang, PhD1,2, Chuncheng Lu, PhD1,2, Minjian Chen, PhD1,2, Heidi Aase, PhD6 and Yankai Xia, PhD1,2 Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 DOI 10.1038/s41424-018-0048-x Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 DOI 10.1038/s41424-018-0048-x Clinical and Translational Gastroenterology Ofﬁcial journal of the American College of Gastroenterology Introduction disease2, which can be distinguished from pathologic jaundice. Physiologic jaundice does not cause serious problems in newborns, but studies have linked it to adverse health outcomes in later life, such as childhood asthma, type 1 diabetes, and impaired visual function3–5. A large population-based longitudinal study in Denmark highlighted the risk for infants with neonatal jaundice (excluding pathologic jaundice resulting from hemolytic diseases) to develop autism and other psychological developmental disorders6, which raises public concerns about the long-term health effects caused by physiologic jaundice. Neonatal jaundice is a common disease that affects up to 60% of newborns1. It is deﬁned as the condition of high total serum bilirubin levels and usually developed in the ﬁrst week of life. Most of the instances of neonatal jaun- dice are considered physiologic without underlying Correspondence: Yankai Xia (yankaixia@njmu.edu.cn) 1State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 211166, China 2Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China Full list of author information is available at the end of the article. These authors contributed equally: Tianyu Dong and Ting Chen Ofﬁcial journal of the American College of Gastroenterology Page 2 of 9 Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 The pathogenesis for neonatal physiologic jaundice can be attributed to the nature for newborns to produce more bilirubin and their limited ability to excrete it2. In the biological process of bilirubin metabolism, gut bacteria has an important role in mediating the transformation of conjugated bilirubin to unconjugated bilirubin7, and then unconjugated bilirubin is further turned into bilinogen to be excreted out of the body. Previous studies have documented the association between early gut micro- biome and child health8–10. Although it is recognized that gut bacteria is essential for bilirubin metabolism, the role of meconium microbiome on the development of phy- siologic jaundice has not been fully clariﬁed. In addition, other known risk factors for neonatal jaundice, including cesarean section and breastfeeding, can inﬂuence the shaping of early gut microbiome11–13. Taking these fac- tors into consideration would help us better understand the relationship between gut microbiome and jaundice. Outcome Measurements and Covariate Data Collection Outcome Measurements and Covariate Data Collection The deﬁnition of neonatal jaundice was based on par- ental answers to the question “Has your child ever been diagnosed with neonatal jaundice by a doctor” in the questionnaire ﬁnished at the 42 days’ interview. If the answer was ‘yes,’ parents were asked to provide detailed information about time of diagnosis, duration, types of jaundice, medication, and hospitalization, in order to conﬁrm physiologic jaundice. Information about feeding pattern and infant use of antibiotics were also obtained at that time. Clinical data from pregnancy and delivery were obtained from the hospital database. Premature rupture of membranes and gestational diabetes mellitus were reported to affect maternal and fetal gut microbiome14,15. Subclinical hypothyroidism in pregnancy was commonly found and involved autoimmunity16,17, which might inﬂuence gut microbiota (Table 1). Thus, these three factors were taken into consideration. Other covariates included maternal characteristics recorded in ques- tionnaires at each interview: smoking and alcohol use during pregnancy, antibiotics use during pregnancy, reproductive history, and child feeding pattern. Here we conducted a nested case–control study with a total of 301 newborns to illuminate the association between meconium microbiome and subsequent diag- nosis of neonatal jaundice. Stratiﬁcation analysis based on delivery mode and feeding pattern was also included in the study. The aim of our study was to clarify the role of early gut microbiome in the development of neonatal jaundice, thereby providing a knowledge base for potential preventive and treatment measures relevant to early gut microbiome interventions. Experimental Design This work was approved by the Ethical Committee of Nanjing Medical University before the study. The study population was drawn from the NMU Mother and Child Cohort Study (Phase II), a prospective birth cohort designed to study prenatal risk factors on pregnancy and child health outcomes. Participants in the present study were mother–infant pairs recruited in early pregnancy from afﬁliated hospitals with Nanjing Medical University between February 2014 and November 2015. The exclu- sion criteria included the following: 1. Age < 20 or > 45 years old; 2. Unwilling to register or deliver in these core hospitals; 3. No intention of long-term residence in the research area during pregnancy. All participants were regularly followed up at each trimester and at 42 days after delivery. Signed informed consent was obtained from the mothers at enrollment. The whole study was con- ducted in accordance with the Declaration of Helsinki of the World Medical Association. Ofﬁcial journal of the American College of Gastroenterology Introduction of these, 12 resulted from assisted reproductive technol- ogy, 8 children had birth defects, 18 had neonatal diseases other than jaundice during the ﬁrst 42 days after birth, 57 did not provide meconium samples, 11 had incomplete clinical or questionnaire data, and 36 newborns had pathologic jaundice. Pathologic jaundice is mainly caused by underlying diseases such as hemolytic diseases and liver diseases, which were not the focus of this study. Using a nested case–control design, eligible cases were identiﬁed and then matched with healthy controls by centers. Finally, a total of 301 mother–infant pairs remained in the present study. Sample Collection and DNA Extraction Meconium samples were all ﬁrst feces samples, which were collected by well-trained nurses from diapers into sterile tubes within 12 h after birth. These samples were immediately stored at −80 °C until DNA extraction. Total DNA was extracted from meconium samples using the QIAamp Fast DNA Stool Mini Kit according to manufacturer’s instructions (Qiagen, Germany). All experiments were carried out on a sterile bench. DNA concentration and purity was determined by a NanoDrop 2000 (Thermo Fisher Scientiﬁc, Wilmington, DE). Microbiome Analysis Relative abundance of each bacterial level from phylum to genus was calculated in QIIME 1.9.1 64 bit. Shannon index was calculated to assess ɑ-diversity within samples. β-Diversity was visualized by PCoA (principal coordinates analysis) plot based on unweighted and weighted unifrac distance matrix. LEfSe (linear discriminant analysis effect size) was used to discover the features contributing to the most variation between control and case groups (LDA (linear discriminant analysis) > 2.5). Predicted metagenome of the 16S rRNA OTU data was obtained by PICRUSt and then was categorized by function according to KEGG Orthology. 16S rRNA Gene Sequencing and Quality Control 16S rRNA Gene Sequencing and Quality Control Hypervariable region V3 of the 16 S rRNA gene was ampliﬁed and then sequenced by HiSeq2500 PE250 plat- form in Novogene Bioinformatics Technology Co. Ltd (Beijing, China). After removing barcode and primer sequences, raw reads were merged using FLASH software Of the 542 pregnant women invited to the present study, 515 (95.02%) agreed to participate. During the follow-up, 72 pregnant women were lost, giving a follow- up rate of 86.02%. In the present study, 142 mother–infant pairs were removed from further analysis; Ofﬁcial journal of the American College of Gastroenterology Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Page 3 of 9 Page 3 of 9 Table 1 Characteristics of the study population Variablesa Controls Cases P-value (n = 160) (n = 141) Maternal age (year) 29.37 ± 3.91 29.48 ± 4.02 0.80 Center Nanjing 128 (80.0) 109 (77.3) 0.58 Suzhou 32 (20.0) 32 (22.7) Antibiotics to mother 4 (2.5) 7 (5.0) 0.36 Maternal smoking 8 (5.0) 2 (1.4) 0.11 Maternal alcohol consumption 7 (4.4) 6 (4.3) 1.00 Multipara 41 (25.6) 38 (27.0) 0.80 Premature rupture of membranes 35 (21.9) 39 (27.7) 0.28 Gestational diabetes mellitus 33 (20.6) 29 (20.6) 1.00 Subclinical hypothyroidism in pregnancy 34 (21.3) 21 (14.1) 0.18 Gestational age (day) 277.46 ± 6.46 274.79 ± 6.99 < 0.001* Gender Male 80 (50.0) 74 (52.5) 0.73 Female 80 (50.0) 67 (47.5) Delivery mode Vaginal 103 (64.4) 100 (70.9) 0.27 Cesarean 57 (35.6) 41 (29.1) Birth weight (g) 3488.63 ± 381.64 3351.99 ± 341.90 < 0.01 Apgar score 1 min 9.97 ± 0.21 9.90 ± 0.56 0.16 5 min 9.99 ± 0.08 9.99 ± 0.17 0.59 Feeding pattern Breast milk 87 (54.4) 90 (63.8) 0.02* Mixed 63 (39.4) 50 (35.5) Formula 10 (6.2) 1 (0.7) Antibiotics to newborn 1 (0.6) 3 (2.1) 0.34 aData was presented as mean ± SD or n (%). Boldface indicates statistical signiﬁcance (p < 0.05, p < 0.01, *p < 0.001). Table 1 Characteristics of the study population Statistical Analysis All statistical analyses were carried out using R v3.4.0. For clinical data and characteristics, comparison of con- tinuous univariate variables and ratios were performed by Student’s t-test, Fisher’s exact test, and χ2-test, respec- tively. For microbiome data, difference of Shannon index and relative abundance of Biﬁdobacterium pseudolongum was examined by Mann–Whitney test. Relative propor- tions of functional genes between control and case groups were compared by Welch’s t-test with corrected P-value in STAMP 2.1.3. Logistic regression models were employed for multivariable analysis and calculating odds ratio (OR) with corresponding 95% conﬁdence interval (CI) adjusting for potential confounders. As the relative abundance of deﬁned features by LEfSe were low (< 0.001) and outliers existed, these continuous variables were transformed into binary variables (low abundance and high abundance) based on the median of the total samples before including in the logistic regression models. Het- erogeneity test was performed by “meta” R package. Spearman’s correlation analysis between relative abun- dance and Shannon index was conducted using cor.test function in R software. A P-value < 0.05 was considered signiﬁcant. Characteristics of Study Population A total of 141 eligible infants with physiologic jaundice were identiﬁed in the cohort. The eligible infants were matched with 160 controls without diagnosis of jaundice or any other neonatal diseases in their ﬁrst month of life. The incidence of physiologic jaundice in the sample was 46.84%. Demographic characteristics of the infants and their mothers are shown in Table 1. Among all partici- pants, 11 mothers used antibiotics during pregnancy and 4 infants used antibiotics after birth. Through univariate analysis, we found that gestational age and birth weight of the cases were signiﬁcantly lower than those of the con- trols (P < 0.001 and P < 0.01, respectively). Feeding pat- terns also varied between the two groups (P = 0.02). No Data was presented as mean ± SD or n (%). Boldface indicates statistical signiﬁcance (p < 0.05, p < 0.01, p < 0.001). Alpha Diversity of Early Gut Microbiome and Risk of Neonatal Jaundice A logistic regression model was employed to assess the impact of ɑ-diversity of early gut microbiome on the development of physiologic jaundice. Shannon index was included in the model to represent ɑ-diversity of meco- nium microbiome. Higher birth weight, longer gestational age and formula feeding (compared with breastfeeding) were all associated with lower risk of physiologic jaundice after adjusting for covariates (OR 0.9992, 95% CI 0.9985–0.9999; OR 0.94, 95% CI 0.90–0.98; and OR 0.094, 95% CI 0.005–0.559, respectively) (Table 2). However, Shannon index was not associated with the outcome (OR 0.94, 95% CI 0.83–1.07) (Table 2). Finally, we conducted LEfSe to discover distinctive features at all levels. According to the threshold LDA > 2.5, ﬁve features were found to be signiﬁcantly different between controls and cases. Four features were more abundant in the control group (Pasteurellales, B. pseu- dolongum, Pasteurellaceae, and Clostridiaceae) and only one feature was more abundant in the case group (Cupriavidus) (Fig. 3). We further conducted stratiﬁcation analysis by delivery mode to control its potential impacts on microbiome. The results of the logistic regression analysis suggested that higher Shannon indices were signiﬁcantly associated with lower risk of physiologic jaundice after adjusting for potential confounders (OR 0.72, 95% CI 0.52–0.98) in infants delivered by cesarean section (Table 3). For vaginal delivered infants, no such association was observed (OR 0.98, 95% CI 0.83–1.15) (Table 3). Heterogeneity test indicated moderate heterogeneity between vaginal and cesarean subgroups (τ2 = 0.03, H = 1.68, I2 = 64.40%, P = 0.09) (Table 3). Differences of microbiome between controls and cases in cesarean infants y y The meconium microbiome was dominated by Firmi- cutes and Proteobacteria (Fig. 1a). Large variation in the microbiome constitution among individuals could be observed (Fig. 1a). β-Diversity of all the samples divided by case and control group was visualized in PCoA plot based on unweighted unifrac distance. However, samples from the two groups were not separated observably (Fig. 1b). Similar results were found in PCoA plot based on weighted unifrac distance (Supplementary Fig. 1a, 1b). We also compared ɑ-diversity using Shannon index and the difference was not signiﬁcant (P = 0.64) (Fig. 1c). As delivery mode was acknowledged as an important factor associated with newborn microbiome, we divided the samples into vaginal and cesarean groups. No sig- niﬁcant differences in ɑ-diversity or β-diversity were observed between the two groups either (P = 0.95) (Fig. 1d, e). It was an interesting ﬁnding that ɑ-diversity of the microbiome might be a protective factor against jaundice only in cesarean-born infants. Thus, we further compared the microbiome between controls and cases in the cesarean group. No signiﬁcant difference was observed in the incidence rates of the vaginal and the cesarean group (49.26% and 41.84%, respectively; P = 0.27). Stacking area map generated from proportion of bacteria at the phylum level showed similar results with that found in the total population (Fig. 2a). Large variations between individuals in abundancy of bacteria could be observed and differ- ences might also exist for the less abundant bacteria (Fig. 2a). No marked separation was found in PCoA plot based on unweighted unifrac distance (Fig. 2b). Similar results were also found in PCoA plot based on weighted unifrac distance (Supplementary Fig. 1c).The Shannon indices were lower in the cases compared with controls, but the P-value was not signiﬁcant (P = 0.20) (Fig. 2c). PICRUSt was used to predict metagenome of the 16S rRNA data followed by function categorizing. Compared with the control group, aldosterone-regulated sodium reabsorption and D-Glutamine and D-glutamate metabo- lism were both signiﬁcantly decreased (corrected P < 0.01 and P = 0.035, respectively) (Fig. 2d, e). (V1.2.7, http://ccb.jhu.edu/software/FLASH/). QIIME 1.9.1 (V1.2.7, http://ccb.jhu.edu/software/FLASH/). QIIME 1.9.1 64 bit was used to obtain quality-ﬁltered reads and to cluster them into operational taxonomic units (OTUs) with Greengenes OTUs (16 S) v13_8 as the reference18. Finally, a total of 17,997,184 (mean 59,791.309 ± 9581.170; Min 29,699, Max 85,436) sequences remained and each sample was normalized to 29699 sequences for further analysis. Ofﬁcial journal of the American College of Gastroenterology Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Page 4 of 9 jaundice whether the infant was exclusively breastfed or not (OR 0.98, 95% CI 0.83–1.17, OR 0.81, 95% CI 0.63–1.03, respectively) (Table 3). jaundice whether the infant was exclusively breastfed or not (OR 0.98, 95% CI 0.83–1.17, OR 0.81, 95% CI 0.63–1.03, respectively) (Table 3). signiﬁcant differences were observed in infant gender, maternal age at delivery, delivery mode, smoking status, or alcohol consumption between the groups. Ofﬁcial journal of the American College of Gastroenterology Relative abundance of Biﬁdobacterium pseudolongum and risk of neonatal Jaundice Notably, B. pseudolongum exhibited the highest LDA score in LEfSe (LDA = 4.08) (Fig. 3). In order to assess the effects of the B. pseudolongum on the outcome in new- borns from cesarean section, we categorized the relative abundance of B. pseudolongum into more abundant and less abundant groups based on the median in the total sample, and included it in the logistic regression model. The results showed a signiﬁcantly lower risk of physio- logic jaundice in the more abundant group compared with the less abundant group after adjusting for confounders (OR 0.24, 95% CI 0.07–0.68) (Table 4). As no differences in the relative abundance of B. pseudolongum were found between vaginal and cesarean groups (P = 0.21), we would We also stratiﬁed the study population by feeding pat- terns. As the sample size of formula feeding infants was limited, we divided all subjects into exclusive breast- feeding and non-exclusive breast-feeding groups. Results showed that the Shannon index was not associated with Ofﬁcial journal of the American College of Gastroenterology Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Page 5 of 9 Fig. 1 Variability and diversity of the meconium microbiome. a Constitution of microbiota among individuals sorted by relative abundance of Firmicutes. b PCoA plot based on unweighted unifrac distance by controls and cases. c Boxplot of Shannon index by controls and cases. d PCoA plot based on unweighted unifrac distance by delivery mode. e Boxplot of Shannon index by delivery mode Ofﬁcial journal of the American College of Gastroenterology Fig. 1 Variability and diversity of the meconium microbiome. a Constitution of microbiota among individuals sorted by relative abundance of Firmicutes. b PCoA plot based on unweighted unifrac distance by controls and cases. c Boxplot of Shannon index by controls and cases. d PCoA plot based on unweighted unifrac distance by delivery mode. e Boxplot of Shannon index by delivery mode Ofﬁcial journal of the American College of Gastroenterology Ofﬁcial journal of the American College of Gastroenterology Dong et al. Discussion Despite high incidence of neonatal physiologic jaundice, newborns with the condition may not be treated properly due to a misunderstanding of the term “physiologic” as not serious, opposed to “pathologic”. Dennery et al.2 reviewed that bilirubin was cellular-toxic and neurotoxic. Many studies have highlighted the association between neonatal jaundice and psychological disorders, including autism spectrum disorder and attention deﬁcit hyperactivity dis- order6,19. Considering the potential health effects, more attention should be paid to control the incidence. also like to know whether this association only existed in cesarean-born infants or not. Therefore, we employed logistic regression models again in both vaginal born infants and the total sample. As shown in Table 4, more abundance of B. pseudolongum was signiﬁcantly asso- ciated with the outcome in the total sample, while no association was found for vaginal born infants (OR 0.55, 95% CI 0.31–0.95, OR 0.68, 95% CI 0.34–1.34, respec- tively). Heterogeneity test suggested moderate hetero- geneity between vaginal born and cesarean section subgroups (τ2 = 0.34, H = 1.60, I2 = 61.20%, P = 0.11) (Table 4). In this study, we used a nested case–control design, and enrolled a total of 301 participants with parental-reported diagnosis of physiologic jaundice. Results showed that higher birth weight, longer gestational age, and formula feeding were all associated with lower risk of physiologic jaundice after adjusting for covariates, which were con- sistent with previous report20,21. When stratiﬁcation analysis by delivery mode was performed, we observed a signiﬁcant association between higher Shannon index and lower risk of jaundice in cesarean-born infants. It has been reported that higher ɑ-diversity was associated with lower risk of atopic eczema, neonatal sepsis, and necrotizing enterocolitis10,22,23. Our study suggested that higher ɑ- diversity of gut microbiome could also be a protective factor for cesarean-born infants at risk of jaundice. This was an interesting ﬁnding as it was not found in vaginal born infants. Jakobsson et al.24 conducted a time-series study to describe the dynamics of gut microbiome Relative abundance of Biﬁdobacterium pseudolongum and risk of neonatal Jaundice Clinical and Translational Gastroenterology (2018) 9:182 Page 6 of 9 Table 2 Association of selected variables and risk of physiologic jaundice Variables ORa 95% CI P-value Maternal age (year) 1.00 0.93–1.09 0.91 Gestational age (day) 0.94 0.90–0.98 <0.01* Antibiotics to mother 2.01 0.53–8.61 0.32 Premature rupture of membranes 1.27 0.70–2.33 0.43 Gestational diabetes mellitus 0.97 0.51–1.82 0.92 Pregnancy combined with hypothyroidism 0.71 0.36–1.38 0.31 Delivery mode Vaginal 1.00 Ref. Ref. Cesarean 0.66 0.37–1.17 0.16 Birth weight (g) 0.9992 0.9985–0.9999 0.04* Feeding pattern Breast milk 1.00 Ref Ref Mixed 0.71 0.42–1.19 0.19 Formula 0.094 0.005–0.559 0.03* Antibiotics to newborn 3.71 0.41–82.86 0.29 Shannon 0.94 0.83–1.07 0.36 aAdjusted for center, delivery mode, gender, feeding pattern, gestational age, birth weight, premature rupture of membranes, gestational diabetes mellitus, subclinical hypothyroidism in pregnancy, smoking, alcohol, maternal age, 1 min apgar score, 5 min apgar score, multipara, antibiotics to mother, and antibiotics to newborn Boldface indicates statistical signiﬁcance (p < 0.05, p < 0.01) CI conﬁdence interval, OR odds ratio Table 2 Association of selected variables and risk of physiologic jaundice Table 3 Stratiﬁcation analysis on the association between Shannon index and risk of physiologic jaundice by delivery mode and feeding patterns Variables Shannon index Heterogeneity OR 95% CI P-value I2% P-value Delivery modea Vaginal 0.98 0.83–1.15 0.80 64.40 0.09 Cesarean 0.72 0.52–0.98 0.04 Exclusive breastfeedingb Yes 0.98 0.83–1.17 0.86 36.70 0.21 No 0.81 0.63–1.03 0.10 aAdjusted for center, gender, feeding pattern, gestational age, birth weight, premature rupture of membranes, gestational diabetes mellitus, subclinical hypothyroidism in pregnancy, smoking, alcohol, maternal age, 1 min apgar score, 5 min apgar score, multipara, antibiotics to mother, and antibiotics to newborn bAdjusted for center, delivery mode, gender, gestational age, birth weight, premature rupture of membranes, gestational diabetes mellitus, subclinical hypothyroidism in pregnancy, smoking, alcohol, maternal age, 1 min apgar score, 5 min apgar score, multipara, antibiotics to mother and antibiotics to newborn Boldface indicates statistical signiﬁcance (p < 0.05). Boldface indicates statistical signiﬁcance (p < 0.05). Correlation between relative abundance of B. pseudolongum and ɑ-diversity The left panel indicated higher abundance in the control group, and the right panel indicated higher abundance in the case group. B. pseudolongum is the abbreviation that stands for B. pseudolongum Fig. 3 Bar plot of the LDA Score (log10) between control and case groups in cesarean infants. The left panel indicated higher abundance in the control group, and the right panel indicated higher abundance in the case group. B. pseudolongum is the abbreviation that stands for B. pseudolongum Fig. 3 Bar plot of the LDA Score (log10) between control and case groups in cesarean infants. The left panel indicated higher abundance in the control group, and the right panel indicated higher abundance in the case group. B. pseudolongum is the abbreviation that stands for B. pseudolongum j Then we focused on the microbiome of cesarean-born infants. Five features were discovered by LEfSe, and the relative abundance of B. pseudolongum, which exhibited the higherst LDA score, was signiﬁcantly associated with jaundice. Similar results were found in the total popula- tion, but not in the vaginal born infants. In addition, correlation analysis showed that relative abundance of B. pseudolongum was signiﬁcantly correlated with ɑ- diversity. B. pseudolongum belongs to the Biﬁdobacter- ium, and was ﬁrst isolated from an anemic Kenyan infant25. Biﬁdobacterium strains were identiﬁed to exert antimicrobial activity against potential pathogenic microorganisms in infant gut26,27, which could beneﬁt the colonization of healthy bacteria28. Despite the limited information about detailed function of B. pseudolongum, we speculate that it may act like other known Biﬁdo- bacterium strains by defending pathogens to promote the maturation of early gut microbiome and ﬁnally lower the risk of jaundice. However, due to the single time point of sample collection in this study, we could only conﬁrm the correlation between B. pseudolongum and ɑ-diversity, but not causality. We should notice that clinicians are using probiotic supplementation including Biﬁdobacterium to prevent and treat neonatal jaundice around the world29. Two meta-analyses on randomized controlled trials of probiotics therapy for the management of neonatal jaundice conservatively concluded that probiotic supple- mentation could be effective in treating neonatal jaun- dice29,30. Large population-based longitudinal time-series studies are needed to conﬁrm the causal association among B. pseudolongum, ɑ-diversity and neonatal jaundice. Table 4 Association of relative abundance of B. pseudolongum and risk of physiologic jaundice Relative abundancea OR 95% CI P-value Pooled Low 1 Ref. Ref. Correlation between relative abundance of B. pseudolongum and ɑ-diversity High 0.55 0.31–0.95 0.03* Vaginal Low 1 Ref. Ref. High 0.68 0.34–1.34 0.27 Cesarean Low 1 Ref Ref High 0.24 0.07–0.68 < 0.01** aAdjusted for center, gender, feeding pattern, gestational age, birth weight, premature rupture of membranes, gestational diabetes mellitus, subclinical hypothyroidism in pregnancy, smoking, alcohol, maternal age, 1 min apgar score, 5 min apgar score, multipara, antibiotics to mother, and antibiotics to newborn Boldface indicates statistical signiﬁcance (p < 0.05, p < 0.01). Table 4 Association of relative abundance of B. pseudolongum and risk of physiologic jaundice Boldface indicates statistical signiﬁcance (p < 0.05, **p < 0.01). diversity of infants by delivery mode in the ﬁrst 2 years after birth. They found delayed colonization of microbiota in cesarean-born infants compared with vaginal born infants, although no signiﬁcant difference in ɑ-diversity was observed in the ﬁrst week. We speculate that for cesarean-born infants, delayed colonization also delays the duration of immature meconium microbiome, and those with lower diversity of meconium microbiome were put into higher risk for jaundice compared with those with high diversity of meconium microbiome. For vaginal born infants with relatively lower diversity of microbiome, rapid colonization of bacteria compensated deﬁciency of diversity, which diluted the effects of low diversity on the development of jaundice. This ﬁnding could also explain the reason why cesarean-born infants were at higher risk to develop jaundice compared with vaginal born infants in a previous study13. diversity of infants by delivery mode in the ﬁrst 2 years after birth. They found delayed colonization of microbiota in cesarean-born infants compared with vaginal born infants, although no signiﬁcant difference in ɑ-diversity was observed in the ﬁrst week. We speculate that for cesarean-born infants, delayed colonization also delays the duration of immature meconium microbiome, and those with lower diversity of meconium microbiome were put into higher risk for jaundice compared with those with high diversity of meconium microbiome. For vaginal born infants with relatively lower diversity of microbiome, rapid colonization of bacteria compensated deﬁciency of diversity, which diluted the effects of low diversity on the development of jaundice. This ﬁnding could also explain the reason why cesarean-born infants were at higher risk to develop jaundice compared with vaginal born infants in a previous study13. The present study included some limitations. First, the outcome measures were based on parental reports, which could result in potential recall bias. Second, the sample size was relatively small. Correlation between relative abundance of B. pseudolongum and ɑ-diversity Spearman’s correlation analysis was used to explore the correlation between relative abundance of B. pseudo- longum and ɑ-diversity. Results showed that relative abundance of B. pseudolongum was signiﬁcantly corre- lated with ɑ-diversity (P < 0.01). Ofﬁcial journal of the American College of Gastroenterology Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Page 7 of Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Page 7 of 9 Fig. 2 Variability and diversity of the meconium microbiome of cesarean infants. a Constitution of microbiota among cesarean newborns sorted by relative abundance of Firmicutes. b PCoA plot based on unweighted unifrac distance by controls and cases. c Boxplot of Shannon index by controls and cases. d, e Boxplot of proportion of functional genes by controls and cases Fig. 2 Variability and diversity of the meconium microbiome of cesarean infants. a Constitution of microbiota among cesarean newborns sorted by relative abundance of Firmicutes. b PCoA plot based on unweighted unifrac distance by controls and cases. c Boxplot of Shannon index by controls and cases. d, e Boxplot of proportion of functional genes by controls and cases Ofﬁcial journal of the American College of Gastroenterology Ofﬁcial journal of the American College of Gastroenterology Page 8 of 9 Page 8 of 9 Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Page 8 of 9 Fig. 3 Bar plot of the LDA Score (log10) between control and case groups in cesarean infants. The left panel indicated higher abundance in the control group, and the right panel indicated higher abundance in the case group. B. pseudolongum is the abbreviation that stands for B. pseudolongum LEfSe identiﬁed three features that were signiﬁcantly different between controls and cases in all subjects (LDA > 2.5) (Supplementary Fig. 2). There was only one feature more abundant in the case group (Clostridium perfrin- gens). Vítek et al.21 collected stool samples from 5-day-old newborns and isolated a strain of C. perfringens capable of reducing bilirubin to urobilinoids, which supported cur- rent ﬁndings. It was highly possible that the increased abundance of C. perfringens was the feedback to high bilirubin levels in jaundice infants. As C. perfringens is acknowledged as an etiologic agent and was harmful to human health, its role in the pathogenesis of neonatal jaundice should be clariﬁed in future studies. Fig. 3 Bar plot of the LDA Score (log10) between control and case groups in cesarean infants. Ofﬁcial journal of the American College of Gastroenterology Guarantor of the article: Yankai Xia. 17. Peeters, R. P. Subclinical hypothyroidism. N. Engl. J. Med. 376, 2556–2565 (2017). Speciﬁc author contributions: T.D. contributed to the conception and design of the study, drafted the initial manuscript, and reviewed and revised the manuscript. T.C. drafted the initial manuscript, and reviewed and revised the manuscript. R.A.W. revised the manuscript critically for important intellectual content. X.W. performed the initial data analysis and interpreted data. W.H. performed the initial data analysis, interpreted data. Y.L. coordinated and supervised data and bio-sample collection. Y.Z. and M.C. coordinated and supervised data and bio-sample collection. C.L. and Y.X. contributed to the conception and design of the study, and reviewed and revised the manuscript critically. H.A. revised the manuscript critically for important intellectual content. 18. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010). 19. Wei, C. C. et al. Neonatal jaundice and increased risk of attention-deﬁcit hyperactivity disorder: a population-based cohort study. J. Child Psychol. Psy- chiatry 56, 460–467 (2015). y 20. Maisels, M. J. & Kring, E. Length of stay, jaundice, and hospital readmission. Pediatrics 101, 995–998 (1998). 21. Tiker, F., Gurakan, B. & Tarcan, A. Serum bilirubin levels in 1-month-old, healthy, term infants from southern Turkey. Ann. Trop. Paediatr. 22, 225–228 (2002). Financial support: This work was supported by the National Natural Science Foundation of China (81630085), Qing Lan Project of Jiangsu Province, Six Talent Peaks Project of Jiangsu Province (JY-052), Second Level of Training Object of Jiangsu Province “333” Project, the Key Project for High-level Visiting Scholars (gxfxZD2016158), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17 1257). 22. Madan, J. C. et al. Gut microbial colonisation in premature neonates predicts neonatal sepsis. Arch. Dis. Child. Fetal Neonatal Ed. 97, F456–F462 (2012). 23. Warner, B. B. et al. Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study. Lancet (Lond., Engl.) 387, 1928–1936 (2016). Scholars (gxfxZD2016158), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_1257). 24. Jakobsson, H. E. et al. Decreased gut microbiota diversity, delayed Bacter- oidetes colonisation and reduced Th1 responses in infants delivered by caesarean section. Gut 63, 559–566 (2014). Potential competing interest: None. Potential competing interest: None. Publisherʼs note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 25. Vazquez-Gutierrez P., et al. Correlation between relative abundance of B. pseudolongum and ɑ-diversity The effects caused by early microbiome in the total sample could be smoothed out. Third, we only had meconium samples at a single time point and could not conﬁrm the causal association between B. pseudolongum and ɑ-diversity. Time-series sample collection design is expected in the future. Page 9 of 9 Page 9 of 9 Dong et al. Clinical and Translational Gastroenterology (2018) 9:182 Study Highlights 3. Huang, L. et al. 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https://openalex.org/W3199275875	https://europepmc.org/articles/pmc8492940?pdf=render	English	null	Clinical Correlates of Deliberate Self-Harm Among Migrant Trauma-Affected Subgroups	Frontiers in psychiatry	2,021	cc-by	9,764	Clinical Correlates of Deliberate Self-Harm Among Migrant Trauma-Affected Subgroups Francesca Baralla 1, Martina Ventura 2, Nikolay Negay 3, Anteo Di Napoli 2, Alessio Petrelli 2, Concetta Mirisola 2 and Marco Sarchiapone 1,2 1 Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy, 2 National Institute for Health, Migration and Poverty, Rome, Italy, 3 Department of Psychiatry and Narcology of Asfendiyarov, Kazakh National Medical University, Almaty, Kazakhstan Introduction: Studies have shown that exposure to potentially traumatic events (PTEs) during the migration process has serious consequences on mental health. Migrants with a history of PTEs are more likely to inﬂict deliberate self-harm (DSH), a spectrum of behavior that includes non-suicidal self-injury (NSSI). With reference to a nonclinical sample of trauma-exposed migrants, this study aims to explore the prevalence of DSH and to assess the association with sociodemographic characteristics and clinical conditions, with particular attention to post-traumatic stress disorder (PTSD) symptoms, resilience capabilities, and feelings of hopelessness. Edited by: Suraj Bahadur Thapa, University of Oslo, Norway Suraj Bahadur Thapa, University of Oslo, Norway Reviewed by: Amit Shrira, Bar-Ilan University, Israel Crosby Alyse Modrowski, Lifespan, United States Correspondence: Francesca Baralla francesca.baralla@unimol.it Reviewed by: Amit Shrira, Bar-Ilan University, Israel Crosby Alyse Modrowski, Lifespan, United States Methods: A sample of migrants underwent a baseline evaluation at an outpatient department of the National Institute for Health, Migration and Poverty (INMP). Migrants with a history of torture, rape, or other severe forms of psychological, physical, or sexual violence were invited to undergo screening at the Institute’s Mental Health Unit. Trauma-exposed migrants completed a series of self-report questionnaires that assessed the presence of PTSD, depression, anxiety, suicidal ideation, resilience, and feelings of hopelessness, in addition to DSH. Univariate and multivariate log-binomial regression models were used to test the association of age and clinical characteristic of migrants with DSH. Prevalence ratio (PR) with 95% conﬁdence intervals (95% CI) and p-values were estimated. Correspondence: Francesca Baralla francesca.baralla@unimol.it Specialty section: This article was submitted to Mood and Anxiety Disorders, a section of the journal Frontiers in Psychiatry Received: 24 January 2020 Accepted: 18 August 2021 Published: 22 September 2021 Citation: Baralla F, Ventura M, Negay N, Di Napoli A, Petrelli A, Mirisola C and Sarchiapone M (2021) Clinical Correlates of Deliberate Self-Harm Among Migrant Trauma-Affected Subgroups. Front. Psychiatry 12:529361. doi: 10.3389/fpsyt.2021.529361 Received: 24 January 2020 Accepted: 18 August 2021 Received: 24 January 2020 Accepted: 18 August 2021 Published: 22 September 2021 Results: A total of 169 migrants (76.9% males), aged between 18 and 68 years, M = 28.93; SD = 8.77), were selected. Of the sample, 26.6% were frequently engaging in some form of DSH, and 30.2% were diagnosed with at least one trauma-related disorder. DSH behaviors were most common in single and unemployed migrants as well as in subjects with post-traumatic stress symptoms, feelings of anxiety, hopelessness, low capability of resilience, and suicidal ideation. Taking into account age and hopelessness, we found that PTSD and low resilience capabilities were associated with a higher risk of DSH [PR adj: 2.21; 95% CI: (1.30–3.75) and PR adj: 2.32; 95% CI: (1.16–4.62), respectively]. Published: 22 September 2021 ORIGINAL RESEARCH published: 22 September 2021 doi: 10.3389/fpsyt.2021.529361 INTRODUCTION However, very few studies investigated the ethnic and cultural meanings and functions of self-harm in voluntary or forced migrants, in particular taking into account the speciﬁcs of displacement and the complex characteristics of trauma in the context of asylum seekers. Self-harm is related to psychiatric and personality disorders (6–8) and is particularly widespread, especially in new social and relational contexts, like virtual environments (8). Self-harm is the most important risk factor for suicide (9) irrespective of the extent, the type, or the motive behind the suicidal intent and therefore may also include suicidal ideation (SUI) (10). SUI is widely acknowledged to be a major risk factor for suicide and seems to be particularly prevalent among ﬁrst- and second-generation migrants (11). There are data that show also the association between self- harm and suicide attempt (SA) (12–14). Literature has shown that patients with suicidal intent constitute a more severe group, and self-injuring patients with SUI diﬀer from patients who have not attempted suicide, in terms of greater severity of psychopathology in the former (15). Non-suicidal self-injury (NSSI), which was proposed as a new diagnosis in the Diagnostic and Statistical Manual of Mental Disorders in 2013 (5th ed.) (16), is a prevalent behavioral problem associated nevertheless with poor outcomes and reduced life expectancy (17). Both NSSI and SA are distinct behavioral phenomena that often co-occur within individuals and form a continuum of self-damage that can be related to suicidal behavior (18, 19). Speciﬁcally, more recent ﬁndings (20) have shown that NSSI increases the risk of transitioning from suicide ideation to a SA. Some studies have suggested that trauma-related symptoms may play an important role in the development and maintenance of self-harm. As underlined by Ford and Gomez (21), a large body of studies have highlighted the mediating eﬀect of trauma on acts of self-harm. Recently, Sami and Hallaq (22) documented that self-harm is frequently a sequela of prolonged exposure to emotional and physical violence, and post-traumatic stress disorder (PTSD) increases the risk of engaging in self-injurious behaviors (SIBs). Given the important mechanistic role of trauma symptomatology in SIB [e.g., (23)], considering in addition the functional role of SIB in a person’s ability to cope with trauma, self-injury has been considered as an eﬀective yet maladaptive The development of intervention to reduce the psychological burden of migrants would be aided by a better understanding of what aﬀects PTSD. INTRODUCTION strategy to alter one’s internal feelings and to alleviate, manage, or eliminate negative emotions and feelings of tension (24). Deliberate self-harm (DSH) refers to the intentional direct (e.g., destruction of one’s own body tissue without suicidal intent) and indirect damage to an individual’s body (e.g., severe substance abuse, overdosing, or ingestion of sharp implements), including suicidal behavior (1). Common forms of DSH (2) result in an alteration or damage to body tissue and can refer to multiple methods (3), including skin cutting, burning, scratching, banging or hitting of body parts, and interfering with wound healing (4, 5). There has been a long-standing interest in the relationship between the exposure to potentially traumatic events (PTEs) and SIB among migrants [e.g., (1, 25, 26)]. The condition of migration is a potential risk factor for engaging in self-harming behaviors (27), especially among minors (28), by virtue of their higher vulnerability to diﬀerent pre-migratory, migratory, and post-migratory stress factors, which increase the risk of mental health (29). Ethnic and racial variations in the rates of self- harm have been observed worldwide, with higher prevalence reported among Asian British males and Black females (30). Individuals who have higher levels of ethnic identity and sense of belonging would be less likely to engage in NSSI (31), while migrants with a complex PTSD, exposed to multiple and chronic trauma, especially in interpersonal contexts have been found to present with more severe anger, aggression, and self-harm (26). Among ethnic minorities, self-harm represents one of the most important predictor factors for suicide behaviors (32). The high prevalence of self-harm and suicidal behavior among migrants may therefore be considered as a consequence of the traumatogenic nature of stressful events that can occur in the migrant’s country of origin, as well as in their host country (33). Fleeing from warfare and persecution may be considered as a series of traumatic events that can occur pre-, peri-, and post- migration and that may diﬀer in their intensity and duration (34). The variation in rates of post-traumatic psychopathology is related to a variety of factors, speciﬁcally to the nature and the intensity of the cumulative PTEs that migrants experience. Studies have shown the eﬀect of “systemic trauma” (23) faced by immigrant populations, which is related to experiences of violence, loss, oppression, and displacement (21). Citation: Baralla F, Ventura M, Negay N, Di Napoli A, Petrelli A, Mirisola C and Sarchiapone M (2021) Clinical Correlates of Deliberate Self-Harm Among Migrant Trauma-Affected Subgroups. Front. Psychiatry 12:529361. doi: 10.3389/fpsyt.2021.529361 Baralla F, Ventura M, Negay N, Di Napoli A, Petrelli A, Mirisola C and Sarchiapone M (2021) Clinical Correlates of Deliberate Self-Harm Among Migrant Trauma-Affected Subgroups. Front. Psychiatry 12:529361. doi: 10 3389/fpsyt 2021 529361 September 2021 \| Volume 12 \| Article 529361 Frontiers in Psychiatry \| www.frontiersin.org Baralla et al. DSH in Trauma-Affected Migrants Conclusion: Given the association between trauma exposure and DSH among migrants, exploring the presence of DSH behavior within the immigrant community is crucial for the implementation of measures to develop intervention in a clinical setting. Conclusion: Given the association between trauma exposure and DSH among migrants, exploring the presence of DSH behavior within the immigrant community is crucial for the implementation of measures to develop intervention in a clinical setting. Keywords: migration trauma exposure, deliberate self-harm behavior, post-traumatic stress (PTS), resilience, non-suicidal self-injury (NSSI) Frontiers in Psychiatry \| www.frontiersin.org INTRODUCTION In particular, in this study, we took into consideration the construct of hopelessness that has received growing attention in the ﬁelds of SIB. Hopelessness can be deﬁned as a trans-diagnostic psychological construct, characterized by rigid and persistently negative expectations about the future and a helplessness to challenge such thoughts (35). It is a phenomenon that it is still understudied in migrant populations. Recent ﬁndings (36) suggest that potentially hopelessness can develop into a worsening September 2021 \| Volume 12 \| Article 529361 Frontiers in Psychiatry \| www.frontiersin.org 2 DSH in Trauma-Affected Migrants Baralla et al. decline of mental health that can lead to increased self-harm and suicide. Care Unit of INMP, migrants with a history of torture, rape, or other severe forms of psychological, physical, or sexual violence were approached for participation. Migrants were invited to undergo screening at the Mental Health Unit of INMP, which led to clinical–diagnostic assessments. Migrants involved in the study signed a form consenting to their inclusion in clinical research and received information about voluntary participation, conﬁdentiality, and protection of personal data. All participants were also informed about mental health services and counseling activities, which they could access if need be. Current psychiatric diagnoses were established using clinical interviewing procedures similar to the Structured Clinical Interview for DSM-5 disorders (SCID-5- CV) (49) conducted by trained and supervised clinical research assistants. The screening interview was divided into multiple modules covering background information like demographic characteristics, lifestyle and behavior, trauma history, lifetime history of mental health, and family history of mental disorders. Participants also completed a series of self-report questionnaires, including those described in the Measurements section of this paper, which were randomly ordered to mitigate for order eﬀects. Another key issue, typically related to the migration process and regarded as a “protective” factor, is resilience (37). Resilience is the ability to positively cope with adverse situations and to maintain positive outcomes in the complex interplay between risk and vulnerability factors (38–40). Emerging data suggest that migration research could beneﬁt from the use of a strengths- based approach, including the resilience construct, for a more thorough understanding of migrant experiences (41, 42). Indeed, it has been argued that resilience represents an essential element of epidemiological and prevention research, which aims to promote wellbeing and improve mental health in migrants (43). METHODS The six-item questionnaire (52) was a modiﬁed and shortened version of the Deliberate Self-Harm Inventory (DSHI) (5) that assesses lifetime history of self-harm behavior using a 4-point Likert scale (never; one to two times; three to four times; and ﬁve or more times). This inventory is based on the deﬁnition of DSH (5) as a direct destruction or alteration of body tissue, without conscious suicidal intent. DSHI measures lifetime presence and frequency of the following self-injury behaviors: self-cutting, INTRODUCTION This is due to the impact that resilience has on how migrants adapt to the migration process and the acculturation experiences in their host countries (44, 45). Depression and anxiety are psychiatric disorders frequently observed in general populations and are reported to be highly prevalent among migrants, both voluntary migrants and asylum seekers (46). In particular, ﬁrst-generation migrants reported considerably more depression, generalized anxiety, and panic attacks in the past 4 weeks and SUI than did second-generation migrants (11). Regarding asylum seekers, signiﬁcant diﬀerences in depression and anxiety scores on psychometric instruments among migrants with a history of detention vs. those without were observed (47). Measurements Migrants completed a self-report questionnaire that assessed exposure to adverse experiences relating to the migration process and/or a trauma history. Furthermore, the presence of the following was ascertained: PTSD, depression and anxiety, resilience, and feelings of hopelessness, DSH, SA and SUI. The research protocol was built ad hoc with the aim of gathering relevant information about the migrants’ clinical features. After clinical assessment, the participants were asked to complete the following self-administered questionnaires and scales. Moreover, a wide range of determinants, connected to migrant’s application for asylum, such as uncertainty regarding the outcome, slowness of the procedures, and social isolation, can increase anxiety and depression, representing trigger factors for self-harm (8). Gaining a better evaluation of self-injury behavior (NSSI and SA), which has been demonstrated to have an association with PTSD [e.g., (48)], is a necessary step in helping healthcare providers to identify and intervene and thus reduce the psychological burden of trauma-exposed migrants. Research on the presence of DSH, related or not to SA, in vulnerable groups such as migrants is required and necessary in order to formulate preventative measures in clinical and treatment settings. Measure of Potentially Traumatic Events To obtain information about the type of PTEs that had occurred during an individual’s lifetime, and/or during the migration process, we used the Harvard Trauma Questionnaire (HTQ) (50). Section Introduction of the HTQ is a checklist with 17 items developed as a cross-culturally valid instrument to measure the kind and variety of PTEs. Examples of items are lack of food and water, loss of a loved one, rape, torture, brainwashing, imprisonment, and combat situations. As in a previous study (51), the reporting format was modiﬁed, removing the options “witnessed” and “heard about.” The response format allowed participants to indicate when the event had happened; and time periods were coded as in infancy/pre-migration (I/PM) and/or adulthood/during migration (A/DM). The current study aims to explore the frequency of DSH and document the prevalence of PTSD, depression, and anxiety in a nonclinical convenience subgroup of trauma-aﬀected migrants. It also aims to evaluate the eﬀect of PTSD, resilience capabilities, and feeling of hopelessness on DSH. We hypothesized that PTSD would be associated with an engagement in DSH, while low resilience and feelings of hopelessness would likely act as negative factors that interfere with the adaptation process. Participants This study involved PTE-exposed migrants who were selected from consecutive admission to the National Institute for Health, Migration and Poverty (INMP) Ambulatory Care Unit between 2017 and 2018. The research was approved by the institutional review board (IRB) of the National Institutes of Health (Prot. PRE/17). Following admission to the Ambulatory Frontiers in Psychiatry \| www.frontiersin.org September 2021 \| Volume 12 \| Article 529361 3 DSH in Trauma-Affected Migrants Baralla et al. self-burning, self-punching, self-scratching, self-carving, self- biting, and self-banging (head and/or other body parts), as well as preventing wounds from healing and skin damage by other methods. In this study, consistent with past studies (52), four indicators of DSH were created. First, in relation to the “history of DSH,” a score of “1” was given to participants who reported a history of DSH behavior. This binary variable was created to indicate a positive screening result for DSH and to identify migrants who may need targeted interventions. For the second variable, “frequency of DSH,” a score of “1” was given to participants who reported having engaged in DSH behavior three or more times (frequent DSH), while a score of “0” was given to participants who reported engaging in DSH behavior twice or less (infrequent DSH). In relation to DSH methods, in line with past studies [e.g., (52)], the third variable DSH-type behavior was deﬁned as a “cutting type” or “non-cutting type”: the “cutting type” consisted of behaviors related to the ﬁrst, second, third, and fourth items in the DSHI (e.g., “ever intentionally cut wrist, arms, or other area(s) of body, or stuck sharp objects into skin such as needles, pins, staples”), while other forms of DSH were classiﬁed as the “non-cutting type” (e.g., “ever intentionally banged your head or punched yourself to the extent that you caused a bruise to appear”). Finally, the variables relating to the participants who reported having harmed themselves so severely as to have warranted hospitalization (“hospitalization” = 1 vs. “no hospitalization” = 0) were dichotomized. associated with an elevated risk of suicide, and we used the scores ≥2 of the screening part (items 1–5) to identify participants with SUI (SUI = 1 vs. NO SUI = 0). Migrants with positive answers on the Beck Scale for Suicidal Ideation (BSSI) were referred to the medical staﬀof INMP for further evaluation. Zung Self-Rating Anxiety Scale A short self-administered 20-item version of the scale (60), covering both aﬀective and somatic symptoms, focuses on the most common general anxiety disorders. Each response is rated on a 1- to 4-point scale, from “none or little of the time” to “most of the time.” There are 20 questions with 15 increasing anxiety level questions and ﬁve decreasing anxiety level questions. Overall scores range from 20 to 80: scores from 20 to 44 indicate a normal range; from 45 to 59, mild anxiety; from 60 to 74, moderate anxiety; and from 75 and above, severe anxiety. In this study, a cutoﬀscore of 45 (≥) was used to indicate the presence of anxiety-related symptomatology. Beck Scale for Suicidal Ideation This is a 19-item scale that assesses a person’s current intensity of thoughts, behaviors, and plans to commit suicide (53). A self- reporting version of the scale was introduced by Beck et al. (54). Each item consists of three alternatives that describe diﬀerent intensities of SUI, which are rated on a 3-point scale from 0 to 2. Participants are instructed to choose the particular statement of each group that is most applicable to them. Total scores are calculated by summing the 19 ratings and can range from 0 to 38, with higher values indicating a greater risk of suicide. Beck and Steer (55) do not distinguish between diﬀerent degrees of suicidal risk. In line with previous studies (56), very low total scores can be Measure of Self-Destructive Behaviors To determine a lifetime history and/or recent episodes of other self-destructive behaviors, such as SAs and substance use, respondents were asked to report both their lifetime and past year use of drugs (marijuana, cocaine, heroin, inhalants, methamphetamine, and hallucinogens), alcohol, and episodes of attempted suicide, and/or a family history of attempted suicide and/or suicide. Participants responded to each question with a “yes” (=1) or “no” (=0), and answers were classiﬁed according to whether or not they reported substance use behaviors, SAs, and/or a family history of SAs. In the case of positive responses related to previous SA(s), to ensure that the answers to (“Have you tried to take your own life?”) were correctly understood, clinicians explained to participants that questions are aimed to assess behavior related to the goal of taking one’s own life. Positive answers were identiﬁed as emergency cases and referred to a psychiatrist for further evaluation and targeted interventions. Participants Posttraumatic Stress Disorder Checklist for DSM-5 This is a self-report measure that assesses the 20 DSM-5 symptoms of PTSD in the updated version of the PTSD Checklist (PCL). The PCL-5 (57) contains 20 items rated on a 5-point Likert-type scale (available online at: www.ptsd.va.gov), with scores ranging from “not at all” (0) to “extremely” (4), resulting in a symptom severity score ranging from 0 to 80. Factor analysis identiﬁed four factors related to the DSM-5 model of PTSD; and they included re-experiencing (RE), hyperarousal (HY), avoidance (AV), and negative feelings (NF), with a three- dimensional factor solution related to the DSM-5 deﬁnition. DSM-5 symptom cluster severity scores can be obtained by summing the scores for the items within a given cluster (cluster B = from 1 to 5; cluster C = 6 and 7; cluster D = from 8 to 14; cluster E = from 15 to 20). According to Lang et al. (58), in this study, an overall cutoﬀscore of 33 (≥) was used to indicate the presence of post-traumatic stress symptoms. Zung Self-Rating Depression Scale This is a self-administered 20-item survey (59) that is used in a variety of settings as a screening tool, covering the common aﬀective, psychological, and somatic characteristics of depression. Each question is framed in terms of positive and negative statements, and each item is scored on a scale ranging from 1 to 4 points (from “a little of the time” to “most of the time”); hence, overall scores range from 25 to 100: scores from 25 to 49 indicate a normal range; 50–59, mild depression; 60–69, moderate depression; and 70 and above, severe depression. The scores provide indicative ranges for depression severity. In this study, a cutoﬀscore of 50 (≥) was used to indicate the presence of depressive symptoms. Frontiers in Psychiatry \| www.frontiersin.org Data Analysis A descriptive analysis of the sample was carried out. Categorical variables are presented as absolute numbers (n) and percentage frequencies (%). Sociodemographic and clinical characteristic of the study population were described. The frequencies of PTEs and the period of occurrence (I/PM and/or A/DM) were evaluated to better characterize the migrants included in the sample. In order to evaluate relationships between continuous clinical variables, the correlation matrix was calculated. As shown in Table 2, the most frequent PTEs occurred during migration (A/DM) were serious injury (49.7%), imprisonment (41.4%), being close to death or lack of food and water (both 37.9%), and the lack of shelter (36.1%). The latter was the most common (31.4%) during the infancy period (I/PM), followed by the lack of food and water (30.2%) and rape or sexual abuse (18.3%). Overall, almost all migrants (94%) in the sample reported suﬀering a trauma during the migration process, whereas less than a half (43%) declared to having suﬀered a trauma during the infancy period. When we analyzed the relation between period of trauma occurrence and DSH, we did not ﬁnd any statistically signiﬁcant diﬀerences in the proportion of subjects with DSH: 28% of subjects who had a trauma injure themselves regardless of when their trauma occurred (data not shown). The outcome under study was deﬁned as a dichotomous variable, using the information “history of DSH” during a lifetime (1 or more episode) scored by the DSHI. The distribution of DSH behavior was analyzed in the entire sample and for diﬀerent sociodemographic and clinical characteristics. Diﬀerences between categories were assessed using chi-square tests or Fisher’s exact test, as appropriate. In order to evaluate determinants of DSH, in a ﬁrst step, separate univariate analyses were performed for DSH outcome and all the variables of interest, by using log-binomial regression models. In particular age, anxiety, PTSD, depression, SUI, hopelessness, and lower resilience were tested; prevalence ratios (PRs) with 95% conﬁdence intervals (95% CI) and p-values were estimated. Then, we would like to investigate the joint role of age, PTSD, low resilience, and hopelessness on DSH. We ﬁrst investigated the presence of interactions between hopelessness and other three covariates, but no statistically signiﬁcant results were found. Finally, multivariate log-binomial regression models were performed to investigate the eﬀect of these covariates on DSH. Connor-Davidson Resilience Scale 2 This is an abbreviated two-item version of the rating scale for the assessment of resilience, created to reduce administration time (61). The two items of the 25-item Connor-Davidson Resilience (CD-RISC) scale (62) used in this case were item 1 (“Able to adapt to change”) and item 8 (“Tend to bounce back after illness or hardship”). The CD-RISC2 suﬃciently represents the original September 2021 \| Volume 12 \| Article 529361 Frontiers in Psychiatry \| www.frontiersin.org 4 DSH in Trauma-Affected Migrants Baralla et al. measure, and the CD-RISC2 can be used in place of the 25-item CD-RISC. Test–retest reliability analysis, convergent validity, and divergent validity demonstrated signiﬁcant correlation (ranging from r = 0.27 to 0.66) with both the 25-item CD-RISC version and the individual items of the CD-RISC. In this study, a cutoﬀscore of 5.5 (≤) was used to indicate participants with lower resilience resources (named “Lower resilience”) (61). The empirical and joint evaluation of the maximum value of VIF, of the absence of very small eigenvalues and of low condition- index values, suggests that problems of multicollinearity can be considered negligible and do not aﬀect the stability of the parameters estimation. All analyses were performed using SAS R⃝ System version 9.3 (66). RESULTS This scale (63) includes 20 items that are answered as true or false; and total scores can range from 0 to 20. Cronbach’s alpha coeﬃcient of the scale in the general population ranged from 0.82 to 0.93 (64). This scale evaluates three major aspects of hopelessness: an individual’s feelings about the future, the loss of motivation, and future expectations. The Beck Hopelessness Scale (BHS) results were dichotomized, using a cutoﬀscore of 9, to diﬀerentiate hopeless (1 = presence) from not hopeless subjects (65). The sample included 169 migrants with permanent residency in Italy aged between 18 and 68, M = 28.9; SD = 8.77), selected from consecutive admission to the Mental Health Unit of INMP. Table 1 presents the sociodemographic and clinical characteristics of the sample. Respondents were mainly males (76.9%), and 80.5% were young adults (aged 18–35 years); most of them were single (73.4%) and unemployed (77.5%), and about a half had a lower or upper secondary education (49.1%). The 78.7% migrated from Africa (mostly from Western Africa, 67.5%) and 21.9% <1 year ago. Of the migrants, 30.2% had at least one trauma-related disorder, 22.5% at least one mood disorder, and 11.8% at least one substance use disorder. Anxiety disorders and adjustment disorder were both present in 7.1% of the study population. Among participants for whom data were available, 18 (10.7%) declared a family history of mental disorders. Frontiers in Psychiatry \| www.frontiersin.org Data Analysis Two models were run, without and with hopelessness, in order to evaluate the mediating role of this factor on PTSD and low resilience. For all analyses, statistical signiﬁcance was predetermined at p < 0.05, 0.01, or 0.001. Moreover, we also evaluated multicollinearity, to exclude the possibility that correlation between covariates could inﬂuence the model results. To this purpose we calculated the variance inﬂation factor (VIF) and the eigenvalues obtained through a principal component analysis conducted on the matrix of the covariates. Of the sample, 26.6% declared a DSH episode in their lifetime (Table 3) with an age of onset of DSH ranging from 18 to 57 years, M = 29; SD = 8.65). To be engaged in DSH was most common in single (or alone) and unemployed migrants. Moreover, in subjects with post-traumatic stress symptoms, feelings of anxiety, hopelessness, low capability of resilience and SUI, and DSH behaviors were found to be more frequent. The results of univariate regression models for testing the association between each variable of interest and DSH are presented in Table 4. Anxiety, PTSD, SUI, hopelessness, and low resilience were signiﬁcantly associated with a two-fold (or more) higher risk of engaging in DSH behaviors. In Table 5, Model 1a shows the results of the multivariate regression model, where the eﬀect of age, PTSD, and low September 2021 \| Volume 12 \| Article 529361 5 DSH in Trauma-Affected Migrants Baralla et al. TABLE 1 \| Sociodemographic and clinical characteristics of the sample. n = 169 SOCIODEMOGRAPHIC n (%) Age groups 18–35 136 (80.5%) 36–68 33 (19.5%) Gender Male 130 (76.9%) Female 39 (23.1%) Area of origin Africa 133 (78.7%) America 8 (4.7%) Asia 15 (8.8%) Europe 13 (7.8%) Social status Single 124 (73.4%) Married 29 (17.2%) Other (separated, divorced, and widowed) 16 (9.4%) Religious Yes 154 (91.1%) No 15 (8.9%) Occupational status Unemployed 131 (77.5%) Employed or student 38 (22.5%) Education Illiterate 20 (11.8%) Primary 41 (24.3%) Lower/upper secondary 83 (49.1%) Bachelor’s degree or equivalent 20 (11.8%) Duration of stay in Italy (months) ≤12 37 (21.9%) >12 132 (78.1%) CLINICAL CHARACTERISTICS Trauma-related disorders 51 (30.2%) Mood disorders 38 (22.5%) Anxiety disorders 12 (7.1%) Adjustment disorder 12 (7.1%) Borderline personality disorder 2 (1.2%) Dysthymic disorder 2 (1.2%) Insomnia disorder 7 (4.1%) Substance use disorder 20 (11.8%) Family history of mental disorders 18 (10.7%) No. and % do not add up to tot./100 owing to multiple responses in categories. Data Analysis Diagnoses related to the evaluation performed during intake. 169 %) 0.5%) 9.5%) 6.9%) 3.1%) 8.7%) 7%) .8%) .8%) 3.4%) 7.2%) .4%) 1.1%) .9%) 7.5%) 2.5%) TABLE 2 \| PTEs and period of occurrence (I/PM and/or A/DM). PTEa I/PM, n (%) A/DM, n (%) Lack of food and water 51 (30.2%) 64 (37.9%) No access to medical care 5 (3%) 45 (26.6%) Lack of shelter 53 (31.4%) 61 (36.1%) Imprisonment – (–) 70 (41.4%) Serious injury 1 (0.6%) 84 (49.7%) Torture 1 (0.6%) 58 (34.3%) Brainwashing – (–) 7 (4.1%) Rape or sexual abuse 31 (18.3%) 22 (13%), Isolation from others 2 (1.2%) 40 (23.7%) Being close to death – (–) 64 (37.9%) Forced separation 1 (0.6%) 52 (30.8%) Murder of family or friend 4 (2.4%) 46 (27.2%) Unnatural death of family/friend 5 (3%) 45 (26.6%) Murder of stranger or strangers 1 (0.6%) 49 (29%) Lost 1 (0.6%) 16 (9.5%) Kidnapped 1 (0.6%) 35 (20.7%) Combat situation 1 (0.6%) 57 (33.7%) PTEs, potentially traumatic events; I/PM, infancy/pre-migration; A/DM, adulthood/during migration. aNo. and % do not add up to tot./100 owing to multiple responses in categories. TABLE 2 \| PTEs and period of occurrence (I/PM and/or A/DM). DISCUSSION This study aimed to explore the frequencies of DSH and its correlates among a nonclinical convenience migrant trauma- exposed subgroup. We have evaluated the relationship between DSH and some sociodemographic characteristics and clinical conditions. The results show high prevalence of DSH in the sample. We also found increased frequencies of PTSD, as well as depressive and other anxiety symptoms. The results show that trauma-related symptoms increase the risk of engaging in SIB. The data highlight that lower resilience resources correspond to an increase in DSH. Moreover, we also considered the feeling of hopelessness as a negative self-view characterized by rigid and persistently negative expectations about the future, which appears to have implications for post-traumatic-related symptoms. Epidemiological data suggest that trauma-exposed subjects who engage in DSH present a highly negative self-view (67). In addition, PTSD may impair the integrity of the self and result in negative expectations regarding the self and the world (68). Previous studies have provided empirical support for the existence of a relationship between traumatic experiences and self-injury (25) and have suggested that trauma-related symptoms may play an important role in the development and continuation of SIB. Other studies suggest that PTE rather than post-traumatic symptoms could be more strongly related to self-injury (48). Taken together, PTE and PTSD may play an important role in the development and continuation of DSH. No. and % do not add up to tot./100 owing to multiple responses in categories. Diagnoses related to the evaluation performed during intake. resilience on DSH was tested. A strong association between PTSD (PR adj = 3.75 [1.77–7.94]), low resilience (PR adj = 3.91 [1.63–9.41]), and DSH was observed. Hopelessness (Model 1b) contributes to explaining part of the relation between PTSD and resilience with DSH. In fact, when including the variable hopelessness in the multivariate model, the eﬀect of PTSD (PR adj = 2.21 [1.30–3.75]) and low resilience (PR adj = 2.32 [1.16–4.62]) on DSH decreased. The issue of self-injury behavior is considered a pervasive public health burden that has received increasing attention from researchers (27, 69–71). In particular, DSH was considered one of the most important predictor factors for suicide among ethnic September 2021 \| Volume 12 \| Article 529361 Frontiers in Psychiatry \| www.frontiersin.org 6 DSH in Trauma-Affected Migrants Baralla et al. TABLE 3 \| Characteristics of migrants who injure themselves. DISCUSSION All the samples (N = 169) With DSH (N = 45, 26.6%) p N N (%) SOCIODEMOGRAPHIC Age groups 18–35 136 37 (27.2%) ns 36–68 33 8 (24.2%) Gender Male 130 33 (25.4%) ns Female 39 12 (30.8%) Civil status Single 124 33 (26.6%) <0.05 Married 29 4 (13.8%) Other (separated, divorced, widowed) 16 8 (50.0%) Religious Yes 154 42 (27.3%) ns No 4 1 (25.0%) Occupational status Unemployed 131 43 (32.8%) <0.01 Employed or student 38 2 (5.3%) Education Illiterate 20 5 (25.0%) ns Primary 41 13 (46.7%) Lower/upper secondary 83 21 (25.3%) Bachelor’s degree or equivalent 20 4 (20.0%) CLINICAL CHARACTERISTICS PTSD (PCL-5) Yes 73 30 (41.1%) <0.001 No 96 15 (15.6%) Anxiety (SAS) Yes 103 35 (34.0%) <0.01 No 66 10 (15.2%) Depression (SDS) Yes 96 30 (31.3%) ns No 73 15 (20.5%) Suicidal ideation (BSSI 1–5) Yes 49 22 (44.9%) <0.001 No 120 23 (19.2%) Hopelessness (BHS) Yes 110 38 (34.5%) <0.001 No 59 7 (11.9%) Low resilience (CD-RISC2) Yes 105 37 (35.2%) <0.001 No 64 8 (12.5%) Lifetime substance use (yes) Yes 20 7 (35.0%) ns No 135 32 (23.7%) Bold values indicate statistical signiﬁcance at p < 0.05, p < 0.01, and p < 0.001. DSH, deliberate self-harm; PTSD, post-traumatic stress disorder; PCL-5, Posttraumatic TABLE 4 \| Univariate log-binomial regression models for DSH behavior. Variable* PR [95% CI—inf] [95% CI—sup] p-value Age-group (ref. 36+) 1.12 0.58 2.18 ns Anxiety (SAS) 2.24 1.19 4.22 <0.01 PTSD (PCL-5) 2.63 1.53 4.51 <0.001 Depression (SDS) 1.52 0.89 2.61 ns Suicidal ideation (BSSI-5) 2.34 1.45 3.79 <0.001 Hopelessness (BHS) 2.91 1.39 6.11 <0.01 Low resilience (CD-RISC2) 2.82 1.40 5.67 <0.01 Bold values indicate statistical signiﬁcance at p < 0.05. DSH, deliberate self-harm; PR, prevalence ratio; SAS, Zung Self-Rating Anxiety Scale; PTSD, post-traumatic stress disorder; PCL-5, Posttraumatic Stress Disorder Checklist for DSM-5; SDS, Zung Self-Rating Depression Scale; CD-RISC2, Connor-Davidson Resilience Scale 2. “Absence” is considered as reference category. TABLE 5 \| Multivariate log-binomial regression models for DSH behavior. Variable PR adj [95% CI—inf] [95% CI—sup] p-value Model 1a Age group (ref. 36+) 1.16 0.44 3.04 ns PTSD (PCL-5) 3.75 1.77 7.94 <0.01 Low resilience (CD-RISC2) 3.91 1.63 9.41 <0.001 Model 1b** Age group (ref. DISCUSSION 36+) 1.21 0.67 2.19 ns PTSD (PCL-5) 2.21 1.30 3.75 <0.01 Low resilience (CD-RISC2) 2.32 1.16 4.62 <0.01 Hopelessness (BHS) 2.01 0.95 4.22 ns Bold values indicate statistical signiﬁcance at p < 0.05, p < 0.01, and p < 0.001. DSH, deliberate self-harm; PR, prevalence ratio; PTSD, post-traumatic stress disorder; PCL-5, Posttraumatic Stress Disorder Checklist for DSM-5; CD-RISC2, Connor-Davidson Resilience Scale 2; BHS, Beck Hopelessness Scale; VIF, variance inﬂation factor. Where not explicit, “absence” is considered as reference category. VIF: Age group (1.031); PTSD (1.079); Resilience (1.066); Hopelessness (1.110). minorities (32). Our results indicate that DSH behaviors are associated with SAs and SUI. Data suggest that DSH may increase the risk of SUI and behavior. A previous investigation conducted by Kalt et al. (72) in detentions centers showed a correlation between trauma and violence experienced during the migration processes and some psychopathological conditions like PTSD, psychosomatic symptomatology, SAs, and self-harm behavior. However, authors mention that these data should be considered with caution, since such behavior are not regularly reported. Moreover, there are still very few studies speciﬁcally focused on self-harm practices in asylum seekers and refugees in Europe (8). According to Berry (73), in the context of transactional stress and coping models, migration processes invoke a vicious cycle of trauma and isolation that is inﬂuenced by an individual’s resources to cope with stress, as well as by personal and social resources Assessing trauma and trauma- TABLE 3 \| Characteristics of migrants who injure themselves. DISCUSSION All the samples (N = 169) With DSH (N = 45, 26.6%) p N N (%) SOCIODEMOGRAPHIC Age groups 18–35 136 37 (27.2%) ns 36–68 33 8 (24.2%) Gender Male 130 33 (25.4%) ns Female 39 12 (30.8%) Civil status Single 124 33 (26.6%) <0.05 Married 29 4 (13.8%) Other (separated, divorced, widowed) 16 8 (50.0%) Religious Yes 154 42 (27.3%) ns No 4 1 (25.0%) Occupational status Unemployed 131 43 (32.8%) <0.01 Employed or student 38 2 (5.3%) Education Illiterate 20 5 (25.0%) ns Primary 41 13 (46.7%) Lower/upper secondary 83 21 (25.3%) Bachelor’s degree or equivalent 20 4 (20.0%) CLINICAL CHARACTERISTICS PTSD (PCL-5) Yes 73 30 (41.1%) <0.001 No 96 15 (15.6%) Anxiety (SAS) Yes 103 35 (34.0%) <0.01 No 66 10 (15.2%) Depression (SDS) Yes 96 30 (31.3%) ns No 73 15 (20.5%) Suicidal ideation (BSSI 1–5) Yes 49 22 (44.9%) <0.001 No 120 23 (19.2%) Hopelessness (BHS) Yes 110 38 (34.5%) <0.001 No 59 7 (11.9%) Low resilience (CD-RISC2) Yes 105 37 (35.2%) <0.001 No 64 8 (12.5%) Lifetime substance use (yes) Yes 20 7 (35.0%) ns No 135 32 (23.7%) Bold values indicate statistical signiﬁcance at p < 0.05, p < 0.01, and p < 0.001. DSH, deliberate self-harm; PTSD, post-traumatic stress disorder; PCL-5, Posttraumatic Stress Disorder Checklist for DSM-5; SAS, Zung Self-Rating Anxiety Scale; SDS, Z ng Self Rating Dep ession Scale BSSI Beck Scale fo S icidal Ideation BHS Beck TABLE 4 \| Univariate log-binomial regression models for DSH behavior. TABLE 3 \| Characteristics of migrants who injure themselves. LE 4 \| Univariate log-binomial regression models for DSH behavior TABLE 5 \| Multivariate log-binomial regression models for DSH behavior. Variable PR adj [95% CI—inf] [95% CI—sup] p-value Model 1a Age group (ref. 36+) 1.16 0.44 3.04 ns PTSD (PCL-5) 3.75 1.77 7.94 <0.01 Low resilience (CD-RISC2) 3.91 1.63 9.41 <0.001 Model 1b** Age group (ref. 36+) 1.21 0.67 2.19 ns PTSD (PCL-5) 2.21 1.30 3.75 <0.01 Low resilience (CD-RISC2) 2.32 1.16 4.62 <0.01 Hopelessness (BHS) 2.01 0.95 4.22 ns Bold values indicate statistical signiﬁcance at p < 0.05, p < 0.01, and p < 0.001. DSH, deliberate self-harm; PR, prevalence ratio; PTSD, post-traumatic stress disorder; PCL-5, Posttraumatic Stress Disorder Checklist for DSM-5; CD-RISC2, Connor-Davidson Resilience Scale 2; BHS, Beck Hopelessness Scale; VIF, variance inﬂation factor. Where not explicit, “absence” is considered as reference category. Frontiers in Psychiatry \| www.frontiersin.org DISCUSSION *VIF: Age group (1.031); PTSD (1.079); Resilience (1.066); Hopelessness (1.110). minorities (32). Our results indicate that DSH behaviors are associated with SAs and SUI. Data suggest that DSH may increase the risk of SUI and behavior. A previous investigation conducted by Kalt et al. (72) in detentions centers showed a correlation between trauma and violence experienced during the migration processes and some psychopathological conditions like PTSD, psychosomatic symptomatology, SAs, and self-harm behavior. However, authors mention that these data should be considered with caution, since such behavior are not regularly reported. Moreover, there are still very few studies speciﬁcally focused on self-harm practices in asylum seekers and refugees in Europe (8). According to Berry (73), in the context of transactional stress and coping models, migration processes invoke a vicious cycle of trauma and isolation that is inﬂuenced by an individual’s resources to cope with stress, as well as by personal and social resources. Assessing trauma and trauma- related symptoms in migrants can contribute to a better understanding of the psychological correlates of self-injury, September 2021 \| Volume 12 \| Article 529361 Frontiers in Psychiatry \| www.frontiersin.org 7 DSH in Trauma-Affected Migrants Baralla et al. speciﬁcally enabling further examination of trauma symptoms as an underlying mechanism. particularly in trauma-exposed migrants, is a necessary step in helping healthcare professionals to identify the phenomenon and to intervene. In particular, the exposure to catastrophic stress experiences related to persecution, war, or organized violence, circumstances where escape is unfeasible due to physical, psychological, environmental, or social constraints, may result in complex trauma or complex PTSD (CPTSD) (74, 75) (ICD-11; DSM- 5). The construct of the CPTSD has drawn attention to the psychological consequences of interpersonal, prolonged, repeated, and extreme traumatization (74, 76). Many symptoms founded in our sample (e.g., negative self-views and negative expectations about the self and the world) could be related to CPTSD, which can be highly prevalent among immigrants and refugees [e.g., (77)]. It is important to evaluate PTSD and CPTSD, because trauma and complex trauma-related symptomatology may correlate with diﬀerent conditions and require distinct interventions in mental healthcare with immigrant and refugees [e.g., (78)]. Such ﬁndings underlined the relevance of ensuring eﬀective assessment of trauma- exposed migrants at the ﬁrst stages, in order to reduce their psychological burden (79). DISCUSSION In 2018, the increasing number of migrants in Europe has requested strategic actions like the “Migration and Health Programme” introduced by the WHO European Region to support the healthcare professionals in providing more prompt and robust responses to the needs of migrants. The high prevalence of DSH in trauma-exposed migrants highlights the importance of routine assessments of these behaviors among this population. It is important that clinicians and physicians, including those in family medicine and primary care settings, are familiar with the association between PTE, PTSD, and DSH in order to implement programs focusing on prevention. Finally, we would like to emphasize the need for future research to investigate the diﬀerent prevalence of self-harm across cultures, as well as its cultural meanings and functions, in particular focusing on qualitative studies, which are better able to explore the subjective meanings of the asylum seekers’ experiences and the deep functions of self-harm practices. AUTHOR CONTRIBUTIONS MS conceived of the present study, participated in its design and coordination, and helped to revise the manuscript. FB devised the larger study from which this subset of data is drawn, wrote the paper, and helped to statistical analysis and to revise the manuscript. NN and CM conceived and designed the study, and wrote the paper. MV helped devise the larger study from which this subset of data is drawn, revised the manuscript, and conducted the statistical analysis. AP and ADN revise the statistical analysis and helped to draft the paper. All authors read and approved the ﬁnal manuscript. The identiﬁcation of DSH and/or SA would be particularly useful given the danger of these behaviors and the reduced life expectancy associated with them. Gaining a better understanding of what percentage of those who self-harm also attempt suicide, ETHICS STATEMENT The studies involving human participants were reviewed and approved by Comitato Etico Istituto Superiore della Sanità (Prot. PRE/17). The patients/participants provided their written informed consent to participate in this study. Our study presents some limitations. First, the study was a cross-sectional design, and the sample is a nonclinical convenience sample, mainly made up of males, not representative of migrant populations. Therefore, migrants with diﬀerent levels of severity of psychiatric symptomatology may not be included in our sample. Furthermore, this study did not have a large enough sample to allow the ﬁnding to be generalized to general migrant populations. Finally, our decision to dichotomize most of the variables to obtain more robust estimates and to make the interpretation easier may have produced an information loss about collected data, albeit modest. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. 7. Brown RC, Plener PL. Non-suicidal self-injury in adolescence. Curr Psychiatry Rep. (2017) 19:20. doi: 10.1007/s11920-017-0767-9 6. Haw C, Hawton K, Houston K, Townsend E. Psychiatric and personality disorders in deliberate self-harm patients. Br J Psychiatry. (2001) 178:48–54. doi: 10.1192/bjp.178.1.48 7. Brown RC, Plener PL. Non-suicidal self-injury in adolescence. Curr Psychiatry Rep. (2017) 19:20. doi: 10.1007/s11920-017-0767-9 8. Gargiulo A, Tessitore F, Le Grottaglie F, Margherita G. Self-harming behaviours of asylum seekers and refugees in Europe: a systematic review. Int J Psychol. (2021) 56:189–98. doi: 10.1002/ijop.12697 9. Owens D, Horrocks J, House A. Fatal and non-fatal repetition of self-harm. Systematic review. Br J Psychiatry. (2002) 181:193–9. doi: 10.1192/bjp.181.3.193 10. Hawton K, Saunders KE, O’Connor RC. Self-harm and suicide in adolescents. Lancet. (2012) 379:2373–82. doi: 10.1016/S0140-6736(12)60 322-5 11. Beutel ME, Junger C, Klein EM, Wild P, Lackner PJ, Blettner M, et al. Depression, anxiety and suicidal ideation among 1st and 2nd generation REFERENCES (2014) 25:63–71. doi: 10.1093/eurpub/cku121 12. 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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. 66. SAS Institute Inc. Base SAS R⃝9.3 Procedures Guide. Cary, NC: SAS Institute Inc. (2011). 67. Weismoore JT, Esposito-Smythers C. The role of cognitive distortion in the relationship between abuse, assault, and non-suicidal self- injury. J Youth Adolesc. (2009) 39:281–90. doi: 10.1007/s10964-009- 9452-6 Copyright © 2021 Baralla, Ventura, Negay, Di Napoli, Petrelli, Mirisola and Sarchiapone. 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 Psychiatry \| www.frontiersin.org September 2021 \| Volume 12 \| Article 529361 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. 68. Janoﬀ-Bulman R. Assumptive worlds and the stress of traumatic events: applications of the schema construct. Soc Cogn. (1989) 7:113–36. doi: 10.1521/soco.1989.7.2.113 69. Plener PL, Fegert JM. Nonsuicidal self-injury: a condition for further study. Child Adolesc Psychiatry Ment Health. (2015) 9:30. doi: 10.1186/s13034-015-0067-2 Frontiers in Psychiatry \| www.frontiersin.org September 2021 \| Volume 12 \| Article 529361 10
https://openalex.org/W4241808480	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/F3FE4D85019710DE41CF3BA41989C512/S095560360000194Xa.pdf/div-class-title-good-practice-of-ect-administration-div.pdf	English	null	[No Title]	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	2,003	cc-by	1,223	Columns Correspondence Columns Correspondence the area. However, we stand by our conclusions. November 2002, 26, 414^418). It mirrored the results of a recent audit we did on atypical, high dose and co-prescribing of antipsychotics, as well as concurrent anticholinergic prescribing. Drug card information on all general adult inpatients, aged 18^65, prescribed an antipsychotic on a regular basis was collected during a single visit in April 2000. Re-audit occurred in June 2001, and on this occasion adult day hospital patients were included. also depends on the individual’s response to the muscle relaxant and therefore may not accurately reflect the duration of seizure activity in the brain. It is preceisely because we wished to understand the heterogeneity of results from good services and good studies that we undertook our work. We examined 20 components of home-based care in experimental and control services of published studies. These included, but were not restricted to, ACT studies. Our results (Burns et al, 2001; Catty et al, 2002) show that the clear distinction between ACTand other forms of intensive home-based care that Professor Stein and colleagues insist on, and which Marshall and colleagues (Marshall et al, 1997; Marshall & Lockwood, 1998) used, simply will not withstand scrutiny. Many of the ACT teams did not have all the ‘ACT’char- acteristics and many of the ‘non-ACT’ teams had more of them. In short, the groups overlap. It is for this reason that we conducted our regression analysis to determine which components are asso- ciated with outcome. Many of these ‘effective ingredients’ were present in later study control services. We would argue that this is why big differences are no longer being found in US or European studies. Using motor seizures as a measure of effectiveness of the ECT was an excellent idea in the days of unmodified ECT. It must not be forgotten that muscle relaxants are used precisely to stop the convulsions, and therefore it would be erroneous to use the absence of motor seizures as evidence of inadequate stimulation. Overall, 231 inpatients received a regular prescription for an antipsychotic drug in 2000 and 321 patients in 2001 (213 in-patients and 99 day hospital patients). Columns Correspondence There were high levels of atypical antipsychotic prescribing (49%, 95% confidence interval [CI] 42^55 in 2000 and 52%, 95% CI 46^57 in 2001), co-prescribing (20%, 95% CI 15^26 in 2000 and 25, 95% CI 20^30 in 2001) and above British National Formulary (2002) limits prescribing (15%, 95% CI 11^20 in 2000 and 17%, 95% CI 12^21 in 2001) when compared to total antipsychotic prescribing. Furthermore, there were high levels of anticholinergic prescribing with atypical antipsychotics alone (21, 95% CI 13^31 in 2000 and 18%, 95% CI 12^26 in 2001). Either electroencephalogram (EEG) monitoring, or if resources do not allow this, use of the simple cuff method would be the best way of monitoring the duration of seizures. The fact the re- stimulation resulted in induction of adequate seizures in only 25% of cases demonstrates that it may not always be the best course of action. Lalla & Milroy (1996) have published a comprehensive review of the literature about seizure duration in the practice of ECT. Their inference from the review is that there is a lack of good evidence to support the contention that longer ECT seizures are more efficacious. LALLA, F. R. & MILROY,T. (1996) The current status of seizure duration inthe practice of electroconvulsive therapy. CanadianJournal of Psychiatry, 41, 299^304. None of this is to take away from the epoch-making impact of Stein and Test’s original study. If we are to progress in our understanding of community care, however, we must be prepared to test more sharply-focused questions with increasingly well-designed studies. These prescribing patterns increase the risk of side-effects and negate the cost benefits of atypical antipsychotics. They also run counter to 1993 Royal College of Psychiatrists guidelines on antipsychotic prescribing and World Health Organization anticholinergic prescribing guidelines (World Health Organization, 1990; Barnes, 1990). This audit appears to indicate problems in dissemination and a lack of widespread knowledge of current guidelines. Prabhat Mahapatra Consultant Psychiatrist, Care Principles, Dover Road, Barham, Canterbury, CT4 6PW Prabhat Mahapatra Consultant Psychiatrist, Care Principles, Dover Road, Barham, Canterbury, CT4 6PW BURNS,T., KNAPP, K., CATTY, J., et al (2001) Home treatment for mental health problems: a systematic review. HealthTechnologyAssessment, 5,1^139. Dr Mahapatra is correct in his assertion that the method of ECT administration in the audit described was common practice at that time. Indeed in one survey (Robertson et al, 1997), 58% of clinics in Scotland did not use the cuff technique. Columns Correspondence Dr Mahapatra is correct in his assertion that the method of ECT administration in the audit described was common practice at that time. Indeed in one survey (Robertson et al, 1997), 58% of clinics in Scotland did not use the cuff technique. CATTY, J., BURNS,T., KNAPP, K., et al (2002) Home treatment for mental health problems: a systematic review. Psychological Medicine, 32, 383^401. BARNES,T. R. (1990) CommentontheWHOconsensus statement: prophylactic use of anticholinergics in patients on long-term neuroleptic treatment. British Journal of Psychiatry,156, 412. MARSHALL, M. & LOCKWOOD, A. (1998) Assertive CommunityTreatment for People with Severe Mental Disorders. Oxford:The Cochrane Library. One of the purposes of audit was to examine the effects of a different ECT machine and further training on rates of missed fits. A significant reduction in missed fits was observed. , GRAY, A., LOCKWOOD, A., et al (1997) Case Managementfor PeoplewithSevereMentalDisorder. Oxford:The Cochrane Library. BRITISH NATIONAL FORMULARY (2002) BNF 43, March. London: British Medical Association & Royal Pharmaceutical Society. As discussed in my paper, stimulus titration and EEG monitoring could be considered in future practice and further audit could be carried out. ROYALCOLLEGEOFPSYCHIATRISTS (1993) Consensus Statement on the Use of High DoseAntipsychotic Medication. Council Report CR26. London: Royal College of Psychiatrists. Tom Burns Professor of Community Psychiatry, Jocelyn Catty Research Fellow, Social and Community Psychiatry, Department of General Psychiatry, St George’s Hospital Medical School, JennerWing, CranmerTerrace, London, SW17 0RE Tom Burns Professor of Community Psychiatry, Jocelyn Catty Research Fellow, Social and Community Psychiatry, Department of General Psychiatry, St George’s Hospital Medical School, JennerWing, CranmerTerrace, London, SW17 0RE WORLD HEALTH ORGANIZATION (1990) Prophylactic Use of Anticholinergics in Patients on Long-term NeurolepticTreatment. A Consensus Statement. Geneva:World Health Organization. ROBERTSON, C., FREEMAN, C. P. L. & FERGUSON, G. (1997) ECT in Scotland. Psychiatric Bulletin, 21, 699^702. ROBERTSON, C., FREEMAN, C. P. L. & FERGUSON, G. (1997) ECT in Scotland. Psychiatric Bulletin, 21, 699^702. Good practice of ECT administration Tom MacEwan Consultant in Old Age Psychiatry, Old Age Psychiatry Directorate, Royal Cornhill Hospital, Clerkseat Building, Aberdeen, AB25 2ZH EmmaWhicher Senior House Officer, Jane Riley Pharmacist, Leeds Community Mental Health Trust,TheAndrew Sims Centre, High Royds Hospital, Bradford Road, Menston LS29 6AQ The audit cycle described by MacEwan (Psychiatric Bulletin, September 2002, 26, 337^339) demonstrates a common error in the practice of electroconvulsive therapy (ECT) administration. Audit of antipsychotic and anticholinergic prescribing: Leeds CMHT 2000-1 From couch to coach Patients who had brief or absent seizures were re-stimulated with higher charges in the practice described by MacEwan. What has not been taken into account is that duration of convulsions The Football Association (FA) launched its strategy ‘‘Psychology for Football’’ at the Pride Park Stadium, Derby, on We read with interest the paper by Harrington et al (Psychiatric Bulletin, 156 https://doi.org/10.1192/pb.27.4.156-a Published online by Cambridge University Press
https://openalex.org/W4313586098	https://file.techscience.com/files/iasc/2023/TSP_IASC-36-2/TSP_IASC_28481/TSP_IASC_28481.pdf	English	null	Sensor-Based Gait Analysis for Parkinson’s Disease Prediction	Intelligent automation and soft computing/Intelligent automation & soft computing	2,023	cc-by	7,570	Sensor-Based Gait Analysis for Parkinson’s Disease Prediction Sathya Bama B* and Bevish Jinila Y Sathyabama Institute of Science and Technology, Chennai, 600119, Tamil Nadu, India *Corresponding Author: Sathya Bama B. Email: sathyabama2022@outlook.com Received: 10 February 2022; Accepted: 11 April 2022 Abstract: Parkinson’s disease is identiﬁed as one of the key neurodegenerative disorders occurring due to the damages present in the central nervous system. The cause of such brain damage seems to be fully explained in many research studies, but the understanding of its functionality remains to be impractical. Speciﬁcally, the development of a quantitative disease prediction model has evolved in recent decades. Moreover, accelerometer sensor-based gait analysis is accepted as an important tool for recognizing the walking behavior of the patients during the early prediction and diagnosis of Parkinson’s disease. This type of minimal infrastructure equipment helps in analyzing the Parkinson’s gait properties without affecting the common behavioral patterns during the clinical practices. Therefore, the Accelerometer Sensor-based Parkinson’s Disease Identi- ﬁcation System (ASPDIS) is introduced with a kernel-based support vector machine classiﬁer model to make an early prediction of the disease. consequently, the proposed classiﬁer can easily predict various severity levels of Parkinson’s disease from the sensor data. The performance of the proposed classiﬁer is com- pared against the existing models such as random forest, decision tree, and k-near- est neighbor classiﬁers respectively. As per the experimental observation, the proposed classiﬁer has more capability to differentiate Parkinson’s from non-Parkinson patients depending upon the severity levels. Also, it is found that the model has outperformed the existing classiﬁers concerning prediction time and accuracy respectively. Keywords: Brain disorders; gait analysis; Parkinson’s disease; support vector machine classiﬁer; healthcare system Intelligent Automation & Soft Computing DOI: 10.32604/iasc.2023.028481 Article Intelligent Automation & Soft Computing DOI: 10.32604/iasc.2023.028481 Article Intelligent Automation & Soft Computing DOI: 10.32604/iasc.2023.028481 Article ech T Press Science 1 Introduction According to recent research studies, Parkinson’s disease is considered to be the second most common chronic neurodegenerative disease in the world. This particular disease can be characterized by primary motor dysfunctions such as tremors, stiffness, bradykinesia, and postural instability in elderly people [1]. In addition, some of the secondary motor symptoms such as micrographia, dysphagia, gait, speech, dystonia, and precision grip impairments may also worsen the life quality of people [2]. Nowadays clinicians show more emphasis on non-motor symptoms such as mood dysfunction, hyposmia, cognitive This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2086 IASC, 2023, vol.36, no.2 IASC, 2023, vol.36, no.2 impairments, pain, and so on. Some of the described motor symptoms are not speciﬁc and they are not associated with Parkinson’s disease as well. However, the assessment of non-motor symptoms is more crucial in clinical practice and has a direct impact on the progression of Parkinson’s disease [3]. Usually, the person affected by Parkinson’s disease could be above the age of sixty and may walk with a slower and shufﬂing gait speed. Progressively, there is a need to evaluate and monitor the increasing changes occurring in motor dysfunction using wearable sensors such as accelerometers, magnetometers, and gyroscopes. Spatio-temporal parameters such as gait velocity, cadence, time, distance, stride length, and step length are seemed to be the major descriptions of the gait cycle [4]. The severity of these Parkinson’s symptoms may vary considerably throughout the day which needs continuous monitoring and evaluation for a longer period. Moreover, prolonged stays in the hospital may lead to more ﬁnancial costs, and also the evaluation could not imitate the real motor symptoms of patients with Parkinson on every day. Importantly, this type of increased burden and ﬁnancial issues are the most challenging tasks to be considered among the biomarkers to effectively monitor and diagnose Parkinson’s patients with proper medical care [5]. To overcome the above limitations, various healthcare examinations are imposed by the clinicians to measure Parkinson’s disease symptoms without increasing the expenses and complexities. These types of early identiﬁcation and diagnosis could overcome the complications of late clinical treatment which can alleviate the unwanted ﬁnancial cost, social demand, family burden, etc. 1 Introduction The current research studies have focused on developing computer-assisted technologies for Parkinson’s disease-related prediction, monitoring process, and diagnosis in the aspects of checking voice disorder, facial expression, handwriting dynamics, freezing-of-gait, and anxiety depression [6]. To check the voice disorder, speech processing and recognition are explored in the cloud-based framework to enable remote healthcare applications for Parkinson’s disease patients [7]. Facial emotion recognition is identiﬁed in Parkinson’s patients by analyzing the cognitive impairment and giving more preference to mouth and eye movement behaviors [8]. The facial expression and movement analysis rely mainly on a video-based system to keep track of the facial movements recorded in successive video frames over a speciﬁed period of time [9]. Handwriting dynamics are explored using machine learning techniques to assess various static and dynamic spiral tests conducted over the patients [10]. Preferably gait analysis is used to classify a Parkinson’s person easily from healthy persons based on gait pattern recognition by deploying sensors under the foot [11]. Similarly, the gait analysis can be done through the deployment of accelerometer sensors in the hip, knee, and ankle. The experimental analysis of human motion can be captured through the subsequent observation of the position, velocity, and acceleration of independent coordinates of the body base [12]. All the above healthcare systems help in improving the quality of life by providing classiﬁcation, prediction, and rehabilitation monitoring on Parkinson’s patients. According to the recent research studies [13–15], the most important issues are highlighted for enhancing the healthcare systems; (a) insufﬁcient prediction and prevention mechanisms for freezing of gait, (b) lack of consistent monitoring and validation of wearable sensor information, (c) lack of optimal placement of wearable sensor for continuous monitoring, (d) inappropriate feature selection scheme for supporting disease prediction and diagnosis, (e) lack of appropriate behavioral assessment scheme to identify the cognitive impairments of Parkinson patients. Therefore, the proposed research study has focused on addressing the above-mentioned research issues to enhance the performance of the Parkinson’s disease prediction and diagnosis process. Since Parkinson’s disease is a progressive disorder, a proposed research study has aimed to offer symptomatic relief and slow down the progression of the disease among elderly people. 2 Related Work Many research studies exploit wearable sensors for promoting remote monitoring and diagnosis of Parkinson’s disease by effectively quantifying the motor symptoms [16,17]. To discriminate against Parkinson’s patients, a low-cost smartphone is used to practice the voice, gait, posture, and ﬁnger tapping exercises at least four times a day [18]. Moreover, several researchers have been dependent on machine learning and deep learning techniques in order to develop remote healthcare systems for robust prediction and diagnosis. In the machine learning context, a novel continuous wavelet transform-based complex plot was introduced during the gait analysis for the diagnosis of idiopathic Parkinson’s disease. During experimentation, an artiﬁcial neural network classiﬁer model was explored along with the temporal features so as to recognize the severity stages of the disease [19]. A Gaussian neural network classiﬁer was used in a recent research study for identifying Parkinson’s by detecting the freezing of gait information as a major symptom that can acquire the variation of knee angle or inter-foot distance [20]. Similarly, a rule-based processing classiﬁer was used to differentiate the normal and abnormal freezing of gait patterns by processing the data observed from the sensor placed on the shank [21,22]. In particular, it can easily describe the freezing of gait with a short stride, tremor, and motor block to give a warning message for severe gait and near fall situations. To further differentiate the Parkinson’s tremor from the essential tremor, a convolutional long and short- term memory network classiﬁer was explored by learning both the postural and resting positions of hand tremor [23]. In order to classify the severity of Parkinson’s symptoms in a proper way, a Convolutional Neural Network classiﬁer was employed with a tremor assessment system for processing the wearable sensor devices [24]. Apart from these techniques, a probabilistic classiﬁer model was used along with the ﬁsher discriminant ratio-based feature selection method to observe the gait patterns without any statistical errors [25]. Moreover, the discriminative feature selection helps in generating more accurate prediction results than the existing classiﬁer models. In a deep learning context, gait abnormal pattern detection was explored using the deep neural network- based framework and the wearable inertial sensors [26]. It helps to achieve more prediction accuracy and less loss function value than the existing convolution neural network-based classiﬁer model. 1 Introduction The key contributions of this research study include; (a) the novel architecture of PDIDS to predict the various severity levels of Parkinson’s disease IASC, 2023, vol.36, no.2 2087 and choose an appropriate diagnosis method pertinent to levels; (b) a feature selection mechanism is applied to select the appropriate features during disease prediction; (c) a probabilistic classiﬁer model is proposed for classiﬁcation of various levels of Parkinson disease by improving the prediction time and accuracy. The rest of the research study is organized into ﬁve sections. The next section can give a complete literature survey on various feature selection mechanisms and classiﬁer models. In Section 3, a detailed description of the proposed PDIDS architecture is given with the formulation of gait recognition, feature selection, and classiﬁcation problems. Section 4 gives complete real-time experimentation and results of the proposed research study. The ﬁnal section could explain the conclusion of the proposed research study and the future development of the Parkinson’s healthcare system. 3 Architecture of Accelerometer Sensor Based Parkinson’s Disease Identiﬁcation System The proposed research study has introduced the layered architecture of ASPDIS as given in Fig. 1. It consists of a sensor and cloud computing layer to implement the accelerometer sensor-based gait analysis for real-time Parkinson’s disease-related prediction and treatment monitoring activity. For carrying out the process, tri-axial accelerometer sensors are placed on the hip, knee, and ankle location of the human body for observing various sequences of acceleration signals with respect to X, Y, and Z axes. These signals are periodically captured in the raspberry pi computing device and ofﬂoaded to remote the cloud servers available in the cloud computing layer for immediate processing and diagnosis. Also, the health status information will frequently be updated on the patient smartphone display device. After live observation of accelerating the data, the proposed healthcare service deployed in the cloud layer will apply the kernel- based support vector machine classiﬁer model to analyze the gait patterns. Based on the previously trained medical data, the classiﬁer can easily discriminate the patient with and without Parkinson’s disease. In the meantime, subsequent variations in health parameters are updated in the patient medical history to help the practitioner during future prediction and diagnosis. According to the severity level, the healthcare service will invoke the appropriate rehabilitation monitoring and assessment facilities for the patients. Furthermore, it can provide telemedicine suggestions and prescriptions to the appropriate patients based on the expert decisions available online. Accordingly, the formulation of gait cycle recognition and disease prediction using the proposed classiﬁer is mathematically represented with the corresponding cycle-speciﬁc to parameters. 2 Related Work A temporal layer was introduced to model the sequential sensor data to learn the complete changes occurring due to the hidden unit present in the recurrent neural network classiﬁer model [27,28]. This model requires the consistent update of current states which may be difﬁcult to train and explore the sequence of long-time activities of sensor data. In the case of deep neural networks, more hidden layers are presented than any other classiﬁer model. It mainly helps in learning the human activities from a large amount of data [29]. A deep convolutional neural network classiﬁer was used for the detection of freezing of gait patterns from the acceleration signals captured from various patients [30]. Mostly, gait activity recognition becomes more challenging due to problematic and imbalanced real-time datasets received from wearable sensors. For overcoming the 2088 IASC, 2023, vol.36, no.2 IASC, 2023, vol.36, no.2 challenges, an ensemble of deep long, and short-term memory network classiﬁers were used to combine the set of learners during the time of classiﬁcation [31]. An active deep learning model was introduced to improve the accuracy and minimize the time during the recognition of human gait activities [32,33]. The major limitations of these machine and deep learning studies have included high-cost sensors installation and computational resources for gait analysis. Moreover, foot pressure sensor-based measurements cannot be sufﬁcient to acquire the Parkinson’s gait pattern. There is a need to incorporate angular motions of the human body by placing the sensors in the joints of the knee, hip, and ankle pants. The study of healthy and Parkinson’s persons needs to be done in different walking styles to judge the deviation among two classes. Therefore, the proposed research study has planned for making gait analysis on human subjects by deploying tri-axial accelerometer sensors on the hip, knee, and ankle positions. 3.1 Formulation of Gait Cycle Recognition IASC, 2023, vol.36, no.2 2090 xt ¼ xt 1 n X n t¼1 xt (2) xtþT ¼ xtþT 1 n X n t¼1 xtþT (3) xt ¼ xt 1 n X n t¼1 xt (2) xtþT ¼ xtþT 1 n X n t¼1 xtþT (3) xt ¼ xt 1 n X n t¼1 xt (2) xtþT ¼ xtþT 1 n X n t¼1 xtþT (3) xt ¼ xt 1 n X n t¼1 xt xtþT ¼ xtþT 1 n X n t¼1 xtþT (2) (3) Next, the anisotropy measurement is deﬁned in Eq. (4) to quantify the degree of time series data variation towards the positive or negative value. Finally, the biphasicity score BS of three-dimensional signal is given by the Eq. (5). The set of time functions characterizing the gait signal is given in the form of delta function such as f1 tð Þ ¼ cxd tð Þ, f2 tð Þ ¼ cyd tð Þ, and f3 tð Þ ¼ czd tð Þ. AN ¼ Pn t¼1 sgn xt ð Þ Pn t¼1 sgn xt ð Þ j j (4) BS ¼ 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 2 2cy cx 2cy þ cx m þ 1 2 2cz cx 2cz þ cx m m s (5) AN ¼ Pn t¼1 sgn xt ð Þ Pn t¼1 sgn xt ð Þ j j (4) BS ¼ 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 2 2cy cx 2cy þ cx m þ 1 2 2cz cx 2cz þ cx m m s (5) Let cx, cx, and cx denotes the corresponding cycles of axes X, Y, and Z. Let cx, cx, and cx denotes the corresponding cycles of axes X, Y, and Z. The acceleration magnitude of the signal can be obtained through the ﬁltering of component ax tð Þay tð Þaz tð Þ using the Eq. (6). In order to identify the human fall rotation, it is more sufﬁcient to make transformations on x and y axes in the two-dimensional coordinate as shown in Eq. (7). 3.1 Formulation of Gait Cycle Recognition A gait cycle speciﬁes the time period of one of the foot contacts with the ground to the next foot. So, to get the complete gait cycle out of sensor data, the time of each gait event must be determined according to the maximum scale of interest Tmax. To measure the acceleration signals from the subject, a pbleal accelerometer sensor is ﬁxed in the front of the center to represent the ﬁxed belt. The orientation of acceleration axes X, Y, and Z represents the medial/lateral, superior/inferior, and anterior/posterior respective forward opens values of these axes correspond to the life, upward and forward opens elevations. It is important to consider the sequence of three-dimensional output signal points xtytzt ½ the received from the tri-axial athe the ccelerometer sensors during gait monitoring process. Then, the gait event detection can be done through the measurement of normalized cross-correlation (NCC), anisobiphasicd biphasicity score (BS). The similarity between any two sequences with respect to time period T is characterized as a normalized cross-correlation function given by Eq. (1). 2089 IASC, 2023, vol.36, no.2 Figure 1: Architecture of ASPDIS Figure 1: Architecture of ASPDIS Figure 1: Architecture of ASPDIS Figure 1: Architecture of ASPDIS NCC T ð Þ ¼ Pn t¼1 xtxtþT þ ytytþT þ ztztþT ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pn t¼1 x2 t þ y2 t þ z2 t Pn t¼1 x2 tþT þ y2 tþT þ z2 tþT q (1) NCC T ð Þ ¼ Pn t¼1 xtxtþT þ ytytþT þ ztztþT ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pn t¼1 x2 t þ y2 t þ z2 t Pn t¼1 x2 tþT þ y2 tþT þ z2 tþT q (1) Here, the average value of points xt and xtþT are computed from the base points as deﬁned by Eqs. (2) and (3) respectively. Similarly, the average value of points yt, zt, ytþT and ztþT are also computed from the corresponding base points. These types of scalar quantity-based measurements provide a robust way of setting the coordinate system. Likewise, the mean value of the acceleration signal is observed to be high for dynamic activities such as hand movement and walking patterns. In case of low value of acceleration signal, static activities such as standing and sitting patterns are observed. 3.1 Formulation of Gait Cycle Recognition kCF a tð Þ ð Þ ¼ max a tð Þ ð Þ ﬃﬃﬃ 1 k r Pk t¼1 a tð Þ ð Þ2 CC a tð Þl i; a tð Þm j ¼ a tð Þl i a tð Þm j al i am j SD a tð Þl i SD a tð Þm j kCF a tð Þ ð Þ ¼ max a tð Þ ð Þ ﬃﬃﬃ 1 k r Pk t¼1 a tð Þ ð Þ2 (14) CC a tð Þl i; a tð Þm j ¼ a tð Þl i a tð Þm j al i am j SD a tð Þl i SD a tð Þm j (15) where i and j denotes the number of accelerometer sensors, and l ¼ Parkinson0s and m ¼ xtytzt ½ denotes the ario s a es of accelerometer (14) (15) where i and j denotes the number of accelerometer sensors, and l ¼ Parkinson0s and m ¼ xtytzt ½ denotes the various axes of accelerometer. 3.1 Formulation of Gait Cycle Recognition ar tð Þ ¼ ax tð Þ2 þ ay tð Þ2 þ az tð Þ2 h i0:5 (6) h ¼ tan1 yv xv 180 p (7) ar tð Þ ¼ ax tð Þ2 þ ay tð Þ2 þ az tð Þ2 h i0:5 h ¼ tan1 yv xv 180 p (6) (7) An integration of acceleration signals is explored to authenticate the subject during continuous monitoring and diagnosis using the Eq. (8). An integration of acceleration signals is explored to authenticate the subject during continuous monitoring and diagnosis using the Eq. (8). I ¼ arcsin az ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ax tð Þ2 þ ay tð Þ2 þ az tð Þ2 q 0 B @ 1 C A (8) (8) where, t ¼ 1; 2; . . . ; k be the number of observations recorded at various time periods. Then, the absolute distance metric is measured to ﬁnd the similarity score between the gait samples p and q as given by the Eq. (9). dist p; q ð Þ ¼ X n i¼1 pi qi j j dist p; q ð Þ ¼ X n i¼1 pi qi j j (9) (9) The complexity of gait movement can be estimated in terms of signal spectrum energy as deﬁned by the Eq. (10). The complexity of gait movement can be estimated in terms of signal spectrum energy as deﬁned by the Eq. (10). The complexity of gait movement can be estimated in terms of signal spectrum energy as deﬁned by the Eq. (10). E a tð Þ ð Þ ¼ Pk t¼1 a tð Þ2 k (10) E a tð Þ ð Þ ¼ Pk t¼1 a tð Þ2 k (10) where, a tð Þ be the tth amplitude of the acceleration signal and k represents the number of spectrum lines. Further, the periodicity can be estimated as entropy measurement as expressed by the Eq. (11). where, a tð Þ be the tth amplitude of the acceleration signal and k represents the number of spectrum lines. Further, the periodicity can be estimated as entropy measurement as expressed by the Eq. (11). where, a tð Þ be the tth amplitude of the acceleration signal and k represents the number of spectrum lines. Further, the periodicity can be estimated as entropy measurement as expressed by the Eq. (11). 3.1 Formulation of Gait Cycle Recognition 2091 IASC, 2023, vol.36, no.2 IASC, 2023, vol.36, no.2 EN ¼ X k t¼1 p tð Þlog2 p tð Þ ð Þ (11) EN ¼ X k t¼1 p tð Þlog2 p tð Þ ð Þ (11) Let p tð Þ denotes the probability of occurrence of amplitude spectrum a tð Þ of the respective acceleration signal. The lower of the entropy value represents the periodicity of the investigated signal. The range of acceleration signals can be represented as standard deviation as expressed by the Eq. (12). For estimating the dynamics of changes occurring in the acceleration signal, a kurtosis parameter is deﬁned by the Eq. (13). SD a tð Þ ð Þ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 k 1 X k t¼1 a tð Þ a ð Þ2 v u u t k a tð Þ ð Þ ¼ m4 a tð Þ ð Þ SD a tð Þ ð Þ2 3 SD a tð Þ ð Þ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 k 1 X k t¼1 a tð Þ a ð Þ2 v u u t (12) k a tð Þ ð Þ ¼ m4 a tð Þ ð Þ SD a tð Þ ð Þ2 3 (13) (12) (13) Let m4 ð Þ be the fourth central movement. A ratio of maximum signal to the rms values gives the crest factor denoting the signal impulsiveness as expressed by the Eq. (14). Also, a correlation coefﬁcient parameter is illustrated between the different pairs of the same accelerometer sensor as expressed by the Eq. (15). inson Disease Prediction using Kernel-Support Vector Machine (SVM) Classiﬁer Model Minw;b;e X k i¼1 /i 1 2 X k i¼1 X k j¼1 /i/jyiyjxT i xj (19) Minw;b;e X k i¼1 /i 1 2 X k i¼1 X k j¼1 /i/jyiyjxT i xj X k i¼1 X k j¼1 /i/jyiyjxT i xj (19) (19) where, the parameter w and b can be determined with constraints such as /i 0, Pk i¼1 /iyi ¼ 0, and i ¼ 1; 2; . . . ; k. Here, the unlabelled data samples can be classiﬁed according to the discriminant function given by Eq. (20). where, the parameter w and b can be determined with constraints such as /i 0, Pk i¼1 /iyi ¼ 0, and i ¼ 1; 2; . . . ; k. Here, the unlabelled data samples can be classiﬁed according to the discriminant function given by Eq. (20). C X ð Þ ¼ sgn X k i¼1 /i yixT i x þ b ! (20) C X ð Þ ¼ sgn X k i¼1 /i yixT i x þ b ! (20) As SVM addresses the non-linear data samples, the kernel function is used mostly to map the samples to the other space. There are four different kernel functions available such as liner, polynomial, radial basis and sigmoid for the classiﬁcation of gait. Out of which, the radial basis function is used to map the lower dimensional data sample to the higher dimensional feature space. Therefore, the non-linear discriminate function using the kernel is expressed by the Eq. (21). In order to complete the transformation, the liner, polynomial, radial basis and sigmoid functions are explored in this study as deﬁned by the Eqs. (22)–(25). C X ð Þ ¼ sgn X k i¼1 /i yi k x1; x2 ð Þ þ b ! (21) k x1; x2 ð Þ ¼ hx1; x2i (22) k x1; x2 ð Þ ¼ hx1; x2i þ b ð Þd (23) k x1; x2 ð Þ ¼ eyikx1x2k2 (24) k x1; x2 ð Þ ¼ tan h yihx1; x2i þ b ð Þ (25) C X ð Þ ¼ sgn X k i¼1 /i yi k x1; x2 ð Þ þ b ! inson Disease Prediction using Kernel-Support Vector Machine (SVM) Classiﬁer Model The classiﬁcation of Parkinson disease is made with respect to acceleration signals received from various accelerometer sensors deployed on the hip, knee and ankle of the human body. In order to effectively assess the human gait analysis, the SVM classiﬁer model is explored in this research study. It helps to classify the set of input parameters a ¼ a1; a2; . . . ; a1 f g into two classes yi 2 1; þ1 f g, where the vector ai ¼ xiyizi ½ . The kernel function ’ ai ð Þ helps to transform the input feature space ai into higher dimensional feature space where the linear clariﬁcation exists. A kernel SVM provides the optimal hyper- plane that can help to maximize the required margin among the positive and negative data points. Therefore, the kernel SVM can be formulated with appropriate decision surface as deﬁned by the Eq. (16). f x ð Þ ¼ wTx þ b (16) where, w be the highest dimensional coefﬁcient vector and b be the offset. Aweight factor w and offset value are deﬁned together for hyper-plane separation as expressed by the Eq. (17). The kernel SVM classiﬁer model tries to achieve the optimal hyper-plane by resolving the optimization problem given by the Eq. (18). IASC, 2023, vol.36, no.2 2092 yi wT’ ai ð Þ þ b 1 ei; i ¼ 1; 2; . . . ; k (17) Minw;b;e 1 2 kwk2 þ C X k i¼1 ei (18) yi wT’ ai ð Þ þ b 1 ei; i ¼ 1; 2; . . . ; k Minw;b;e 1 2 kwk2 þ C X k i¼1 ei (17) (18) Let C be the hyper parameter that controls the tradeoff between the classes margin and ei. The sample ai satisﬁes the conditions such as ei 0, yi wT’ ai ð Þ þ b ð Þ 1 ei and 8ai 2 1; 2; . . . ; m f g. As a result, the optimization problem can be converted into a Lagrangian dual problem using multipliers a ¼ a1; a2; . . . ; an ð Þ as formulated by the Eq. (19). inson Disease Prediction using Kernel-Support Vector Machine (SVM) Classiﬁer Model CMCC ¼ T P T N F P F N ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ T P þ F P T P þ F N T N þ F P T N þ F N r (27) CMCC ¼ T P T N F P F N ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ T P þ F P T P þ F N T N þ F P r (27) Let the value of CMCC could range from −1 to +1, where the value of +1 denotes the perfect, −1 denotes the disagreement and 0 denotes the unhealthy predictions of the speciﬁed classiﬁers. Let the value of CMCC could range from −1 to +1, where the value of +1 denotes the perfect, −1 denotes the disagreement and 0 denotes the unhealthy predictions of the speciﬁed classiﬁers. inson Disease Prediction using Kernel-Support Vector Machine (SVM) Classiﬁer Model k x1; x2 ð Þ ¼ hx1; x2i k x1; x2 ð Þ ¼ hx1; x2i þ b ð Þd k x1; x2 ð Þ ¼ eyikx1x2k2 k x1; x2 ð Þ ¼ tan h yihx1; x2i þ b ð Þ C X ð Þ ¼ sgn X k i¼1 /i yi k x1; x2 ð Þ þ b ! (21) k x1; x2 ð Þ ¼ hx1; x2i (22) k x1; x2 ð Þ ¼ hx1; x2i þ b ð Þd (23) k x1; x2 ð Þ ¼ eyikx1x2k2 (24) k x1; x2 ð Þ ¼ tan h yihx1; x2i þ b ð Þ (25) (21) (22) (24) (25) The performance of the kernel-SVM classiﬁer model is measured in terms of disease prediction time, prediction accuracy and Matthews’s correlation coefﬁcient. Here, the prediction time represents the time of patient request submission to the ending response received from the healthcare system. Then, the classiﬁer prediction accuracy denotes the ratio of properly classiﬁed Parkinson patients to the total number of patients observed as given by the Eq. (26). CAcc ¼ T P þ T N T P þ F P þ T N þ F N (26) 2093 IASC, 2023, vol.36, no.2 IASC, 2023, vol.36, no.2 where, T P and T N indicates the amount of true positive and negative rate, F P and F N indicate the amount of false positive and negative rate. Finally, the Matthews’s correlation coefﬁcient can be measured between the predicted and observed classiﬁcation using confusion matrix as deﬁned by the Eq. (27). where, T P and T N indicates the amount of true positive and negative rate, F P and F N indicate the amount of false positive and negative rate. Finally, the Matthews’s correlation coefﬁcient can be measured between the predicted and observed classiﬁcation using confusion matrix as deﬁned by the Eq. (27). 4 Experimental Evaluations To assess the performance of the proposed classiﬁer model against the existing classiﬁers, a benchmarking dataset is taken from the inertial sensor database [34,35]. It consists of forty-ﬁve persons, equally classiﬁed into control, Parkinson and geriatric persons with characteristics as given in Table 1. The persons were asked to present a 40-meter walk and free walk during the recording of the dataset at the sampling rate of 102.4 Hz. In case of 40-meter walk, the person was asked to walk four times 10 meter at the comfortable speed without any obstacle in the hospital environment. Afterwards, the persons were asked to turn 180 degrees along the transverse plane. In case of free walk, the persons were asked to walk for two minutes at a comfortable speed. During the ﬁrst two minutes, the person was asked to walk straight for 20 meters twice the time. In next two minutes, the person was asked to climb the stairs for 30 s. To mimic the scenarios connecting the 20 meters walk and stair hiking, the persons were asked to perform sit-to-stand movement, walk straight, walk in the curves, and walk throughout the doors manually. In order to provide in-depth estimate, the most excellent performing axes of accelerometer sensor data are used as mentioned in Table 2. Table 1: Sampling persons characteristics Categories Control persons Parkinson persons Geriatric persons Test 40 m walk Free walk 40 m walk Free walk 40 m walk Free walk Persons 10 5 10 5 10 5 Strides 485 1286 496 1619 795 1249 Gender 5:5 3:2 5:5 3:2 4:6 2:3 Age 64.0 ± 8.4 64.2 ± 10.0 63.8 ± 9.3 72.8 ± 6.3 81.0 ± 4.1 80.4 ± 5.9 Table 1: Sampling persons characteristics Table 2: Stride results of acceleration data Table 2: Stride results of acceleration data xes Control persons Parkinson persons Geriatric persons X 67 73 47 Y 53 59 11 Z 56 32 44 XY 80 86 56 YZ 79 68 38 XZ 75 77 38 XYZ 85 93 51 2094 IASC, 2023, vol.36, no.2 IASC, 2023, vol.36, no.2 The performance metrics such as prediction time and accuracy were used to evaluate the proposed kernel-based support vector machine classiﬁer model against the existing random forest, decision tree and k-nearest neighbor classiﬁers. The results of various classiﬁer models were observed during the experimentation of persons in 40 meters walk as given in Table 3. 4 Experimental Evaluations It is more evident from the observed results, the proposed kernel-based support vector machine classiﬁer model on 40 meters walk has obtained an average of 21.43%, 22.10% and 22.10% of less prediction time over the existing classiﬁers in the context of control, Parkinson and geriatric persons respectively. Then, the proposed kernel-based support vector machine classiﬁer model on free walk has obtained an average prediction accuracy improvement of 16.02%, 16.70% and 12.99% over the existing classiﬁers in the context of control, Parkinson and geriatric persons respectively. Table 3: Performance measurement on 40 meters walk Table 3: Performance measurement on 40 meters walk Classiﬁer models Control persons Parkinson persons Geriatric persons Prediction time Prediction accuracy Prediction time Prediction accuracy Prediction time Prediction accuracy Random forest 0.036 77.51 0.035 76.41 0.038 85.31 Decision tree 0.611 76.26 0.628 77.21 0.618 79.36 k-Nearest neighbor 0.056 81.11 0.054 82.21 0.058 83.86 Proposed kernel-based support vector machine 0.020 94.32 0.018 95.31 0.017 95.84 Similarly, the performance of various classiﬁer models is observed during the free walk experimentation of persons as shown in Table 4. From the experimental results, it is distinctly understandable that the proposed classiﬁer model has achieved very less average prediction time of 18%, 18.23% and 18.03% over the existing classiﬁers in the aspects of control, Parkinson and geriatric persons respectively. In addition, the proposed classiﬁer model has achieved more prediction accuracy of 11.74%, 11.78% and 12.99% over the existing classiﬁers in terms of control, Parkinson and geriatric persons respectively. In both the cases of 40 meters walk and free walk, the proposed classiﬁer model has outperformed the existing classiﬁer models in the aspects of prediction time and prediction accuracy. In future, the research study can improve the healthcare system by providing a negotiation-based personalized diagnosis mechanism [36,37]. In addition, the research study can be extended to monitor and diagnose the remote patients by exploring real-time human tracking and activity identiﬁcation mechanisms. Further, the detection methodology can be enhanced using deep learning algorithms for the improvement of prediction accuracy. 4 Experimental Evaluations Table 4: Performance measurement on free walk Classiﬁer models Control persons Parkinson persons Geriatric persons Prediction time Prediction accuracy Prediction time Prediction accuracy Prediction time Prediction accuracy Random forest 0.031 79.81 0.028 82.55 0.030 85.31 Decision tree 0.522 75.89 0.528 78.12 0.518 79.36 (Continued) Table 4: Performance measurement on free walk 2095 IASC, 2023, vol.36, no.2 Table 4 (continued) Table 4 (continued) Classiﬁer models Control persons Parkinson persons Geriatric persons Prediction time Prediction accuracy Prediction time Prediction accuracy Prediction time Prediction accuracy k-Nearest neighbor 0.044 80.34 0.042 81.51 0.041 83.86 Proposed kernel-based support vector machine 0.019 90.42 0.017 92.51 0.016 95.84 5 Conclusion The proposed research study has developed the robust and efﬁcient healthcare framework to validate the gait cycle observed from the accelerometer sensors. Therefore, the observed results have shown that the proposed ASPDIS provides more robust and accurate classiﬁcation of persons with and without Parkinson disease. Moreover, the proposed kernel-based support vector machine classiﬁer model can provide more computational efﬁciency and better prediction results while comparing to the existing classiﬁer models. It can also provide consistent performance even with a smaller number of training samples used during the experimentation process. As a result, the proposed classiﬁer has outperformed the others in terms of prediction time and accuracy. In addition, the proposed ASPDIS could be enhanced to perform well for the recognition of daily living activities of humans in the smart hospitals or homes. Still there is a lack in deploying the optimal number of accelerometer sensors over the human body to assess Parkinson disease motor symptoms. So, there is a need to identify the different combinations of sensor placements during the experimentation in order to dramatically improve the performance of the proposed ASPDIS. In future, freezing of gait and slowness of hand movements can be analyzed for the further improvement in the performance of healthcare systems. A personalized gait pattern analysis can be integrated with the healthcare system to detect and track the freezing of gait information in case of individuals with Parkinson disease. Funding Statement: The authors received no speciﬁc funding for this study. Conﬂicts of Interest: The authors declare that they have no conﬂicts of interest to report regarding the present study. [2] C. Raza, R. Anjum and N. A. Shakeel, “Parkinson’s disease: Mechanisms, translational models and management strategies,” Life Sciences, vol. 226, no. 3, pp. 77–90, 2019. [1] N. Amato, S. Caverzasio and S. 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https://openalex.org/W4389560465	https://link.springer.com/content/pdf/10.1134/S1063772923090032.pdf	English	null	Extremely Wide Pairs in the World of Binary Stars	Astronomy reports	2,023	cc-by	5,983	1. INTRODUCTION ocity star (HVS) whose velocity during ejection allows it not only to escape from the vicinity of the SMBH but also to become unbound from the parent galaxy. Depending on the time elapsed since ejection and the ejection velocity, an HVS may remain within the bounds of the Galaxy, reach its periphery, and even escape beyond its boundaries. Jack Hills was the first to propose such a scenario, providing model esti- mates of the escape velocity of one of the BS compo- nents from the vicinity of the SMBH to several thou- sand km/s [2], in order to draw attention to the phe- nomenon of HVSs, which, with their anomalous kinematics, would serve as indirect evidence of the existence of SMBHs. In the accepted classification of binary stars (BSs) based on their semimajor axes, they are divided into close ( ) and wide ( AU) pairs [1]. According to modern GAIA observations, the upper limit for the semimajor axis of wide BS can be extended to 10 000 AU (0.0485 pc). In the case of close BS, these limits are associated with the phase of deep collapse of the initial rotating gas-dust clouds, and in the case of wide systems, it involves the process of star cluster disintegration or the capture of neighboring stars due to close encounters of binary or single stars. ⊙ < 1000 a R ∼100 a In the systems we are studying, the distance between their components can be comparable to the radius of the Galaxy and even larger. It would be more accurate to say that in the past the components of these pairs could belong to a single parent BS, the con- nection with which was severed. Scenarios in which the links of BSs can be broken are well-known: the dis- ruption of unstable triple systems, the double colli- sions of close BSs, their collisions with single stars, supernova explosions in close BSs, and the dynamic capture of a BS by a supermassive black hole (SMBH). Let us focus on the latter scenario. However, history took its course, and S-stars [3] were discovered first. Thanks to the technological rev- olution in adaptive optics and high-angular-resolution spectroscopy, there was an avalanche of S-star discov- eries. ISSN 1063-7729, Astronomy Reports, 2023, Vol. 67, No. 9, pp. 894–901. © The Author(s), 2023. This article is an open access publication. Russian Text © The Author(s), 2023, published in Astronomicheskii Zhurnal, 2023, Vol. 100, No. 9, pp. 792–799. ISSN 1063-7729, Astronomy Reports, 2023, Vol. 67, No. 9, pp. 894–901. © The Author(s), 2023. This article is an open access publication. Russian Text © The Author(s), 2023, published in Astronomicheskii Zhurnal, 2023, Vol. 100, No. 9, pp. 792–799. Extremely Wide Pairs in the World of Binary Stars G. N. Dremovaa,, V. V. Dremova,, and A. V. Tutukovb,** a Russian Federal Nuclear Center, Snezhinsk, Russia b Institute of Astronomy, Russian Academy of Sciences, Moscow, Russia e-mail: G.N.Dryomova@mail.ru e-mail: V.V.Dryomov@vniitf.ru *e-mail: atutukov@inasan.rssi.ru Received April 20, 2023; revised April 30, 2023; accepted May 18, 2023 Abstract—This article raises the question of the possibility of identifying stellar pairs in which one of the com- ponents belongs to the family of near-nuclear central S-stars and the other belongs to a population of hyper- velocity stars (HVSs). In the recent past, they could be genetically linked in a single parent binary star (BS), and today its components are separated by hundreds or more parsecs as a result of dynamic capture of a BS by the field of a supermassive black hole (SMBH). There is interest in the mutual reconstruction of popula- tions of S-stars and HVSs calculated in the context of the classical Hills scenario, based on the principle of supplementing their observational data. The paper is based on a report presented at the astrophysical memo- rial seminar “Novelties in Understanding the Evolution of Binary Stars,” dedicated to the 90th anniversary of Professor M.A. Svechnikov. Keywords: stellar pairs, hypervelocity stars, Hills scenario, statistical analysis DOI: 10.1134/S1063772923090032 1. INTRODUCTION The triumphant outcome of many years of (starting from 1989) observations of S-stars and non- trivial methods of their analysis, proving the presence of a massive central body—SMBH—led to the award of the Nobel Prize for 2020 to Andrea Ghez and Reinhard Genzel. At the moment, there are about 10000 S-stars in the search map of the Galactic Cen- ter, and routine work is still needed to recover the ele- ments of their orbits [4]. Despite the constantly increasing number of discovered central stars, no more than several dozen S-stars remain informatively Due to the redistribution of angular momentum, which is inevitably encountered in the three-body problem, both binary components can end up in new statuses: a central near-nuclear S-star and a hypervel- 894 EXTREMELY WIDE PAIRS 895 valuable, which surprisingly coincides with the statis- tics of discovered HVSs up to the present time. [9]. Variation of the parameters of the approach of BSs to the SMBH allows the extraction of the maximum and average ejection velocities of stars as HVSs depending on the pericentric distance. The reliability of the obtained values is tested during the modeling of BS in the N-body approach, where each of the BS components is defined by a set of structurally gravitat- ing elements that interact with each other [10]. This approach provides an objective view of the survival of a star as it approaches the SMBH. [9]. Variation of the parameters of the approach of BSs to the SMBH allows the extraction of the maximum and average ejection velocities of stars as HVSs depending on the pericentric distance. The reliability of the obtained values is tested during the modeling of BS in the N-body approach, where each of the BS components is defined by a set of structurally gravitat- ing elements that interact with each other [10]. This approach provides an objective view of the survival of a star as it approaches the SMBH. The phenomenon of HVSs allowed a reevaluation of the kinematic structure of the Galaxy and formu- lated the problem of identifying an object with anom- alous kinematics due to central ejection. Since the first HVS, SDSS J090745.0+024507, was discovered in 2005 by Warren Brown and his colleagues [5], the sta- tistics have grown to 40 stars over the years. 1. INTRODUCTION Conversely, for stars brighter than , GAIA provides 3–4 times more accurate measurements of distances and proper motions com- pared to HST. 18m 18m Thus, one could say that by chance, at the present time, observational statistics for both classes (S-stars and HVS) coincide. And from comparing these statis- tics, i.e., from the individually observed HVS [6] and S-stars [7], we can begin to solve the problem of reconstructing S-HVS pairs. It should be understood that these data are equally subject to the effects of observational selection due to the faint magnitude of HVS due to their distance from us by tens and even hundreds of kiloparsecs and the “light pollution” of S-stars from the densely populated Galactic Center (GC). 1. INTRODUCTION Two approaches have been developed in the search for HVSs during this time: to search among bright halo stars, which contrast with the local population of old globular clusters (and therefore likely arriving in the halo with high velocities, since the flight time should be consistent with their age), and among nearby low- mass disk stars, which are the majority. Both approaches have their “uncertainty ceiling” due to, on the one hand, the problem of determining the proper motion of faint, distant halo objects and, on the other hand, the abundance of low-mass disk stars, among which the search involves the routine process of data analysis. Initially enthusiastic about the possibility of an accelerated search for HVSs in its observation pro- gram, the GAIA team encountered difficulties in identifying stars with anomalous kinematics. GAIA gives three times larger errors for stars fainter than compared to HST, so it is more effective to use it for the second approach. Conversely, for stars brighter than , GAIA provides 3–4 times more accurate measurements of distances and proper motions com- pared to HST. 18m 18m Through numerical experiments posed in both the three-body and N-body scenarios, obtained statistics of HVS and S-star populations, considering the segre- gation of their masses, proved to be consistent with their lifetimes, including the time of capture of S stars into the vicinity of the central SMBH [11]. Thus, the Hills scenario [2] is capable of explaining both the phenomenon of HVSs and the accumulation of S-stars, although it is not the only channel for the for- mation of S-stars. The problem of direct identification of S–HVS pairs remains technically unsolved today, posing a challenge to modern astrometry and spectroscopy. Theoretical identification algorithms also face diffi- culties, involving aspects such as synchronizing the evolutionary scales of previously related components. A star that remains in the vicinity of the SMBH may be partially or completely disrupted by its tidal field, while the other companion will continue to evolve over the course of its flight. We can attempt to address these questions based on the analysis of observational data, following the principle of complementing the S-star catalog with HVS data. gives three times larger errors for stars fainter than compared to HST, so it is more effective to use it for the second approach. 2.2. HVS Catalog and Reconstructed S-Stars These two distributions overlap in the range of AU − ~9300 32 600 The observed distribution of S-stars from the cata- log [7] based on the semimajor axes of their orbits (blue histogram in Fig. 2) overlaps with a similar dis- tribution reconstructed from the observational data of the HVS catalog [6] (cyan histogram in Fig. 2), as if they were pairs in parent BSs. These two distributions overlap in the range of AU ( ),which clearly demonstrates the cor- relation between the data from the Brown et al. [6] cat- alog and the Gillessen et al. [7] catalog. − ~9300 32 600 − × ⊙ 6 (2 7) 10 R Neglecting the interaction between the components of BSs compared to their interaction with the SMBH, Neglecting the interaction between the components of BSs compared to their interaction with the SMBH, and assuming that the total energy of the former S-HVS pair is zero at infinity, the ejection velocity of the ejected component can be estimated within the Hills scenario [2] as (Table 1). Using the kinematic criterion for HVSs as stars gravitation- ally unbound from the Galaxy, which, for the Galactic Center region, corresponds to km/s according to Wu et al. [12], it turns out that 22 out of 39 S-stars could have had a companion component that was subsequently ejected as an HVS during the dynamic capture of the parent BS. v 1/2 eject b = (2 ) e veject > 750 ( ),which clearly demonstrates the cor- relation between the data from the Brown et al. [6] cat- alog and the Gillessen et al. [7] catalog. − × ⊙ 6 (2 7) 10 R Figure 2 illustrates the effect of observational selec- tion in the data from both catalogs, manifested in the presence of opposite (mirror-symmetric) maxima. The peak of the observed distribution of S-stars [7] corresponds to the value of the semimajor axis . S-stars on wider orbits (with semimajor axes ) have not been discovered yet; monitoring of the central region has been conducted for just over thirty years, whereas longer observations are required to determine the orbits of S-stars from images. × ⊙ ∼ 6 1 10 R × ⊙ 6 > 7 10 a R The HVS ejection velocity spectrum (light histo- gram in Fig. 2.1. S-Star Catalog and Reconstructed HVSs From the Gillessen et al. catalog [7], 39 S-stars with known orbital periods and angular sizes of the semimajor axis of their orbits around the SMBH were selected. Using estimates of the distance from the Sun to the Galactic Center, kpc, and the mass of the SMBH, , derived from multi-star orbital fitting of 17 S-stars selected based on the significance criterion of gravitational acceleration according to [7], the linear sizes of the S- star orbits and their average orbital velocities were calculated (Table 1). Based on this data, it is possible to calculate the spe- cific binding energy of the S-star, , located in the central field of the SMBH: P a ≈ 0 8.32 R × ⊙ 6 SMBH = 4.28 10 M M 0 '' = 206 265 a A R π vorb = 2 / A P be From the Gillessen et al. catalog [7], 39 S-stars with known orbital periods and angular sizes of the semimajor axis of their orbits around the SMBH were selected. Using estimates of the distance from the Sun to the Galactic Center, kpc, and the mass of the SMBH, , derived from multi-star orbital fitting of 17 S-stars selected based on the significance criterion of gravitational acceleration according to [7], the linear sizes of the S- P a ≈ 0 8.32 R × ⊙ 6 SMBH = 4.28 10 M M '' 2.2. HVS Catalog and Reconstructed S-Stars Similarly, it is possible to perform the reverse reconstruction, i.e., reconstruct the orbital parame- ters of the captured companion, such as the semimajor axis of its orbit around the SMBH, based on observa- tions of HVSs. To date, the most complete catalog of HVSs is Brown et al. [6], which contains information on 39 objects, including their spatial velocity values . Assuming that the kinetic energy of the ejected HVS from the vicinity of the SMBH was balanced by the gravitational energy of the interaction of the pre- sumably remaining companion (S-star) in the central field of the SMBH, it is possible to estimate the semi- major axis of its orbit using the formula . vobs v calc 2 S BH obs = / a G M Similarly, it is possible to perform the reverse reconstruction, i.e., reconstruct the orbital parame- ters of the captured companion, such as the semimajor axis of its orbit around the SMBH, based on observa- tions of HVSs. To date, the most complete catalog of HVSs is Brown et al. [6], which contains information on 39 objects, including their spatial velocity values star orbits and their average orbital velocities were calculated (Table 1). Based on this data, it is possible to calculate the spe- cific binding energy of the S-star, , located in the central field of the SMBH: 0 '' = 206 265 a A R π vorb = 2 / A P be . Assuming that the kinetic energy of the ejected HVS from the vicinity of the SMBH was balanced by the gravitational energy of the interaction of the pre- sumably remaining companion (S-star) in the central field of the SMBH, it is possible to estimate the semi- major axis of its orbit using the formula . vobs v calc 2 S BH obs = / a G M (1) − v 2 orb SMBH b = . 2 M e G A (1) The observed distribution of S-stars from the cata- log [7] based on the semimajor axes of their orbits (blue histogram in Fig. 2) overlaps with a similar dis- tribution reconstructed from the observational data of the HVS catalog [6] (cyan histogram in Fig. 2), as if they were pairs in parent BSs. 2. RECONSTRUCTION OF S–HVS PAIRS BASED ON OBSERVATIONAL DATA Over nearly thirty years of continuous monitoring of the central region of the Galaxy, the orbits of 39 S-stars from the Gillessen et al. catalog [7] have been reconstructed. Using this data, it is interesting to investigate whether these S-stars could have a com- panion in a parent BS system in the past. To begin with, the specific binding energy of the S-star in the central field of the SMBH can be estimated within the framework of the three-body problem in order to con- vert it into the kinetic energy of the possible compan- ion’s ejection, assuming that the total energy of the BS system at infinity becomes zero. The ejection velocity can be then derived from the kinetic energy of the ejection. The obtained velocity spectrum of the pre- sumed ejected companions of S-stars should be com- pared with the spatial velocities of observed HVSs from the Brown et al. catalog [6]. This approach to the reconstruction of S–HVS pairs adheres to the princi- ple of complementarity, where data from one catalog are tested against data from another. A detailed investigation of this problem can be con- ducted based on the possibility of numerical modeling of the capture of BSs into the vicinity of supermassive black holes (SMBHs) in the context of the Hills sce- nario [2]. The capture modeling is carried out in sev- eral stages. First, a simplified layered model of star density in the Galactic Center (0.1–0.01 parsecs) is tested in the mode of paired random encounters [8]. To effectively study the conditions for the capture of BS with subsequent ejection of a star as a HVS, the principle of ensemble representation of initial data is used. This allows for a wide range of parameters for the initial configurations of BS (orbital phase of BS and the inclination of its own orbit relative to the orbit around the SMBH) to be varied in a single calculation ASTRONOMY REPORTS Vol. 67 No. 9 2023 896 DREMOVA et al. second component: in 22 cases as HVSs and in 17 cases while remaining gravitationally bound to the Galaxy. 2.2. HVS Catalog and Reconstructed S-Stars 1), reconstructed from the observed dis- tribution of S-stars based on their average orbital velocities (blue histogram in the same figure), main- tains a correlation with it and also aligns well with the observed velocity spectrum of HVSs from the Brown et al. catalog (pink histogram) [6]. Due to observa- tional selection effects, the distribution of observed HVSs lies within a narrow range of ejection velocities (600–800 km/s). It is likely that the fastest HVSs with velocities exceeding 1000 km/s escape the Galaxy in less than 50 Myr. In this distribution, one notable object stands out with a spatial velocity of ~1755 km/s, known as Koposov’s star [13], discovered within the Southern Spectroscopic Survey S at a distance of kpc from the Galactic Center. It is now used as an etalon test for the scenario of central origin of HVSs. 5 ∼8.6 Let us return to Fig. 1. It shows the reconstructed distribution of HVSs based on the observed S-stars from the catalog [7] (cyan histogram) with an extended maximum in the range of 500–1300 km/s. In reality, the observed HVSs from the catalog [6] have a maximum in the velocity distribution in the range of 600–700 km/s (pink histogram). In cases of low- impulse ejections of one of the components of a BS that ended up in the vicinity of the SMBH, the second component remains on a wider orbit around the SMBH. This means that the peak of the pink histo- gram (600–700 km/s) in the distribution of observed HVS (Fi 1) hift t th l f i th 6 6 10 R The analysis of HVS ejection velocities (Table 1), reconstructed from the observations of S- stars from the catalog [7], confirms the plausibility of the scenario of their capture by a parent BS into the vicinity of the SMBH, followed by the ejection of the veject HVSs (Fig. 1) shifts to the value of in the cyan histogram (Fig. 2), which shows the recon- structed distribution of S-stars based on their semima- × ⊙ ∼ 6 6 10 R ASTRONOMY REPORTS Vol. 67 No. 9 2023 EXTREMELY WIDE PAIRS 897 Table 1. Orbital parameters of observed S-stars from the catalog of Gillessen et al. [7] and the ejection rates of poten- tial evolutionarily paired objects derived from them with an analysis of their status as HVSs under the condition km/s [12] > 750 ASTRONOMY REPORTS Vol. 2.2. HVS Catalog and Reconstructed S-Stars catalog [7] based on their mean ities (blue histogram) with the calculated (cyan histogram) distribution of ejection velocities for the presumed paire and with the observed (pink histogram) distribution of spatial velocities of HVS from the Brown et al. catalog [6]. 100 300 500 700 900 1100 v, km/s 1300 1500 1700 1900 2100 0 5 10 15 N Fig. 1. A comparison of the observed distribution of S-stars from the Gillessen et al. catalog [7] based on their mean orbital veloc- ities (blue histogram) with the calculated (cyan histogram) distribution of ejection velocities for the presumed paired with S-stars and with the observed (pink histogram) distribution of spatial velocities of HVS from the Brown et al. catalog [6]. jor axes. This is why the effect of inverse symmetry is observed. Other causes of the discrepancy between observed and calculated values of semimajor axes may reflect different scenarios for the origin of S-stars unrelated to the disruption of BSs through the Hills mechanism [2]. According to calculations by Loose et al. [15], S-stars could have originated from natural recurrent star formation associated with gas accumulation in the vicinity of the Galactic Center. The origin of S-stars may also be linked to tidal “stripping” of young star clusters during their approach to the central SMBH, as indicated by Fragione et al. [16]. An example of such a cluster is the young and dense Arches cluster, located 25 pc away from the SMBH. The model distribution of S-stars obtained within the framework of the Hills scenario [2] shows the pres- ence of compact orbits with semimajor axes two orders of magnitude smaller, AU ( ) (Fig. 2 from [11]), compared to the observed ones. This discrepancy is primarily associated with the effects of observational selection. S-stars on small orbits are more difficult to detect; so far, the most compact orbit among the detected S-stars is for S 4716 with a semimajor axis AU [14]. Another pos- sible reason is that the gravitational potential of the Galaxy, which is still not precisely known, decelerates HVSs, causing their velocities to be lower than expected from the observed orbits of S-stars. Addi- tionally, the deviation from the model distribution of S-stars in terms of semi-major axes could be related to the conditions for a star to be ejected as an HVS. 2.2. HVS Catalog and Reconstructed S-Stars 67 No. 9 2023 able 1. Orbital parameters of observed S-stars from the catalog of Gillessen et al. [7] and the ejection rates of pote al evolutionarily paired objects derived from them with an analysis of their status as HVSs under the conditi km/s [12] S-star , days , km/s , km/s Status S1 1.0641 60632.5 888 863 HVS S2 0.2244 5844.1 1944 1870 HVS S4 0.6384 28124.7 1149 1112 HVS S6 1.1757 70129.1 848 818 HVS S8 0.7238 33932.3 1080 1044 HVS S9 0.4872 18737.6 1316 1272 HVS S12 0.5342 21513.5 1257 1215 HVS S13 0.4723 17897.5 1336 1293 HVS S14 0.5120 20198.6 1283 1242 HVS S17 0.6365 27978.6 1150 1113 HVS S18 0.4255 15304.2 1407 1363 HVS S19 0.9300 49309.5 955 919 HVS S21 0.3917 13514.5 1467 1420 HVS S22 2.3429 197238.2 601 579 S-star S23 0.4525 16728.7 1369 1317 HVS S24 1.6883 120899.7 707 684 S-star S29 0.7655 36890.8 1050 1015 HVS S31 0.8030 39447.6 1030 1986 HVS S33 1.1750 70129.1 848 818 HVS S38 0.2532 7012.9 1828 1762 HVS S39 0.6617 29622.2 1131 1090 HVS S42 1.6990 122360.8 703 683 S-star S54 2.1462 174227.1 623 609 S-star S55 0.1928 4675.3 2087 2027 HVS S60 0.6934 31813.8 1103 1066 HVS S66 2.6863 242529.9 561 542 S-star S67 2.0138 157425.3 647 626 S-star S71 1.7402 126378.6 697 672 S-star S83 2.6648 239607.9 563 544 S-star S85 8.2270 1307616 318 311 S-star S87 4.9004 599019.8 414 402 S-star S89 1.9333 148293.9 659 639 S-star S91 3.4285 349915.2 496 480 S-star S96 2.6809 241799.5 561 542 S-star S97 4.1493 463875.1 453 434 S-star S145 2.0031 155599.0 652 625 S-star S175 0.7404 35137.6 1066 1033 HVS R34 3.2371 320329.5 511 493 S-star R44 6.9751 997144.8 354 330 S-star ect > 750 ⊙ 6 /10 , A R orb P vorb veject 898 DREMOVA et al. Fig. 1. A comparison of the observed distribution of S-stars from the Gillessen et al. catalog [7] based on their mean orbital veloc- ities (blue histogram) with the calculated (cyan histogram) distribution of ejection velocities for the presumed paired with S-stars and with the observed (pink histogram) distribution of spatial velocities of HVS from the Brown et al. catalog [6]. 100 300 500 700 900 1100 v, km/s 1300 1500 1700 1900 2100 0 5 10 15 N Fig. 1. A comparison of the observed distribution of S-stars from the Gillessen et al. 2.2. HVS Catalog and Reconstructed S-Stars Allowing for ejections with velocities lower than those corresponding to the HVS criterion would result in larger semimajor axes for captured S-stars in the Hills scenario, ensuring agreement with the observed distri- bution. − ∼139 330 − × ⊙ 4 (3 7) 10 R ∼400 A The model distribution of S-stars obtained within the framework of the Hills scenario [2] shows the pres- ence of compact orbits with semimajor axes two orders A detailed kinematic analysis conducted by Ali et al. [17] for 112 S-stars revealed 3D structures of their orbits, which belong to two closely adjacent, oppo- sitely rotating disks inclined at 45° to the Galactic plane. The distribution of orbit inclinations and proper motion vectors contradicts the initial data on thermal eccentricity distribution and chaotic orbit dis- tribution of S-stars. Ordered orbital structures could result from resonant processes in the nuclear disk. One should consider the tidal disruption of S-clus- ter stars in the gravitational field of the SMBH. The ASTRONOMY REPORTS Vol. 67 No. 9 2023 899 EXTREMELY WIDE PAIRS Fig. 2. The observed distribution of S-stars from the Gillessen et al. catalog [7] based on their major semi-axes (blue histogram) and the reconstructed data from HVS observations from the Brown et al. catalog [6] (cyan histogram). 2 4 6 8 10 0 2 4 6 8 10 12 N A/Ro × 106 Fig. 2. The observed distribution of S-stars from the Gillessen et al. catalog [7] based on their major semi-axes (blue histogram) and the reconstructed data from HVS observations from the Brown et al. catalog [6] (cyan histogram). 3. SEARCH FOR S–HVS PAIRS theoretical estimate of their frequency obtained in the homogeneous bulge model [18] is comparable to the frequency of flashes attributed to the accretion activity of the SMBH. According to numerical modeling of tidal disruptions of observed BSs in the central disk cluster at distances from the center of 0.05–0.5 pc, Generozov [19] predicts that a conical stream of ejected HVSs with an angular resolution of 30° was produced in the vicinity of the Galactic Center in the recent past ( Myr ago). These ejected HVSs are cur- rently at galactocentric distances of several tens of kpc. This distribution, along with the results of kinematic analysis of S-cluster stars [17], can facilitate the search for HVSs in the GAIA data. It is worth mentioning the star S5-HVS1 (Koposov’s star) [13], which was con- sidered part of the conical stream in the modeling [19], and its observational data were used as a refer- ence for predictions. ∼5 The main question of the paper remains: is it pos- sible to “assemble” S–HVS pairs from direct observa- tions? Let us attempt to outline an algorithm for “assembling” S–HVS pairs. 1. For S-stars closest to the Galactic center with known orbital elements, reconstruct, within the framework of the Hills scenario [2], the ejection veloc- ities of their potential companions based on their binding energy with the SMBH. Selecting the closest S-stars will satisfy the condition for releasing evolu- tionarily linked companions as HVSs. 2. For the same S-stars, analyze their known orbital elements that define the orientation of their orbital plane in space. Additionally, estimate their spectral class, mass, and age from spectroscopic observations. 3. The search for a candidate among observed HVSs to pair with the selected S-star should consider several necessary conditions: Thanks to the development of image filtering tech- nologies, such as the Lucy–Richardson algorithm, the following S-cluster stars have been discovered to date from the closest to the Sgr A source: S 4715 (a ~ 1190 AU), S 4714 ( AU), S 62 ( AU), S 4711 ( AU), S 4716 ( AU) [20, 14]. It is with such stars, primarily, that the problem of reconstructing S–HVS pairs should be addressed. OPEN ACCESS This article is licensed under a Creative Commons Attri- bution 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 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 other- wise 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/. As for the absence of sufficiency conditions, per- haps they cannot exist in probabilistic problem formu- lations, except in rare cases when it becomes possible to directly observe the process of breaking the BS con- nection with the ejection of an HVS. In perspective, this appears to be an exciting task of reconstructing genetically related components of BSs disrupted by the tidal gravity of a SMBH. The authors declare that they have no conflicts of interest. The authors declare that they have no conflicts of interest. FUNDING This work was supported by ongoing institutional fund- ing. No additional grants to carry out or direct this particu- lar research were obtained. 3. SEARCH FOR S–HVS PAIRS ∼840 a ∼740 a ∼620 a ∼400 a —similar spectral classes between the S-star and the HVS, considering for the “spectral twins” effect, which follows from the conditions of star formation in the parent binary system; —similar spectral classes between the S-star and the HVS, considering for the “spectral twins” effect, which follows from the conditions of star formation in the parent binary system; —matching ages between the S-star and its poten- tial companion (candidate HVS), which follows from the youth of the near-nuclear S-cluster; ASTRONOMY REPORTS Vol. 67 No. 9 2023 DREMOVA et al. 900 —knowledge of the HVS’s velocity and its current distance from the Galactic Center, which will con- strain the establishment of the pair with the selected S- star; tion directions in the form of conical “fountains.” These fountains are caused by the disk-like structure of the S-cluster and its inclination to the Galactic plane [16, 17]. All of these refined models will contrib- ute to solving this problem. —meeting the requirement of the central ejection of the chosen HVS as a candidate for pairing with the S-star, which can be determined by reverse-time inte- gration of the HVS’s trajectory in the Galaxy’s gravi- tational potential (the knowledge of which is a sepa- rate major challenge!). The HVS’s total (radial plus tangential) velocity vector at the time should point toward the Galactic Center; = 0 t —meeting the requirement of the central ejection of the chosen HVS as a candidate for pairing with the S-star, which can be determined by reverse-time inte- gration of the HVS’s trajectory in the Galaxy’s gravi- tational potential (the knowledge of which is a sepa- rate major challenge!). The HVS’s total (radial plus tangential) velocity vector at the time should point toward the Galactic Center; = 0 t CONFLICT OF INTEREST —the spatial velocity vector of the HVS at time , reconstructed through the method of reverse integration of its motion, should lie in the orbital plane of the S-star to which the considered HVS is assumed to be a companion. However, the current spatial velocity vector of the HVS does not necessarily have to lie in the orbital plane of the S-star due to possible deviations in the galactic potential during its journey. = 0 t REFERENCES The problem of reconstructing S-HVS pairs no longer seems fantastical today. The technical progress of the 21st century has shown the smallest gap in his- tory between the development and implementation of methods and technologies that have proven their effectiveness and unveiled new possibilities in a long list of discoveries: HVSs, SMBHs, gravitational waves, and quantum entanglement. 1. H. Kobulnicky and C. 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Modeling tidal deformations of stars in the near- nuclear S-cluster and the ejections of some of them during their pericenter passages near the SMBH pro- vides constraints on the selection of preferential ejec- 5. W. Brown, M. Geller, S. Kenyon, and J. Kutz, Astro- phys. J. 622, 33 (2005). 6. W. Brown, M. Lattanzi, S. Kenyon, and M. Geller, Astrophys. J. 866, 39 (2018). 7. S. Gillessen, P. Plewa, F. Eisenhauer, R. Sari, et al., Astrophys. J. 837, 30 (2017). 8. G. N. Dremova, V. V. Dremov, and A. V. Tutukov, Astron. Rep. 60, 695 (2016). 9. G. N. Dremova, V. V. Dremov, V. V. Orlov, A. V. Tutu- kov, and K. S. Shirokova, Astron. Rep. 59, 1019 (2015). 10. G. Dryomova, V. Dryomov, and A. Tutukov, Baltic Astron. 24, 1 (2015). 11. G. N. Dremova, V. V. Dremov, and A. V. Tutukov, Astron. Rep. 63, 862 (2019). 12. X. Wu, B. Famaey, G. Gentile, H. Perets, and H. Zhao, Mon. Not. R. Astron. Soc. 386, 2199 (2008). ASTRONOMY REPORTS Vol. 67 No. EXTREMELY WIDE PAIRS 19. A. Generozov, Mon. Not. R. Astron. Soc. 501, 3088 (2021). 13. S. E. Koposov, D. Boubert, T. S. Li, G. S. da Costa, et al., Mon. Not. R. Astron. Soc. 491, 2465 (2020). 14. F. Peißker, A. Eckart, M. Zajacek, B. Ali, and S. Brit- zen, Astrophys. J. 933, 49 (2022). 20. F. Peißker, A. Eckart, M. Zajacek, B. Ali, and M. Par- sa, Astrophys. J. 899, 50 (2020). 15. H. Loose, E. Krügel, and A. Tutukov, Astron. Astro- phys. 105, 342 (1982). Translated by M. Chubarova Translated by M. Chubarova REFERENCES 9 2023 ASTRONOMY REPORTS Vol. 67 No. 9 2023 ASTRONOMY REPORTS Vol. 67 No. 9 2023 EXTREMELY WIDE PAIRS 901 ASTRONOMY REPORTS Vol. 67 No. 9 2023 Translated by M. Chubarova 16. G. Fragione, R. Capuzzo-Dolcetta, and P. Kroupa, Mon. Not. R. Astron. Soc. 467, 451 (2017). 17. B. Ali, D. Paul, A. Eckart, M. Parsa, et al., Astrophys. J. 896, 100 (2020). Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 18. G. N. Dremova, V. V. Dremov, and A. 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https://openalex.org/W4220796070	https://digital.csic.es/bitstream/10261/279852/1/Expansion-of-Naturally-Regenerated-Forest.pdf	English	null	Expansion of Naturally Regenerated Forest	Forests	2,022	cc-by	1,582	Editorial Expansion of Naturally Regenerated Forest Josu G. Alday 1,2,* and Carolina Martínez-Ruiz 3,4,* Josu G. Alday 1,2,* and Carolina Martínez-Ruiz 3,4,* Josu G. Alday 1,2,* and Carolina Martínez-Ruiz 3,4,* 1 Joint Research Unit CTFC–AGROTECNIO–CERCA, 25198 Lleida, Spain 2 1 Joint Research Unit CTFC–AGROTECNIO–CERCA, 25198 Lleida, Spain 2 Department of Crop and Forestry Sciences, Universitat de Lleida, Avda. Alcalde Rovira Roure 191, 25198 Lleida, Spain 3 Área de Ecología, Departamento Ciencias Agroforestales, E.T.S. de Ingenierías Agrarias, Universidad de Valladolid, Avda. de Madrid 50, 34071 Palencia, Spain 3 Área de Ecología, Departamento Ciencias Agroforestales, E.T.S. de Ingenierías Agrarias, 3 Área de Ecología, Departamento Ciencias Agroforestales, E.T.S. de Ingeni Universidad de Valladolid, Avda. de Madrid 50, 34071 Palencia, Spain Universidad de Valladolid, Avda. de Madrid 50, 34071 Palencia, Spain 4 Sustainable Forest Management Research Institute, University of Valladolid-INIA, E.T.S.II.AA., Avda. Madrid 50, 34071 Palencia, Spain p * Correspondence: josu.alday@udl.cat (J.G.A.); carolina.martinez.ruiz@uva.es (C.M.-R.) Forests cover 31% of the global land area and are home to most of Earth’s terrestrial biodiversity. Forests supply a wide set of ecosystem services, provide livelihoods, mitigate climate change, and are essential for sustainable food production, contributing to the health of the planet. However, deforestation and forest degradation continue to take place at alarming rates in some parts of the world, whereas in others the trend has been reversed with forests expanding over abandoned agricultural areas. p g g In this era of contrast, improving our knowledge of ecological processes and mecha- nisms involved in the natural regeneration and expansion of forests should be a priority. Factors affecting forest expansion are extensive, such as seed production and dispersal, seedlings establishment, shrubs species colonization, microsites availability, soil conditions, as well as the complex interactions that occur within and between all levels of forest bio- logical diversity (e.g., local, ecosystem, landscape, species, population, and genetic) in the colonization process. Hence, substantial efforts are needed to integrate this knowledge in new management and implementation actions in these two contrasting scenarios: (i) to reverse deforestation and biodiversity loss for the beneﬁt of current and future generations; and (ii) to manage expanding forest over agricultural areas to maximize the conservation value of these new forests and guarantee the ecosystem services they provide. Within this Special Issue, we collected contributions that target directly the effect of these factors, such as microsite availability, boundary effect, light conditions, soil properties, or shrub colonization to improve our understanding of constraints/un-constraints for natural for- est expansion all around the world, while providing practical recommendations for the reforestation of degraded ecosystems. Citation: Alday, J.G.; Martínez-Ruiz, C. Expansion of Naturally Regenerated Forest. Forests 2022, 13, 456. https://doi.org/10.3390/ f13030456 Citation: Alday, J.G.; Martínez-Ruiz, C. Expansion of Naturally Regenerated Forest. Forests 2022, 13, 456. https://doi.org/10.3390/ f13030456 Received: 2 March 2022 Accepted: 13 March 2022 Published: 15 March 2022 Received: 2 March 2022 Accepted: 13 March 2022 Published: 15 March 2022 The Special Issue starts with a manipulative experiment about seedling emergence of two contrasting co-occurring Quercus species under a bioclimatic limit, using shrubs as facilitators. Interestingly, the clear facilitative effect of shrubs on the seedling emergence of Q. ilex and Q. pyrenaica was demonstrated, this being a positive effect over emergence even greater than those caused by acorn size and large herbivore exclusion [1]. Similarly, a study describing the boundary form effect between two patches with contrasting vegetation (mine grassland and adjacent forest) on woody colonization (Quercus species) and forest expansion showed that the tree colonization decreased with increasing distance to the forest and differed depending on the boundary form, with more intense colonization in concave boundaries than in convex boundaries close to the forest [2]. Both manuscripts showed that shrub facilitative processes and edge characteristics have a strong potential to be used in the restoration of Quercus native forests based on natural processes. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The identiﬁcation of the factors controlling the understory and main tree species distribution and seed dispersal are essential to understand natural forest dynamics. As such, some authors using forest inventory data reported the relationships between environmental https://www.mdpi.com/journal/forests Forests 2022, 13, 456. https://doi.org/10.3390/f13030456 Forests 2022, 13, 456 2 of 2 gradients and shrub functional groups distribution patterns and niche characteristics as the ﬁrst step to improve our ability to discuss potential forest conservation management goals or threats due to land-use changes and future climate change [3]. Regarding seeds, an experiment testing whether intraspeciﬁc variation of dispersal potential and the associated traits of seeds (Acer palmatum diaspores) should be inﬂuenced by maternal habitat quality showed that samaras produced by trees from shaded microhabitats had greater dispersal potential, in terms of slower terminal velocity of descent, than those produced in open microhabitats [4]. These ﬁndings indicate that an active maternal control through the morphological allometry of samaras is produced to facilitate species persistence. The main tree species dynamics are subjected to current climate change; therefore, integrated bio-geographic studies of pine forest might help to identify forest areas most susceptible to climate change effects. Here, the results showed that the more fragile and precipitation- poor upland pine settings display a higher vulnerability to the current climate change, resulting in increased tree death and forest ﬁres [5]. These three main results suggest that the dispersal potential and shrubs as facilitative microhabitats could be factors to consider in forest expansion management plans. However, forest sustainability and adaptation to the ongoing climate change can trigger deforestation and forest degradation worldwide. Finally, forest restoration has become one of the most important challenges for ecolog- ical restoration, although the post-disturbance recovery of plant–soil feedback has been rarely assessed or described. Aboveground–belowground interactions between trees and fungi are fundamental in nutrients and carbon cycles [6]. Here, biomass and structure of fungal communities were compared between P. sylvestris, Q. robur and Q. ilex (holm oak) forests and naturally revegetated mined sites to identify similarities in soil/litter inoculum between habitats to enhance tree seedlings’ establishment success in degraded areas [6]. These results showed that tree restoration strategies in degraded sites could use speciﬁc soil/litter inoculum from similar nearby forest areas upon planting/seeding, depending on tree species of interest, to provide a satisfactory soil fungal community over which to start the functional optimization between fungi and tree species dependent on the plant resources needed. Author Contributions: Conceptualization, writing—review and editing, C.M.-R. and J.G.A. All authors have read and agreed to the published version of the manuscript. Funding: Josu G. Alday was supported by a Ramón y Cajal fellowship (RYC-2016-20528) from Spanish Government. Conﬂicts of Interest: The authors declare no conﬂict of interest. 6. Adamo, I.; Dashevskaya, S.; Alday, J.G. Fungal Perspective of Pine and Oak Colonization in Mediterranean Degraded Ecosystems. Forests 2022, 13, 88. [CrossRef] 5. Zhumadina, S.; Chlachula, J.; Zhaglovskaya-Faurat, A.; Czerniawska, J.; Satybaldieva, G.; Nurbayeva, N.; Mapitov, N.; Myrza- galiyeva, A.; Boribay, E. Environmental Dynamics of the Ribbon-Like Pine Forests in the Parklands of North Kazakhstan. Forests 2022, 13, 2. [CrossRef] g g g 2. Martínez-Ruiz, C.; Milder, A.I.; López-Marcos, D.; Zaldívar, P.; Fernández-Santos, B. Effect of the Forest Woody Colonization and Forest Expansion in Degraded Ecosystems. Forests 2021, 12, 773. [CrossRef] 3. Olthoff, A.E.; Martínez-Ruiz, C.; Alday, J.G. Niche Characterization of Shrub Functional Groups along an Atlantic-Mediterranean Gradient. Forests 2021, 12, 982. [CrossRef] 1. Díaz-Hernández, R.; Vicente Villardón, J.L.; Martínez-Ruiz, C.; Fernández-Santos, B. The Effects of Native Shrub, Fencing, and Acorn Size on the Emergence of Contrasting Co-Occurring Oak in Mediterranean Grazed Areas. Forests 2021, 12, 307. [CrossRef] 1. Díaz-Hernández, R.; Vicente Villardón, J.L.; Martínez-Ruiz, C.; Fernández-Santos, B. The Effects of Native Shrub, Fencing, and Acorn Size on the Emergence of Contrasting Co-Occurring Oak in Mediterranean Grazed Areas. Forests 2021, 12, 307. [CrossRef] 2. Martínez-Ruiz, C.; Milder, A.I.; López-Marcos, D.; Zaldívar, P.; Fernández-Santos, B. Effect of the Forest-Mine Boundary Form on Woody Colonization and Forest Expansion in Degraded Ecosystems. Forests 2021, 12, 773. [CrossRef] J.W.; Wang, X.; Dong, Y.; During, H.J.; Xu, X.; Anten, N.P.R. Maternal Environmental Light Conditions ogical Allometry and Dispersal Potential of Acer palmatum Samaras. Forests 2021, 12, 1313. [CrossRef] g g g 2. Martínez-Ruiz, C.; Milder, A.I.; López-Marcos, D.; Zaldívar, P.; Fernández-Santos, B. Effect of the Forest-Mine Boundary Form on Woody Colonization and Forest Expansion in Degraded Ecosystems. Forests 2021, 12, 773. [CrossRef] 4. Chen, B.J.W.; Wang, X.; Dong, Y.; During, H.J.; Xu, X.; Anten, N.P.R. Maternal Environmental Morphological Allometry and Dispersal Potential of Acer palmatum Samaras. Forests 2021, 12, 1313. 4. Chen, B.J.W.; Wang, X.; Dong, Y.; During, H.J.; Xu, X.; Anten, N.P.R. Maternal Environmental Light Conditions Affect the Morphological Allometry and Dispersal Potential of Acer palmatum Samaras. Forests 2021, 12, 1313. [CrossRef] 5. Zhumadina, S.; Chlachula, J.; Zhaglovskaya-Faurat, A.; Czerniawska, J.; Satybaldieva, G.; Nurbayeva, N.; Mapitov, N.; Myrza- 4. Chen, B.J.W.; Wang, X.; Dong, Y.; During, H.J.; Xu, X.; Anten, N.P.R. Maternal Environmental Light Conditions Affect the Morphological Allometry and Dispersal Potential of Acer palmatum Samaras. Forests 2021, 12, 1313. [CrossRef] 5 Zhumadina S ; Chlachula J ; Zhaglovskaya Faurat A ; Czerniawska J ; Satybaldieva G ; Nurbayeva N ; Mapitov N ; Myrza Woody Colonization and Forest Expansion in Degraded Ecosystems. Forests 2021, 12, 773. [CrossRef] 3. Olthoff, A.E.; Martínez-Ruiz, C.; Alday, J.G. Niche Characterization of Shrub Functional Groups along an Atlantic-Mediterranean Gradient. Forests 2021, 12, 982. [CrossRef]
https://openalex.org/W4281616356	https://research-repository.st-andrews.ac.uk/bitstream/10023/25467/1/Fiori_2022_WES_Employmenttransitions_CC.pdf	English	null	Influences on Employment Transitions around the Birth of the First Child: The Experience of Italian Mothers	Work, employment and society	2,022	cc-by	9,445	Work, Employment and Society Work, Employment and Society 1 19 Work, Employment and Society 1–19 Work, Employment and Society 1–19 Influences on Employment Transitions around the Birth of the First Child: The Experience of Italian Mothers © The Author(s) 2022 ttps://doi.org/10.1177/09500170221082479 © The Author(s) 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/09500170221082479 journals.sagepub.com/home/wes ttps://doi.org/10.1177/09500170221082479 © The Author(s) 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/09500170221082479 journals.sagepub.com/home/wes Francesca Fiori University of St Andrews, UK Giorgio Di Gessa University College London, UK Corresponding author: Francesca Fiori, School of Geography and Sustainable Development, University of St Andrews, Irvine Building, North Street, St Andrews KY16 9AL, UK. Email: Francesca.Fiori@st-andrews.ac.uk Abstract This article studies mothers’ employment transitions around childbirth. It argues that leaving employment around childbirth and returning after an interruption might depend on multiple influences: the micro-context of individual and household characteristics, the meso-context of women’s jobs and the macro-context of broader cultural and institutional factors. This conceptual model is tested using data from the Italian Institute of Statistics (ISTAT) ‘Family and Social Subjects (2009)’ survey. The findings confirm that mothers’ transitions out of employment are shaped by micro-characteristics such as education, meso-characteristics such as status and security of prior jobs, and macro geographical and temporal factors. Subsequent returns to employment also reflect micro and macro influences, as mothers born before 1950, with low education, and large families are less likely to return; but they seem less dependent upon prior job characteristics. The research highlights the importance of considering multiple levels of influence to understand the enabling factors of maternal employment. Article 2479 WES0010.1177/09500170221082479Work, Employment and SocietyFiori and Di Gessa 2479 WES0010.1177/09500170221082479Work, Employment and SocietyFiori and Di Gessa 2479 WES0010.1177/09500170221082479Work, Employment and SocietyFiori and Di Gessa Introduction Employment rates of women in Europe have increased substantially over the past few decades but they remain considerably lower than those observed for men. Further, moth- ers have lower employment rates than childless women, particularly if they have young children, as they give up paid work after having a child (EUROSTAT, 2018). Higher rates of female employment are central to the promotion of economic growth in the cur- rent context of an ageing and shrinking workforce, and to women’s economic independ- ence (Nieuwenhuis et al., 2020). Employment breaks and reduced work commitment during the childrearing years might affect mothers’ earnings, future employability and income in later life (Adams et al., 2016; Grimshaw and Rubery, 2015; Muller et al., 2020). Despite policy consensus on the need to promote and support higher levels of maternal employment, the motherhood gap in labour force participation remains com- mon across Western economies (Eurofound, 2016), and the complexity of the mecha- nisms at force is still to be fully understood. Theoretical debates have traditionally emphasised the role of either preferences or constraints – agency or structure – in their attempt to understand why some women continue to work after childbirth, while others abandon employment around the time of family formation. On the one hand, women’s labour market participation is seen as the result of individual choices based on rational considerations of their roles within the household (Becker, 1981) or their preferences for family or work (Hakim, 2000). On the other hand, it is structural, institutional and cultural factors that are thought to explain mothers’ employment behaviours (Crompton and Harris, 1998; Fagan, 2001; McRae, 2003). Both approaches, however, have their limitations: one for its failure to acknowl- edge that individual choices are rarely unconditional or exclusively based on rational considerations or preferences; the other for its inability to account for differences between women within the same cultural or institutional context. Recently, more scholars have suggested that mothers’ moves into and out of the labour market should be regarded as the outcome of intentional choices, taken in a particular context of structural opportunities and constraints and conditioned by the prevailing cultural and normative models defining the expected roles of men and women (Dotti Sani and Scherer, 2018; Duncan et al., 2003; Grimshaw and Rubery, 2015; Kang et al., 2018; Korpi et al., 2013; Solera, 2009). This study contributes to knowledge in two substantive ways. Keywords Work, Employment and Society 00(0) 2 Introduction First, following on from such perspective, it proposes a comprehensive theoretical framework for under- standing employment transitions around the time of childbirth. These transitions are conceptualised as the result of mothers’ individual and household characteristics (micro- context), their work environment (meso-context) and national policies and institutions (macro-context). These contexts – it is argued – might play a different role on transi- tions out and back into the labour market following childbirth. Second, it illustrates the model empirically, by drawing on rich quantitative evidence from the Italian Institute of Statistics (ISTAT) ‘Family and Social Subjects’ survey. The survey reconstructs and contextualises the work and family histories of a nationally representative sample of mothers in Italy who were working before childbirth. The theoretical model, and the 3 3 Fiori and Di Gessa expectations around the role of its distinct contexts of influence, are tested empirically by addressing the following research questions: expectations around the role of its distinct contexts of influence, are tested empirically by addressing the following research questions: 1. To what extent is mothers’ likelihood of leaving employment around the birth of the first child influenced by their personal and household characteristics (micro- context) and their work environments prior to childbirth (meso-context)? 2. And among women who left employment when they became mothers, to what extent does their likelihood of never returning to work respond to the character- istics of their micro and meso-contexts? 3. Lastly, does women’s likelihood of leaving employment and never returning to work vary over time and across regions (macro-context)? 3. Lastly, does women’s likelihood of leaving employment and never returning to work vary over time and across regions (macro-context)? Italy provides an interesting case study. On the one hand, the combination of tradi- tional gender norms, a family-centred welfare regime, and a late and weak tertiarisation of the economy, have long inhibited mothers’ participation in the labour market (Bettio and Villa, 1998; Saraceno, 1994). On the other hand, high levels of employment protec- tion have contributed to greater stability in the labour market, particularly for women employed in the public sector (Breen, 2004; Solera, 2009). The next section reviews empirical studies on mothers’ micro and meso-contexts and sets out expectations about their importance for employment decisions around childbirth. Introduction It also draws a picture of the Italian macro labour market and welfare context and dis- cusses how its development over time and regional variation can contribute to differ- ences in levels of maternal employment. Then, data, methods and empirical results are presented. The final section summarises and discusses the main findings and their implications. The micro-context: The role of individual and household characteristics Theoretical explanations emphasising the role of women’s individual characteristics within both sociological and economic literature agree on education being the most important factor in determining women’s labour force participation. Perhaps less univo- cal are the mechanisms through which education is hypothesised to affect mothers’ employment (Konietzka and Kreyenfeld, 2010). Sociological approaches emphasise how the expansion of mass education has contributed to broader social and cultural changes that have reshaped women’s preferences and life choices, and questioned tradi- tional gender roles (Hakim, 2000). Human capital theories (Becker, 1981) posit that women who have invested time and resources in education and training have more incen- tives to work because of more favourable employment conditions and the expected returns in terms of wages. Women who postpone motherhood have had more time to accumulate human capital and work experience and to establish themselves in the labour market, which might, in turn, increase their chance to remain in the labour market after childbirth (Gustafsson et al., 1996; Miller, 2011). Work, Employment and Society 00(0) 4 00(0) Women’s labour market decisions after childbirth also respond to characteristics and preferences of other household members. For instance, studies on couples report a posi- tive association between men’s egalitarian gender role attitudes and their female part- ners’ participation in employment (Farré and Vella, 2013). Partners’ employment status, occupation and income are also important factors that affect women’s labour market participation (Becker, 1981; Bernardi, 1999; Solera, 2018). When family resources are scarce, particularly in the event of men’s unemployment, women may be compelled to work (Vitali and Arpino, 2016). The relationship between partner’s income and maternal employment, however, might be less univocal: for instance, mothers whose partners earn stable and high income may have the option to reduce their working hours or to abandon employment after the birth of a child, as well as the ability to afford childcare and to remain in employment (Solera, 2018). The availability of extended family networks may also act as an enabling factor of mothers’ labour force participation. For example, by providing childcare or domestic work, grandparents contribute to easing women’s difficulties in combining work and family life and reducing the financial cost of childcare, thus allowing mothers to con- tinue to work (Arpino et al., 2014; Di Gessa et al., 2016). The micro-context: The role of individual and household characteristics Literature also suggests an intergenerational link between fathers’ wealth and education and their daughters’ suc- cessful labour market outcomes (Hellerstein and Morrill, 2011). Moreover, the similarity between mothers’ and daughters’ employment is well established: mothers’ employment may shape daughters’ behaviour and attitudes towards employment and family roles (McGinn et al., 2019). Lastly, family and kinship networks might influence mothers’ decisions in relation to employment by constructing and enforcing norms and expecta- tions around the gendered division of roles and morally approved notions of ‘good moth- erhood’ (Duncan et al., 2003). Thus, mothers’ micro-contexts – defined here as the combination of mothers’ personal and household characteristics – are likely to shape the employment transitions around the time of childbirth, and define the nature (temporary or permanent) of their absence from the labour market. Mothers who invested in human capital and with a supportive house- hold environment will be more likely to remain employed after having a child and to return to employment if they leave around childbirth (Tomlinson et al., 2009). The meso-context: The role of job characteristics Mothers’ participation in the labour market also responds to the opportunities and con- straints of the jobs available to them. According to Desai and Waite (1991), occupations might encourage women’s labour force participation either by making it relatively easy to combine work with motherhood (‘convenience’ of working), or by making it difficult to stay away from work (‘costs’ of not working). The likelihood to remain in employment after having a child thus varies depending on the type of job women hold at the time of pregnancy. Generally, the chances of working after childbirth are higher among women in higher-level, non-manual and professional occupations whose higher wages represent an incentive to work after childbirth (Desai and Waite, 1991; Norman, 2020; Saurel-Cubizolles et al., 1999; Wenk and Garrett, 1992). Women in higher-status occupations might also be better able to afford to pay for childcare (Fagan and Norman, 2012). 5 Fiori and Di Gessa 5 Remarkable differences in levels of employment after childbirth are also observed between women working in the public and in the private sector (Lu et al., 2017; Saurel- Cubizolles et al., 1999; Solera and Bettio, 2013). Jobs in the public sector tend to be characterised by a high level of stability and employment protection, which make them particularly attractive for women planning to combine work and motherhood. At the same time, they offer more opportunities for flexible working schedules, including shorter hours or less pressure to work long hours, and more generous parental leave poli- cies. Moreover, jobs in the public sector are often in highly feminised sectors such as health, education and public administration. These conditions yield non-monetary returns that reduce the wage costs of motherhood and increase the convenience of returning to work (Desai and Waite, 1991). Rates of maternal employment are also higher among women with permanent con- tracts (Baxter, 2009; Bratti et al., 2005; Saurel-Cubizolles et al., 1999) and those working in larger firms (Bratti et al., 2005) – reflecting the importance of contractual stability and maternity protection in supporting participation rates among new mothers (Bratti et al., 2005; Solera and Bettio, 2013). The role of part-time employment, on the other hand, is less clear-cut. The meso-context: The role of job characteristics Although most scholars suggest that working part-time is a common strat- egy for combining work and childrearing (Del Boca, 2002; Desai and Waite, 1991), others highlight that part-time work often involves low-paid precarious jobs with poor benefits and might be an indicator of lower motivation to work (Saurel-Cubizolles et al., 1999). Part-time arrangements are also very common among mothers returning to the labour market after a period of inactivity due to caring responsibilities. However, whether this denotes a preference for, rather than an acceptance of, part-time jobs is contended (Rose, 2001). The move to part-time jobs after childbirth might be part of a process of occupa- tional downgrading, whereby mothers accept jobs beneath their capabilities and qualifi- cations, and inferior in status, pay and working hours to those held at the time of pregnancy (Tomlinson et al., 2009). Indeed, it has been argued that once women leave employment after their first child is born, the characteristics of their past occupation become irrelevant for their future employment choices (Desai and Waite, 1991; Wenk and Garrett, 1992). Given that job characteristics before and after childbirth might not necessarily be aligned, it is thus reasonable to assume that the meso-contexts defined by the character- istics of the jobs held at pregnancy would play a much stronger role on mothers’ transi- tions out of employment following childbirth, than on the subsequent return of those mothers who left employment. Nevertheless, the meso-context before childbirth might still be relevant, as mothers in insecure and low-status positions would have accrued less work-related human capital and therefore face greater challenges if they intend to return to the labour market. The macro-context: A focus on the Italian labour market and institutional setting Women’s decisions regarding employment and motherhood are also defined by opportu- nities and constraints at a broader institutional level, such as the way welfare functions are distributed among the state, the market and the family (Esping-Andersen, 1990). Therefore, mothers’ employment outcomes may vary substantially across countries and Work, Employment and Society 00(0) 6 00(0) over time. Evidence suggests that mothers’ labour force participation tends to be higher in countries with a tax benefits system that rewards dual-earner households, a large share of jobs with part-time and flexible working schedules, and available and affordable childcare services (e.g. Dotti Sani and Scherer, 2018; Korpi et al., 2013; Nieuwenhuis et al., 2012). Similarly, the variation in maternal employment rates over time has been interpreted in light of structural and institutional changes such as economic fluctuations and changes in parental leave regulation or provision of public childcare (Berghammer, 2014; Konietzka and Kreyenfeld, 2010). In Italy, despite its increase over the last 40 years, the employment rate in 2017 for all women aged 15–69 was 49%, still below the corresponding male rate (67%) (ISTAT, 2018) and the level observed among women elsewhere in Europe (63%) (EUROSTAT, 2018). Moreover, the employment rate for mothers aged 25–49 was 56%, much lower than for women of the same age without children (74.3%), as many mothers leave employment to take care of their children (ISTAT, 2018). p y ( ) Several features of the Italian context have contributed over time to this low level of female (and maternal) labour force participation. First, the expansion of the services sec- tor in Italy has been comparatively limited. Whereas in most OECD countries this sector has grown extensively and has represented the main source of employment for women (Nieuwenhuis et al., 2012; Thévenon, 2013), in Italy the development of services has been slow and has contributed little to the growth of female employment (Solera, 2009). Second, the familistic nature of the Italian welfare state (Esping-Andersen, 1990; Saraceno, 1994), with low levels of public expenditure for families and children and the expectations that families should provide care and support to their family members, has further hindered the creation of social reproduction jobs, reinforcing a gendered model of society where women are responsible for (unpaid) care and have limited opportunities to take up (paid) employment. The macro-context: A focus on the Italian labour market and institutional setting The childcare infrastructure for children under three is illustrative of the insufficient institutional efforts to promote maternal employment, with the average coverage rate in 2018 being only 25.5%, well below the EU 2002 target of 33% (ISTAT, 2020). The lack of public support is often compensated for by the informal childcare offered by grandparents (Di Gessa et al., 2016). Third, the Italian tax system encourages a gendered division of labour within households. With individuals being the basic unit of taxation and with tax-credit and cash-transfers for children and non-work- ing spouses, the system disincentivises the labour force participation of wives, particu- larly from households at the bottom of the income distribution (Colonna and Marcassa, 2015). Lastly, the high regulation of the Italian labour market during the 1970s and 1980s, while increasing job security and protection for full-time labour market partici- pants, created barriers and restricted employment opportunities for new entrants and/or individuals (and women in particular) looking for temporary or part-time employment within larger firms (Barbieri and Scherer, 2009; Del Boca, 2002). Moreover, it encour- aged the development of smaller, less regulated, family firms, often employing women in informal and irregular employment, and in self-employment, with consequent disad- vantages in terms of income, employment protection and work–life balance (Bettio and Villa, 1998; Del Boca, 2002). Different categories of female workers also benefit from different opportunities to maintain employment throughout childbearing years. Maternity protection introduced in 7 Fiori and Di Gessa the 1970s, and further extended in the 2000s, granted the right to maternity leave for all female employees in both the public and private sector, although contracts within the public sector provide enhanced protection, particularly in terms of pay and leave of absence, and allowing shorter and more flexible working patterns. However, the rights of female workers employed on non-standard short-term contracts or in self-employment, whose incidence on total female employment has grown over the last three decades (Barbieri and Scherer, 2009), are more limited. For instance, women employed with so- called atypical contracts – who now represent the 14% of total female employment (ISTAT, 2018) – often do not meet the criteria (such as length of prior employment) to be granted maternity pay, or have no guarantees of employment following maternity leave (Addabbo et al., 2019). The macro-context: A focus on the Italian labour market and institutional setting ( ) The Italian labour market also differs dramatically at the geographical level, with higher employment rates in the North compared to the South (49% vs 32%, respectively, for women in 2017) (ISTAT, 2018). The North–South divide is a long-standing phenom- enon, dating back to the unification of two very distinct areas, and the consequently dif- ferent paths and paces of development. Whereas abundant natural resources and proximity to foreign markets favoured early mass industrialisation of the Northern regions, Southern regions long remained predominantly agricultural. Consequently, very different employment opportunities and employment conditions have been available in these two parts of the country. Larger proportions of women have entered employment in the industry and manufacturing sector in the North, while the smaller proportion of women in the South have been predominantly employed in agriculture, and then in the public sectors of government, health and education (Daniele and Malanima, 2011). Equally stark are the North–South differences in the provision of childcare services, with coverage rates above 40% in some Northern regions and below 15% in most Southern regions (ISTAT, 2020). More generally, the divide extends to all spheres of social and economic development, and the two parts of the country also differ in their acceptance of (and adherence to) norms on gender roles within the family (Pinnelli and Fiori, 2007), with historically more conservative views towards women’s employment in the South. It can therefore be assumed that mothers’ employment transitions following childbirth in Italy respond also to the macro-context of regional and historical opportunities and constraints. Mothers’ participation in employment will depend on the development of employment protection over time, on structural opportunities of the labour market, and the prevailing gender and family models. Thus, younger women will be more likely to remain employed, and mothers in the South might benefit from employment in sectors facilitating work–family balance while at the same time conforming to more traditional gendered expectations. Moreover, personal, household, or job attributes might play dif- ferent roles across different macro-contexts. The data are drawn from ‘Family and Social Subjects’, a large-scale nationally repre- sentative face-to-face survey carried out by ISTAT in 2009 (ISTAT, 2013). With a Outcome variables Retrospective information on each working episode is used to derive two dichotomous variables synthesising mothers’ work trajectories following the birth of their first child. The first variable is measured for all mothers and captures whether they left employment around the birth of their first child (defined as the period within 2 years before or after the childbirth). Maternity leave is not considered a job interruption, as it is not a formal interruption of the employment relation (for employees) or cessation of trading (for self- employed). The second variable is measured only for mothers who left employment around the birth of the first child, and it distinguishes between those who never returned to work and those who went back to work. Study population The data are drawn from ‘Family and Social Subjects’, a large-scale nationally repre- sentative face-to-face survey carried out by ISTAT in 2009 (ISTAT, 2013). With a Work, Employment and Society 00(0) 8 00(0) response rate of 75%, the survey collects information on a range of demographic and socioeconomic information on 43,850 individuals. The percentage of women in the sam- ple aged 15–64 in employment is 44%, comparable to estimates from the European Labour Force Survey for the same year (46%). The analyses are restricted to female respondents born between 1920 and 1969 (i.e. aged 40 and over at the time of the interview) (N = 13,079), who had had at least one child (N = 11,226) and who were in employment before the birth of their first child (N = 6197). A more recent edition of the survey in 2016 could have allowed the extension of the analyses to women born in the first half of the 1970s, but its release did not include the information necessary to derive age at motherhood or to reconstruct employment circumstances at that time. Given the focus on women’s employment transitions around the birth of the first child, the following were excluded from the analyses: mothers who interrupted their work careers for reasons other than ‘marriage’ or ‘childbirth’; those with no information on the characteristics of their job at the time of the birth of their first child; and mothers who were single when they gave birth to their first child. The final analytical sample consisted of 5454 mothers in (formal or informal) employment before the birth of their first child. Attention is limited to employment interruptions around the birth of the first child as it has the greatest disruptive effect on female labour force participation (Desai and Waite, 1991). Employment interruptions for ‘marriage’ reasons are treated as interruptions for the ‘birth of the first child’ due to the correlated occurrence and close timing of the two events among older cohorts of Italian women (Dalla Zuanna and Castiglioni, 1994). The meso-context: Mothers’ job characteristics around the birth of the first child The meso-context: Mothers’ job characteristics around the birth of the first child A series of indicators described mothers’ job characteristics around the time of their first childbirth. Employed respondents were asked what their occupation was, whereas self-employed could self-describe as entrepreneurs, freelance, self-employed without employees, or occasional helpers. Based on these characteristics, a four-category var- iable was created distinguishing between employees in higher (e.g. manager and pro- fessional) or lower (e.g. labourer or apprentice) occupational status, or self-employed in higher (e.g. entrepreneurs and freelance) or lower (e.g. occasional helpers) occupa- tional status. Then, a variable distinguished between full-time and part-time jobs (based on respondents’ account of working time, not on actual hours worked). Based on the economic sector of work, respondents were classified as working in the third sector (health, education or public administration); in manufacturing; in agriculture, hunting and fishing; or in other sectors (including predominantly those in retail trade – no finer classifications were possible because of low numbers of mothers employed in sectors such as construction or transport). Employees also provided information about their contract duration (fixed or open-ended); sector (public or private); and contractual agreement (formal contract or verbal agreement). Table A2 (online Appendix) reports the percentage distributions of the characteristics of the mothers’ working context. – no finer classifications were possible because of low numbers of mothers employed in sectors such as construction or transport). Employees also provided information about their contract duration (fixed or open-ended); sector (public or private); and contractual agreement (formal contract or verbal agreement). Table A2 (online Appendix) reports the percentage distributions of the characteristics of the mothers’ working context. The micro-context: Individual and household characteristics As personal and household characteristics, models include: age at motherhood (< 25, 25–29, 30+); total number of years in employment before the birth of the first child; total number of children (1, 2, 3 or 4 and more); and respondents’ and their husbands’ educa- tional attainment. The original survey variable reported the highest level of completed education: none; primary (age 11); lower secondary (age 14); upper secondary (age 19); and higher education. Owing to small numbers of women with no and higher qualifica- tions, the main analyses distinguish only between up to lower secondary qualification and upper secondary qualifications or above. 9 Fiori and Di Gessa As characteristics of the wider family, models include two variables that, by survey design, refer to when respondents were aged 14. These are the employment status of the respondent’s mother (in paid work vs not in paid work, including unemployed, home- makers, or retired) and their father’s occupational status (employed in medium–high occupations such as managers and professionals, or clerical and administrative occupa- tions; employed in low technical and routine occupations; and self-employed, for which no information on occupational status was available). Lastly, women’s residential prox- imity to parents or in-laws at the time of the first marriage was included, distinguishing between women co-habiting with at least one of them, those living in the same munici- pality and those living in a different municipality. The percentage distribution of these variables is reported in Table A1 (in the online Appendix). The macro-context: Mothers’ birth cohort and region of residence To explore the role of the macro-context, the empirical analyses include two variables: geographical area of residence (North versus Centre/South), considered as a proxy of exposure to different cultural and normative contexts, as well as to different contexts of opportunities for female employment; and birth cohort (distinguishing between mothers born in the 1950s and 1960s, and those born in the decades before) to capture differences in labour market opportunities and social policies over time, as well as broader social and normative changes shaping gender and family behaviours (see Table A2 for their per- centage distributions). 10 Work, Employment and Society 00(0) Results Overall, among mothers aged 40 and older who were in paid employment before the birth of their first child, over three-quarters (77%) remained employed when their first child was born, while 10% left employment but only temporarily and 13% never returned to work. The majority of the sample was working full-time, with about 70% employed with an open-ended contract. Statistical analyses Two sets of logistic regressions were used to assess the relationship between micro, meso and macro contextual influences and mothers’ work trajectories. First, we assessed whether these influences relate to a mother’s likelihood to leave paid work around the birth of the first child. Second, focusing only on those mothers who had left the employ- ment, we tested whether they also affect the likelihood to never return to work. The base models only include personal and household-related characteristics. A sec- ond version of the models then comprises also the characteristics of a mother’s working context. To disentangle the specific contribution of the different aspects of mothers’ work context, separate models were estimated for each of the job-related variables described above. The last model specification includes the two macro variables. Micro and meso influences on leaving employment after childbirth Tables 1 and 2 show results from logistic regression models on the likelihood of leaving employment and its associations with personal and household characteristics (Table 1, model 1) and job characteristics (Table 2, models 2A–F). Model 1 (Table 1) shows significant differences between mothers who never left their job and those who did. For instance, low level of education and the total number of chil- dren were significantly associated with higher odds of leaving the labour market. Sensitivity analyses using a finer educational classification confirm the finding (Table A3, online Appendix). Older mothers and those who had a working mother in childhood were significantly less likely to leave employment around the birth of their first child, whereas mothers whose fathers were employed in low positions were more likely to do so. Moreover, compared to mothers who lived with at least one of their parents or par- ents-in-law, respondents who lived away from them were more likely to leave their job when their first child was born. Models 2A–F in Table 2 show the role that each job attribute plays on the likelihood of leaving employment around the birth of the first child, once personal and household characteristics are controlled for (full details in Table A4, online Appendix). Except for the number of hours worked, all job characteristics are significantly associated with the likelihood to leave the labour market. Mothers employed in low positions (compared to those employed in mid–high), in the private sector (compared to public), with a verbal contract (compared to written) and a fixed-term contract (compared to open-ended) who work in manufacturing, agriculture, or other sectors (compared to the third sector) are more likely to leave employment around the birth of their first child. Self-employed 11 Fiori and Di Gessa Table 1. Relationship between the micro-context and leaving employment around childbirth. Fully adjusted odds ratio. Model 1 OR 95% CI Age at motherhood: 15–24 1.01 0.86–1.19 25–29 Ref. 30 and older 0.71 0.58–0.86 Number of years worked 0.99 0.98–1.02 Education: ⩾ upper secondary Ref. ⩽ lower secondary 2.89 2.42–3.47 1 child Ref. 2 children 1.35 1.14–1.58 3 children 1.36 1.11–1.68 4 + children 1.49 1.12–1.97 Respondent’s mother employed at 14 0.70 0.62–0.82 Respondent’s father: employed in mid–high position Ref. Employed low position 1.28 1.01–1.61 Self-employed 1.18 0.93–1.52 No info/dead 1.35 0.97–1.89 Partner’s education: ⩽ lower secondary 0.99 0.84–1.19 Living with parents/in-laws Ref. Micro and meso influences on leaving employment after childbirth Parents in same municipality 0.97 0.82–1.15 Parents in other municipality 1.38 1.13–1.69 N 5454 Notes: Bold for 95% significance level. CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. Notes: Bold for 95% significance level. CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. Notes: Bold for 95% significance level. CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. mothers are less likely to leave employment compared to employees with an open-ended contract and with a formal contract. However, self-employed mothers are more likely to leave compared to those employed in the public sector. Additional analyses investigating the simultaneous effects of multiple work characteristics confirm the findings above (full details in Table A5, online Appendix). mothers are less likely to leave employment compared to employees with an open-ended contract and with a formal contract. However, self-employed mothers are more likely to leave compared to those employed in the public sector. Additional analyses investigating the simultaneous effects of multiple work characteristics confirm the findings above (full details in Table A5, online Appendix). The role of the macro-context Table 4 explores the role of macro variables. Even when individual, household and job characteristics are controlled for, mothers in the Centre–South and those born in the 1950s and 1960s were less likely to leave the labour market compared to mothers in the North and those born in preceding decades. Further, among mothers who left employ- ment, those born in the 1950s and 1960s were less likely to never return. Similar results were also found when small sub-categories of education were excluded (Table A7, online Appendix). Additional analyses (available upon request) further explored whether the role of micro and meso-level characteristics varied across birth cohorts and regions, with consistent findings across macro-contexts. Micro and meso influences on non-returns to the labour market Models on the likelihood to never return to work for mothers who left employment after their first child show no significant association with mothers’ job characteristics. Therefore, Table 3 only shows results from the base model (model 3), while full details on job characteristics can be found in Table A6, models 5A–F (online Appendix). Among those mothers who interrupted their careers, those who are older, less edu- cated, with multiple children are more likely to have never returned to work, whereas those whose mothers were in employment were more likely to return to work. Partner’s education and proximity to parents/in-laws are not associated with the duration of the exit from the labour market. Work, Employment and Society 00(0) 12 Table 2. Relationship between the meso-context and leaving employment around childbirth. Fully adjusted odds ratios. Models OR 95% CI Model 2A Employee: mid–high position Ref. Employee: low position 1.81 1.47–2.21 Self-employed: mid–high position 0.62 0.46–0.84 Self-employed: low position 1.02 0.71–1.47 Model 2B Full-time employment Ref. Part-time employment 0.85 0.67–1.07 Model 2C Government/health/education Ref. Manufacturing/industry 3.33 2.53–4.38 Agriculture 1.38 1.00–1.90 Other sector 3.95 3.05–5.11 Model 2D Employed: open-ended contract Ref. Employed: fixed contract 1.23 1.03–1.47 Self-employed 0.49 0.39–0.61 Model 2E Employed: public sector Ref. Employed: private sector 4.47 3.42–5.82 Self-employed 1.78 1.28–2.46 Model 2F Employed: formal contract Ref. Employed: verbal contract 2.22 1.84–2.67 Self-employed 0.56 0.45–0.69 N 5454 Notes: Bold for 95% significance level. The fully adjusted model controls for the individual and household Notes: Bold for 95% significance level. The fully adjusted model controls for the individual and household variables reported in Table 1. Full models in Table A4 (online Appendix). CI: confidence interval; OR: odds ratio. S F l d S l S b (2009) ISTAT Notes: Bold for 95% significance level. The fully adjusted model controls for the individual and household variables reported in Table 1. Full models in Table A4 (online Appendix). CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. Source: Family and Social Subjects (2009), ISTAT. Discussion and conclusions This study Work, Employment and Society 00(0) 14 contributes to this literature by proposing a holistic theoretical framework that situates mothers’ employment transitions by examining the intersection of the micro-context of their personal and household characteristics; the meso-context of their working environ- ments before childbirth; and lastly the macro-context of broader structural, institutional and cultural forces to which their lives are exposed. This framework has the potential to capture the multidimensionality and the complexity of mechanisms shaping transitions both out and back into the labour market following childbirth and periods of intensive childcare. This model is illustrated by drawing on quantitative longitudinal evidence from Italy – an interesting study context for its distinctive pattern of low female labour market par- ticipation but comparatively stable careers in full-time jobs for the select group of women who enter employment, as opposed to patterns of discontinuous or curtailed participation observed elsewhere in Europe (Breen, 2004; Solera, 2009). Most women who were employed before the birth of their child remained in employment (77%). Of those who left, however, nearly 60% never returned. Empirical analyses confirm that employment transitions should be understood as the result of a complex system of influences acting at different levels. First, women’s micro-contexts are defining factors of employment transitions after childbirth. In line with expectations, women’s moves out of employment first, and then their return to work, respond to personal characteristics – particularly prior labour mar- ket attachment, as measured through investment in education, and seniority – as well as to features of the wider household context – such as residential proximity to the child’s grandparents. Only partners’ characteristics do not exert any influence, although this is possibly due to homogamy between partners (Bernardi, 1999; Solera, 2018). Overall, the findings support the hypothesis that the micro-context acts as a significant influence on both the transition out of employment around childbirth, and later returns to the labour market. The influence of mothers’ micro-context, however, should not conceal the role of the occupational meso-context, which – as expected – largely defines women’s employment behaviour around childbirth. Both the statistical significance and the magnitude of the effects indicate stark differences by occupational status, industry sector and contractual type, confirming both the segmented nature of the Italian labour market and the associ- ated variation in levels of employment protection of different categories of workers. Discussion and conclusions Contemporary policy debates view female employment as crucial for the well-being of women and society at large and call for sustained research examining its enabling fac- tors and variation across countries and social groups. Academic research traditionally 13 Fiori and Di Gessa Table 3. Relationship between the micro-context and non-returns to the labour market after childbirth. Fully adjusted odds ratios. Model 3 OR 95% CI Age at motherhood: 15–24 0.69 0.51–0.92 25–29 Ref. 30 and older 1.87 1.26–2.77 Number of years worked 1.05 1.01–1.09 Education: ⩽ lower secondary 1.75 1.27–2.41 1 child Ref. 2 children 1.46 1.07–1.97 3 children 2.23 1.53–3.26 4 + children 3.55 2.09–6.03 Respondent’s mother employed at 14 0.60 0.46–0.76 Respondent’s father: employed in mid–high position Ref. Employed low position 0.83 0.54–1.28 Self-employed 0.81 0.51–1.28 No info/dead 0.80 0.44–1.45 Partner’s education: ⩽ lower secondary 0.79 0.58–1.07 Living with parents/in-laws Ref. Parents in same municipality 1.03 0.77–1.38 Parents in other municipality 0.90 0.64–1.28 N 1260 Notes: Bold for 95% significance level. Full models in Table A6 (online Appendix). CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. Table 4. Relationship between the macro-context and employment transitions following childbirth. Fully adjusted odds ratios. Table 4. Relationship between the macro-context and employment transitions following childbirth. Fully adjusted odds ratios. Leaving employment Non-return OR 95% CI OR 95% CI Birth cohort: 1920–1949 Ref. Ref. Birth cohort: 1950–1969 0.76 0.67–0.88 0.69 0.53–0.90 North Ref. Ref. Centre and South 0.68 0.59–0.78 1.19 0.93–1.53 N 5454 1260 Notes: Bold for 95% significance level. The fully adjusted model controls for individual, household and job characteristics. CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. Notes: Bold for 95% significance level. The fully adjusted model controls for individual, household and job characteristics. CI: confidence interval; OR: odds ratio. Source: Family and Social Subjects (2009), ISTAT. focuses on the role of either individual agency or macro-structural factors, and only more recent studies acknowledge the importance of looking across several levels of influence to advance understanding of mothers’ labour force participation. Discussion and conclusions Women employed in the predominantly public sectors of education, health and public administration are the least likely to leave employment after the birth of their first child. The contractual security, the enhanced level of maternity protection and the greater flex- ibility of working arrangements within this sector offer some women the privilege to combine work and family (Solera, 2009). However, once women leave employment after their first child, characteristics of their past occupation become irrelevant to their subsequent return to work. As suggested by research on Britain (Tomlinson et al., 2009), this might be explained by a misalignment of the employment contexts before and after interruption during childbearing/rearing years, as mothers downgrade to low-wage, part-time occupations upon their return to the labour market. Future research should extend the longitudinal perspective to investigate which aspects of the meso-contexts of working arrangements post-interruption enable 15 Fiori and Di Gessa the return of mothers to the labour market. Indeed, the employment profiles and trajecto- ries of women returners are an under-researched but extremely relevant topic (Tomlinson et al., 2009). As women who take longer breaks from the labour market have lower socioeconomic status than those with continuous employment, downgrading to even lower pay and shorter hours upon their return to the labour market can exacerbate class inequalities. the return of mothers to the labour market. Indeed, the employment profiles and trajecto- ries of women returners are an under-researched but extremely relevant topic (Tomlinson et al., 2009). As women who take longer breaks from the labour market have lower socioeconomic status than those with continuous employment, downgrading to even lower pay and shorter hours upon their return to the labour market can exacerbate class inequalities. The model’s application to Italian data also supports the importance of the macro- context. As expected, structural characteristics of the labour market and changes in wel- fare provision have created different opportunities for women to combine work and family life. First, younger cohorts who have benefitted from the expansion of public employment in the tertiary sector and enjoyed progressively greater levels of maternity protection (Bettio and Villa, 1998; Del Boca, 2002) were less likely to leave employ- ment, both temporarily and permanently. Second, probabilities to leave the labour mar- ket around the time of childbirth are significantly lower for women living in the Centre–South. Discussion and conclusions This partly reflects the nature of female employment in the South – first, within family-run agricultural businesses (with a weak separation between the produc- tive and domestic sphere) and, second, within the more secure and family-friendly public sector (Daniele and Malanima, 2011). However, Southern women who leave employ- ment are less likely to return. It is plausible that the more traditional norms and cultural expectations on women’s roles that exert a stronger conditioning on their entry into the labour market in the first place (Solera and Bettio, 2013), also hinder the returns of those who abandoned employment upon becoming mothers. Lastly, the study theorised and addressed the potential interplay between levels of influence, specifically exploring whether the role of the micro and meso-contexts varies across birth cohorts and regions of residence. Although in this study the effect of indi- vidual, household and job characteristics on mothers’ employment transitions is similar across macro-contexts, the potential interplay between contexts of influence should not be dismissed. It is plausible that in other country contexts, or when comparing different countries, the role of the micro and meso-context might be more strongly dependent on the broader institutional and cultural context, as suggested by Dotti Sani and Scherer (2018), and that the same set of attributes produce different outcomes in different con- texts. Similarly, future studies could extend the theoretical model and test how micro characteristics interact with the meso-context. For example, they could explore whether education plays a stronger protective role for mothers in precarious and lower-paid occupations. The merits of this research should be weighed against some limitations, mostly imposed by the data. First, the research design did not fully account for potential endo- geneity in the relationship between the birth of the first child and employment experi- ences. More sophisticated analyses could attempt to reduce the bias due to selection of mothers-to-be in employment depending on their family–work orientation. Second, as the survey does not reconstruct individuals’ residential histories, it was not possible to include time-referenced contextual indicators of the mothers’ area of residence, such as regional activity rates or childcare provision. Third, all measurements are self-reported and the possibility of recall bias in the retrospective accounts of work and fertility histo- ries cannot be excluded. ORCID iD Francesca Fiori https://orcid.org/0000-0002-7374-6618 Francesca Fiori https://orcid.org/0000-0002-7374-6618 Discussion and conclusions Lastly, the model could not be tested on more recent cohorts of Work, Employment and Society 00(0) 16 00(0) women (born after 1970) as they had not yet completed their reproductive and employ- ment histories. Their experience would have been of interest as they have been exposed to more flexible and less protected forms of employment following the labour market reforms in the 2000s. Applying the same theoretical model to more recent data would also shed light on the extent to which factors conditioning maternal employment have changed following the impact of the Great Recession of 2008–2009 and the Covid-19 pandemic on the labour market. Nevertheless, the richness of information on women’s life courses allowed a thorough investigation of the complex mechanisms influencing their labour force participation. In conclusion, this study contributes to advancing the understanding of maternal employment by proposing a novel theoretical framework that accounts for the complex interplay of multiple levels of influence on mothers’ employment transitions around childbirth. These, it is argued, are shaped by mothers’ individual and household charac- teristics (micro-context), employment characteristics (meso-context) and broader cul- tural and institutional influences (macro-context). The efficacy of this integrated approach is supported by analyses based on large-scale, high-quality nationally repre- sentative data from Italy. Future research could extend this conceptual model to other countries, to enhance understanding of maternal employment, and its enabling factors, across different cultural and institutional contexts. 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Giorgio is a statistician and demographer by training, and much of his research has focused on applying quantitative social science research methods to the social challenges presented by population ageing. In particular, Giorgio is interested in understanding the social and demographic determinants of healthy ageing to tackle factors that impact older people’s health status and contribute to their increased well- being. Giorgio’s research focuses primarily on complex relationships between social engagement (including paid work, volunteering and caring) and health in mid and later life using longitudinal secondary data and advanced quantitative methodologies. Date submitted March 2020 Date accepted January 2022
W4392237188.txt	https://journals.wlb-stuttgart.de/ojs/index.php/wfr/article/download/10625/10504	de		Rezension von: Bischoff, Johannes E. (Hrsg.), Hugenotten in Franken	Württembergisch Franken	2,024	cc-by	521	in Hugenotten Johannes E. Franken. Zum 31. Bischoff. Sickte: deutschen Hugenottentag Verlag des 1979 in Erlangen hg. von Hugenottenvereins 1979. 117 S. deutschen 111. Im Jahre 1686 wurden auf Veranlassung der Kurfürsten von Brandenburg Hugenotten im Bayreuth aufgenommen. Für sie wurde die neue Stadt Christian-Erlangen Fürstentum gegründet. Der Herausgeber, langjähriger Stadtarchivar in Erlangen, stellt in dem vorliegenden die noch vorhandenen Bändchen und schildert Spuren der Hugenottensiedlungen in 6 Orten dar (S. 18) französischen Teppichwirker in Schwabach und Erlangen eingehend die (S. 56). Der bekannte Hugenottenforscher Wilhelm Beuleke gibt dazu eine Übersicht über Herkunft und Verbleib der hugenottischen Tapetenweber und Gobelinwirker (S. 42). Theo Kiefner behandelt die „Bragelaner” (Savoyer dem Pragelatal) in aus Erlangen 1686/ 88 (S. 82). Die Schrift vermittelt neue und wichtige Informationen. P. Morand: Bilder aus Jagst- und Kochertal. der Bibel. Gotische Fresken und Wu moderne Glasfenster im Tauber-, Bad Mergentheim: Katholische Kurseelsorge 1980. 203 S., zahlr. Farbtafeln. Nach dem großen Erfolg seines Buches „Verborgene Schätze” (vgl. WFr legt der Kurgastseelsorger von Bad Mergentheim einen bildern ausgestatteten zur christlichen Kunst hat Bildband und mittelalterliche Fresken Frankens finden zu moderne sind, gesammelt und Wort sich wechselseitig erhellen. Das ihnen in den die 1979, S. 211) mit hervorragenden FarbHeimat vor. Pater Morand unserer Glasfenster, und ordnet neuen, biblische Anliegen des Buches Kirchen Hohenlohe- Texte ist zu, so daß Bild Seelsorge, Hinführung zur Botschaft der Bibel durch anschauendes Betrachten. Für den Zeitgenossen, dem die „biblischen Geschichten” oft fremd geworden sind, erfüllt das Buch die Funktion einer Biblia Pauperum. Der nach Ortsnamen gegliederte Katalogteil bringt ausführliche Sach- information. Der Kunstfreund wird manches wenig Bekannte entdecken. Gö Bernhard Schemmel: Figuren und Reliefs an Haus und Hof in Franken. Quellen und Forschungen zur Geschichte des Bistums und Hochstifts Würzburg. Band 31. Hg. von Th. Kramer u. K. Wittstadt. Würzburg: Schöningh 1978. 68 S., 334 Abb. Verfasser hat sich hier Der dankenswerterweise einem bisher wenig beachteten Gebiet zugewandt, da dieses durch seinen meist provinziellen Kunstcharakter und durch seine überwiegend volkskundliche und frömmigkeitsgeschichtliche Bedeutung einseitig festgelegt ist. B. Schemmel interpretiert das Thema unter allen Motiven dominieren im katholischen Teil Frankens Allen stehen voran die Hausmadonnen, von nur denkbaren Aspekten. Bei den diejenigen aus dem kirchlichen Bereich. denen bisher nur die kunsthistorisch be- deutendsten Beispiele, beschränkt auf Würzburg, Bamberg und Nürnberg, bearbeitet worden sind. Zu bewundern ist die arbeiter, noch mehr aber unermüdliche Sammeltätigkeit des Verfassers und seiner Mit- die methodische Gliederung und Durcharbeitung des Materials. Denn gerade da, wo weder eine Zeitobergrenze noch eine Qualitätsgrenze feste Normen setzen, ist die Gefahr, die Übersicht zu verlieren, sehr groß. Es ist aber dem Verfasser gelungen, einen charakteristischen, einen „repräsentativen” Querschnitt zu erarbeiten. Das ikonographische Register erschließt eine erstaunliche Vielfalt der Motive, das Ortsregister eine überraschende Vielzahl von Orten, in welchen Figuren und Reliefs gewiesen sind. Hans Bauer: dieser Art nach- Grünenwald Fränkische Hoftore. Steinerne Hoftore und Pforten - ein geschichte fränkischer Ortsbilder. Marktbreit: Siegfried Greß 1979. 95 Beitrag zur BauS., 156 Fotos und viele Zeichnungen. Wenn tore man in ländlicher 296 den erstaunlichen Reichtum diesem Buch betrachtet, Architektur in Franken dann an Formen und Schmuckdetails fränkischer Hof- muß bisher in verwundern, Forschung warum und dieses Grundelement Veröffentlichung so wenig
https://openalex.org/W3043462560	https://peerj.com/articles/9537v0.2/submission	English	null	Peer Review #2 of "Predictive values of stool-based tests for mucosal healing among Taiwanese patients with ulcerative colitis: a retrospective cohort analysis (v0.1)"	null	2,020	cc-by	10,470	Predictive values of stool-based tests for mucosal healing among Taiwanese patients with ulcerative colitis: a retrospective cohort analysis Hsu-Heng Yen Corresp., Equal first author, 1, 2, 3 , Mei Wen Chen Eq Chun Hsu 1 , Yang-Yuan Chen 1 2, 3 , Mei Wen Chen Equal first author, 4, 5 , Yu-Yao Chang Corresp., 6 , Hsuan-Yuan Huang 6 , Tsui- n Equal first author, 4, 5 , Yu-Yao Chang Corresp., 6 , Hsuan-Yuan Huang 6 , Tsui- , Hsuan-Yuan Huang 6 , Tsui- 1 Division of Gastroenterology, Department of Internal Medicine, Changhua christian Hospital, Changhua, Taiwan 2 Institute of Medicine, Chung Shan Medical and Dental College, Taichung, Taiwan 3 General Education Center, Chienkuo Technology University, Changhua, Taiwan 4 Department of Tumor Center, Changhua Christian Hospital, Changhua, Taiwan 5 Information Management, Chien-Kuo Technology University, Changhua, Taiwan 6 Department of Colorectal Surgery, Changhua christian Hospital, Changhua, Taiwan 1 Division of Gastroenterology, Department of Internal Medicine, Changhua christian Hospital, Changhua, Taiwan 2 Institute of Medicine, Chung Shan Medical and Dental College, Taichung, Taiwan 3 General Education Center, Chienkuo Technology University, Changhua, Taiwan 4 Department of Tumor Center, Changhua Christian Hospital, Changhua, Taiwan 5 Information Management, Chien-Kuo Technology University, Changhua, Taiwan 6 Department of Colorectal Surgery, Changhua christian Hospital, Changhua, Taiwan Corresponding Authors: Hsu-Heng Yen, Yu-Yao Chang 6 Department of Colorectal Surgery, Changhua christian Hospital, Changhua, Taiwan Corresponding Authors: Hsu-Heng Yen, Yu-Yao Chang Email address: 91646@cch.org.tw, 177176@cch.org.tw Background/Purpose. Over the past two decades, ulcerative colitis (UC) has emerged in the Asia Paciﬁc area, and its treatment goal has shifted from symptom relief to endoscopic remission. Endoscopy is the gold standard for the assessment of mucosal healing; however, it is an invasive method. Fecal calprotectin (FC) is a non-invasive stool-based inﬂammatory marker, which has been used to monitor mucosal healing status, but it is expensive. By contrast, the immune fecal occult blood test (iFOBT) is a widely utilized stool-based screening tool for colorectal cancer. In this study, we compared the predictive values of iFOBT and FC for mucosal healing in Taiwanese patients with UC. Methods. A total of 50 patients with UC identiﬁed via the electronic clinical database of Changhua Christian Hospital, Taiwan, were retrospectively enrolled from January 2018 to July 2019. Results of iFOBT, FC level, and blood tests as well as Mayo scores were reviewed and analyzed. Colonic mucosa was evaluated using the endoscopic Mayo subscore. Results. The average age of the patients was 46 years, and 62% of the patients were men. Manuscript to be reviewed Predictive values of stool-based tests for mucosal healing among Taiwanese patients with ulcerative colitis: a retrospective cohort analysis Disease distribution was as follows: E1 (26%), E2 (40%), and E3 (34%). Complete mucosal healing (Mayo score = 0) was observed in 30% of patients. Endoscopic mucosal healing with a Mayo score of 0 or 1 was observed in 62% of the patients. Results of FC and iFOBT were compared among patients with and without mucosal healing. Predictive cutoﬀ values were analyzed using receiver operating characteristics curves. iFOBT and FC had similar area under the curve for both complete mucosal healing (0.813 vs. 0.769, respectively, p = 0.5581) and endoscopic mucosal healing (0.906 vs. 0.812, respectively, p = 0.1207). Conclusion. In daily clinical practice, FC and iFOBT do not diﬀer in terms of predictive PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed values for mucosal healing among Taiwanese patients with UC. values for mucosal healing among Taiwanese patients with UC. PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Predictive values of stool-based tests for mucosal healing among Taiwanese patients with 2 ulcerative colitis: A Retrospective Cohort Analysis 3 4 Hsu-Heng Yen1,2,3, May-Wen Chen4,5, Yu-Yao Chang6, Hsuan-Yuan Huang 6, Tsui-Chun Hsu1, 5 Ya-Huei Zeng1, Yang-Yuan Chen1 6 7 1 Division of Gastroenterology, Department of Internal Medicine, Changhua Christian Hospital, 8 Changhua, Taiwan 9 2Institute of Medicine, Chung Shan Medical and Dental College, Taichung, Taiwan 10 3General Education Center, Chienkuo Technology University, Changhua, Taiwan 11 4Department of Tumor Center, Changua Christian Hospital, ChangHua, Taiwan 12 5Department of Information Management, Chien-Kuo Technology University, ChungHua, 13 Taiwan 14 6Department of Colorectal Surgery, Changhua Christian Hospital, Changhua, Taiwan 15 16 Corresponding and reprint requests to: 17 Hsu-Heng Yen 2 ulcerative colitis: A Retrospective Cohort Analysis 3 4 Hsu-Heng Yen1,2,3, May-Wen Chen4,5, Yu-Yao Chang6, Hsuan-Yuan Huang 6, Tsui-Chun H 5 Ya-Huei Zeng1, Yang-Yuan Chen1 6 7 1 Division of Gastroenterology, Department of Internal Medicine, Changhua Christian Hosp 8 Changhua, Taiwan 9 2Institute of Medicine, Chung Shan Medical and Dental College, Taichung, Taiwan 10 3General Education Center, Chienkuo Technology University, Changhua, Taiwan 11 4Department of Tumor Center, Changua Christian Hospital, ChangHua, Taiwan 12 5Department of Information Management, Chien-Kuo Technology University, ChungHua, 13 Taiwan 14 6Department of Colorectal Surgery, Changhua Christian Hospital, Changhua, Taiwan 15 16 Corresponding and reprint requests to: 17 Hsu-Heng Yen 18 E-mail: 91646@cch.org.tw, blaneyen@gmail.com PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 19 Division of Gastroenterology, Department of Internal Medicine, Changhua Christian Hospital, Manuscript to be reviewed 19 Division of Gastroenterology, Department of Internal Medicine, Changhua Christian Hospital, 21 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 22 Abstract 22 Abstract 23 24 Background/Purpose. Over the past two decades, ulcerative colitis (UC) has emerged in the 25 Asia Paciﬁc area, and its treatment goal has shifted from symptom relief to endoscopic 26 remission. Endoscopy is the gold standard for the assessment of mucosal healing; however, it is 27 an invasive method. Fecal calprotectin (FC) is a non-invasive stool-based inflammatory marker, 28 which has been used to monitor mucosal healing status, but it is expensive. By contrast, the 29 immune fecal occult blood test (iFOBT) is a widely utilized stool-based screening tool for 30 colorectal cancer. In this study, we compared the predictive values of iFOBT and FC for mucosal 31 healing in Taiwanese patients with UC. 32 Methods. A total of 50 patients with UC identified via the electronic clinical database of 33 Changhua Christian Hospital, Taiwan, were retrospectively enrolled from January 2018 to July 34 2019. Results of iFOBT, FC level, and blood tests as well as Mayo scores were reviewed and 35 analyzed. Colonic mucosa was evaluated using the endoscopic Mayo subscore. 36 Results. The average age of the patients was 46 years, and 62% of the patients were men. 37 Disease distribution was as follows: E1 (26%), E2 (40%), and E3 (34%). Complete mucosal 38 healing (Mayo score = 0) was observed in 30% of patients. Endoscopic mucosal healing with a 39 Mayo score of 0 or 1 was observed in 62% of the patients. Results of FC and iFOBT were PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) 38 healing (Mayo score = 0) was observed in 30% of patients. Endoscopic mucosal healing with a 38 healing (Mayo score = 0) was observed in 30% of patients. Endoscopic mucosal healing with a 39 Mayo score of 0 or 1 was observed in 62% of the patients. Results of FC and iFOBT were 39 Mayo score of 0 or 1 was observed in 62% of the patients. Results of FC and iFOBT were PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 40 compared among patients with and without mucosal healing. Predictive cutoff values were 41 analyzed using receiver operating characteristics curves. iFOBT and FC had similar area under 42 the curve for both complete mucosal healing (0.813 vs. 0.769, respectively, p = 0.5581) and 43 endoscopic mucosal healing (0.906 vs. 0.812, respectively, p = 0.1207). 44 Conclusion. In daily clinical practice, FC and iFOBT do not differ in terms of predictive values 45 for mucosal healing among Taiwanese patients with UC. 40 compared among patients with and without mucosal healing. Predictive cutoff values were 41 analyzed using receiver operating characteristics curves. iFOBT and FC had similar area under 42 the curve for both complete mucosal healing (0.813 vs. 0.769, respectively, p = 0.5581) and 43 endoscopic mucosal healing (0.906 vs. 0.812, respectively, p = 0.1207). 46 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 47 Introduction 48 49 Ulcerative colitis (UC) is a chronic inflammatory disease of the colon (Wei et al. 2017; Yen et a 50 2017; Yen et al. 2019); without adequate treatment and monitoring of the disease, it may lead to 51 complications such as bleeding, perforation, and development of malignancy (Wei et al. 2017). 52 The state-of-the-art treatment goal for UC has shifted from clinical remission with symptom 53 control to endoscopic remission using the treat-to-target strategy (Jackson & De Cruz 2019; 54 Rubin et al. 2019; Wei et al. 2017). Fecal calprotectin (FC) is a non-invasive fecal marker 55 commonly used in Western countries to determine mucosal healing (Freeman et al. 2019; 56 Motaganahalli et al. 2019); however, FC is expensive compared with the immune fecal occult 57 blood test (iFOBT) and is not reimbursed for clinical use in Taiwan. By contrast, colonoscopy 58 remains the gold standard for the assessment of colonic mucosal status, enables screening for 59 colitis-associated malignancies (Wei et al. 2017; Yen et al. 2017), and costs less in Asia than in 60 Western countries (Chang et al. 2020; Yen & Hsu 2019). However, colonoscopy is invasive and 61 patients may show slight reluctance to undergo this procedure (Lin et al. 2015; Yen & Hsu 62 2019). Moreover, colonoscopy may induce worsening of UC or result in complications (Wei et 63 al. 2017). 47 Introduction 64 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 65 Quantitative iFOBT has replaced guaiac-based measures of stool hemoglobin concentrations 66 owing to its better performance in colorectal cancer screening. Recent studies conducted in 67 Canada (Ma et al. 2017), Japan (Nakarai et al. 2013), Korea (Ryu et al. 2016), and China (Shi et 68 al. 2017) used iFOBT to predict mucosal healing among patients with UC (Dai et al. 2018). 69 Meanwhile, only limited data are available on the comparison of the relative predictive values of 70 iFOBT and FC for mucosal healing (Kim et al. 2020; Naganuma et al. 2020; Ryu et al. 2019; 71 Takashima et al. 2015). In 2018, with the aim of improving clinical practice, we began 72 performing iFOBT and FC at our institution for disease monitoring of patients with 73 inflammatory bowel disease (IBD). In the present study, we aimed to compare the predictive 74 values of iFOBT and FC for mucosal healing in Taiwanese patients with UC. 75 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 94 for >6 months; (b) tested by both iFOBT and FC within 1 month prior to colonoscopy; (c) had 95 medical records of clinical symptoms, partial Mayo score, and laboratory test results, and (d) had 96 undergone colonoscopy with documentation of the endoscopic Mayo scoring system. The Mayo 97 score and laboratory test data were obtained 1 month before colonoscopy. 98 99 The requirement for informed consent for data extraction was waived by the institutional review 100 board because of the retrospective design of the study and the minimal risk involved. Mayo 101 scores, including stool frequency, rectal bleeding, endoscopy findings, and overall clinical 102 evaluation, were used to evaluate UC severity using 0–3 points for each component. The partial 103 Mayo score comprised three non-endoscopic variables. An endoscopic Mayo score of 0 indicates 104 complete mucosal healing (CMH), and an endoscopic Mayo score of 0 or 1 on colonoscopy 105 indicates endoscopic mucosal healing (EMH). The primary endpoint of this study was to 106 compare the predictive values of stool-based tests for the mucosal status, i.e., CMH and EMH. 107 The secondary endpoint was to compare the correlation between endoscopy activity and stool- 108 based tests and blood-based systematic inflammatory markers. The study complied with the 109 World Medical Association Declaration of Helsinki for medical research involving human 110 subjects including research on identifiable human material and data The study was approved by 94 for >6 months; (b) tested by both iFOBT and FC within 1 month prior to colonoscopy; (c) had 95 medical records of clinical symptoms, partial Mayo score, and laboratory test results, and (d) had 96 undergone colonoscopy with documentation of the endoscopic Mayo scoring system. The Mayo 97 score and laboratory test data were obtained 1 month before colonoscopy. 94 for >6 months; (b) tested by both iFOBT and FC within 1 month prior to colonoscopy; (c) had 95 medical records of clinical symptoms, partial Mayo score, and laboratory test results, and (d) had 96 undergone colonoscopy with documentation of the endoscopic Mayo scoring system. The Mayo 97 score and laboratory test data were obtained 1 month before colonoscopy. 97 score and laboratory test data were obtained 1 month before colonoscopy. 98 99 The requirement for informed consent for data extraction was waived by the institutional review 100 board because of the retrospective design of the study and the minimal risk involved. Manuscript to be reviewed Manuscript to be reviewed 76 Materials & Methods 77 78 The medical records of patients diagnosed with UC between January 2018 and July 2019 at 79 Changhua Christian Hospital, Taiwan, were retrospectively reviewed. From January 2018, 80 patients diagnosed with IBD, including UC and Crohn’s disease, received integrated hospital 81 care by a trained IBD nurse, who recorded clinical symptoms using the Mayo scoring system for 82 UC severity during each outpatient clinic visit. Patients with UC underwent screening 83 colonoscopy for the evaluation of disease activity and monitoring of malignancy. Laboratory 84 tests were conducted annually during the follow-up period. Starting from 2018, prior to 85 colonoscopy, stool samples were collected for an immunochemical occult test to evaluate and 86 compare the usefulness of iFOBT and FC for evaluating IBD activity in clinical practice. 87 Samples for iFOBT were analyzed using the HM-JACK system (Kyowa Medex, Shizuoka, 88 Japan), which is a fully automated quantitative iFOBT system. The HM-JACK system can 89 accurately measure fecal hemoglobin concentrations within a range of 7–400 ng/mL. Samples for 90 FC were analyzed using the commercially available Quantum Blue® fCAL test (Buhlmann 91 Laboratories AG, Schonenbuch, Switzerland). 92 93 Patients enrolled in the present study met the following inclusion criteria: (a) diagnosed with UC 93 Patients enrolled in the present study met the following inclusion criteria: (a) diagnosed with UC 93 Patients enrolled in the present study met the following inclusion criteria: (a) diagnosed with UC Manuscript to be reviewed Mayo 101 scores, including stool frequency, rectal bleeding, endoscopy findings, and overall clinical 102 evaluation, were used to evaluate UC severity using 0–3 points for each component. The partial 103 Mayo score comprised three non-endoscopic variables. An endoscopic Mayo score of 0 indicates 104 complete mucosal healing (CMH), and an endoscopic Mayo score of 0 or 1 on colonoscopy 105 indicates endoscopic mucosal healing (EMH). The primary endpoint of this study was to 106 compare the predictive values of stool-based tests for the mucosal status, i.e., CMH and EMH. 107 The secondary endpoint was to compare the correlation between endoscopy activity and stool- 108 based tests and blood-based systematic inflammatory markers. The study complied with the 109 World Medical Association Declaration of Helsinki for medical research involving human 110 subjects including research on identifiable human material and data. The study was approved by 111 the Institutional Review Board of Changhua Christian Hospital (approval number: CCH IRB Manuscript to be reviewed Manuscript to be reviewed 112 190814). 113 114 Statistical analysis 115 The extracted data were organized using Microsoft Excel and analyzed using MedCalc Statistical 116 Software version 19.16 (MedCalc Software bvba, Ostend, Belgium; https://www.medcalc.org; 117 2020). Continuous data are expressed as means and standard deviations or as medians and 118 interquartile ranges for normally and non-normally distributed data, respectively. Categorical 119 variables are presented as numbers and percentages. The mean values of normally distributed 120 variables were compared by an independent sample’s Student’s t-test. Mann–Whitney U-test and 121 Kruskal–Wallis test were performed to compare the mean values of 2 and ≥3 groups of variables, 122 respectively, with non-normal distributions. The frequencies of categorical variables were 123 compared using Pearson’s χ2 or Fisher’s exact test, as appropriate. Spearman’s rank correlation 124 was performed to determine the correlation between fecal test data and UC severity as reflected 125 by the Mayo score. Receiver operating characteristic (ROC) curve analysis was conducted to 126 determine the best cutoff values of iFOBT and FC levels for predicting mucosal healing. All P- 127 values of <0.05 were considered statistically significant. 127 values of <0.05 were considered statistically significant. 128 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 129 Results 130 131 Clinical features of patients with UC 132 During the study period, a total of 102 patients with UC received treatment at the hospital and 50 133 met the inclusion criteria. The clinical characteristics of all patients are presented in Table 1. The 134 average age of the patients was 46 years, and the majority of them were men (31 men, 19 135 women). The median duration of UC was 3.5 years. Based on the Montreal classification, the 136 diseases observed included proctitis (E1, 26%), left-sided UC (E2, 40%), and extensive UC (E3, 137 34%). Further, there were 8% current smokers, 24% ever-smokers, and 68% non-smokers. Oral 138 5-aminosalicylic acid (5-ASA) was administered to 94% of the patients, followed by rectal 5- 139 ASA (54%), steroids (34%), and immune modulators (24%). CMH with an endoscopic Mayo 140 score of 0 was achieved in 30% of the patients, whereas EMH)with an endoscopic Mayo score of 141 ≤1 was achieved in 62%. 142 143 Comparison of patients with and without mucosal healing 129 Results 130 131 Clinical features of patients with UC 132 During the study period, a total of 102 patients with U 133 met the inclusion criteria. The clinical characteristics 134 average age of the patients was 46 years, and the majo 135 women). The median duration of UC was 3.5 years. B 136 diseases observed included proctitis (E1, 26%), left-si 137 34%). Further, there were 8% current smokers, 24% e 138 5-aminosalicylic acid (5-ASA) was administered to 9 139 ASA (54%), steroids (34%), and immune modulators 140 score of 0 was achieved in 30% of the patients, where 141 ≤1 was achieved in 62%. 142 143 C i f i i h d i h l h 129 Results 143 Comparison of patients with and without mucosal healing 143 Comparison of patients with and without mucosal healing 144 In our cohort, 30% (15/50) of the patients exhibited CMH as evaluated by colonoscopy; the 145 remaining patients had endoscopic Mayo scores of 1 (33.3%), 2 (18.52%), and 3 (22.22%) 146 (Table 2). Age, sex, disease distribution, and drugs used did not differ between patients with PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 147 CMH and those without CMH. Compared with patients without CMH, those with CMH had 148 lower iFOBT (P = 0.003) and FC (P = 0.0028) values. 147 CMH and those without CMH. Compared with patients without CMH, those with CMH had 148 lower iFOBT (P = 0.003) and FC (P = 0.0028) values. 149 EMH, as indicated by an endoscopic Mayo score of 0 or 1 on colonoscopy, was achieved in 65% 150 (Table 3). Age, sex, disease distribution, and drugs used did not differ between those with EMH 151 and those without EMH. Compared with patients without EMH, those with EMH had lower 152 iFOBT (P <0.001) and FC (P = 0.0002) values. 154 Relative predictive values of iFOBT and FC for mucosal healing 155 Both iFOBT and FC showed moderate correlations with the total endoscopic Mayo score (Fig. 156 1). The correlation among stool-based tests (FC and iFOBT), blood inflammatory markers (CRP 157 and ESR), and endoscopically evaluated UC activity was analyzed using Spearman’s rank 158 correlation coefficient (Table 4). Compared with FC, ESR, and CRP, iFOBT exhibited a higher 159 correlation with the endoscopic Mayo score. As illustrated in Fig. 2, an iFOBT criterion of ≤30 160 ng/mL had 93.33% sensitivity and 71.43% specificity in terms of predicting CMH, whereas an 161 iFOBT criterion of ≤43 ng/mL had 80.65% sensitivity and 100% specificity in predicting EMH. 162 As illustrated in Fig. 3, an FC criterion of ≤156 μg/g had 86.67% sensitivity and 62.86% 163 specificity in terms of predicting CMH, whereas an FC criterion of ≤156 μg/g had 74.19% 164 sensitivity and 84.21% specificity in predicting EMH. As illustrated in Fig. 4, the ROC curve P J i i PDF \| (2020 05 48530 1 1 NEW 18 J 2020) 155 Both iFOBT and FC showed moderate correlations with the total endoscopic Mayo score (Fig. 156 1). The correlation among stool-based tests (FC and iFOBT), blood inflammatory markers (CRP 157 and ESR), and endoscopically evaluated UC activity was analyzed using Spearman’s rank 158 correlation coefficient (Table 4). Compared with FC, ESR, and CRP, iFOBT exhibited a higher 159 correlation with the endoscopic Mayo score. As illustrated in Fig. 2, an iFOBT criterion of ≤30 160 ng/mL had 93.33% sensitivity and 71.43% specificity in terms of predicting CMH, whereas an 161 iFOBT criterion of ≤43 ng/mL had 80.65% sensitivity and 100% specificity in predicting EMH. Manuscript to be reviewed 162 As illustrated in Fig. 3, an FC criterion of ≤156 μg/g had 86.67% sensitivity and 62.86% 163 specificity in terms of predicting CMH, whereas an FC criterion of ≤156 μg/g had 74.19% 164 sensitivity and 84.21% specificity in predicting EMH. As illustrated in Fig. 4, the ROC curve PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) 166 higher AUCs for both CMH and EMH than FC values. Manuscript to be reviewed 165 analysis of the ability to predict mucosal healing showed that iFOBT results tended to have 166 higher AUCs for both CMH and EMH than FC values. 166 higher AUCs for both CMH and EMH than FC values. 168 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 169 Discussion 170 171 In the present study, we compared the predictive values of iFOBT and FC for mucosal healing 172 among Taiwanese patients with UC in clinical practice. Our findings were consistent with those 173 of previous studies, which demonstrated that iFOBT and FC had similar predictive values for 174 predicting EMH among patients with UC (Kim et al. 2018; Kim et al. 2020; Ma et al. 2017; 175 Naganuma et al. 2020; Ryu et al. 2019; Takashima et al. 2015). iFOBT might be used as an 176 alternative non-invasive monitoring tool for patients with UC, particularly in Taiwan, where 177 other fecal tests such as FC level are not widely available and are more expensive. 178 179 The incidence of IBD is increasing worldwide, particularly in the Asia Pacific region (Jung 180 2020; Wei et al. 2013; Yen et al. 2019). Our recent national cohort study from Taiwan (Yen et a 181 2019) reported a six-fold increase to 12.8/100,000 in the prevalence of UC over the past 15 182 years. The treatment goal for IBD has shifted from clinical remission to biochemical remission, 183 endoscopic remission, and histological healing (Rubin et al. 2019; Wei et al. 2017). Patients wit 184 UC are typically evaluated using clinical symptoms based on the Mayo scoring system. 185 However, subjective reports of symptoms such as bowel frequency may not correlate well with 186 the endoscopy findings. Uncontrolled but asymptomatic inflammation may increase the risk of 169 Discussion 170 171 In the present study, we compared the predictive values of iFOBT and FC for mucosal healing 172 among Taiwanese patients with UC in clinical practice. Our findings were consistent with those 173 of previous studies, which demonstrated that iFOBT and FC had similar predictive values for 174 predicting EMH among patients with UC (Kim et al. 2018; Kim et al. 2020; Ma et al. 2017; 175 Naganuma et al. 2020; Ryu et al. 2019; Takashima et al. 2015). iFOBT might be used as an 176 alternative non-invasive monitoring tool for patients with UC, particularly in Taiwan, where 177 other fecal tests such as FC level are not widely available and are more expensive. 185 However, subjective reports of symptoms such as bowel frequency may not correlate well with 186 the endoscopy findings. Uncontrolled but asymptomatic inflammation may increase the risk of PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 187 disease relapse or subsequent development of complications(Peyrin-Biroulet et al. 2015). 188 Colonoscopy has been the gold standard for the evaluation of mucosal status; however, it is 189 invasive and less acceptable by patients compared with non-invasive tests such as blood and 190 stool tests. In the present study, 25% of the patients with an endoscopic Mayo score of 3 for 191 severe colonic inflammation reported no rectal bleeding and/or had normal or near-normal stool 192 frequencies. Therefore, relying on patient-reported symptoms may underestimate the severity of 193 colonic inflammation. 194 Calprotectin is a calcium-binding protein, which is mainly found in neutrophils (Wei 2016). FC 195 has been correlated with mucosal inflammation and has been used as a surrogate biomarker for 196 evaluating IBD activity in Western countries (Kim et al. 2020; Lin et al. 2015; Takashima et al. 197 2015). Although non-invasive, FC is not readily available in Asia and it costs the same as or 198 more than colonoscopy in Taiwan (Lin et al. 2015; Wei et al. 2017); therefore, its use in our 199 clinical practice has been limited. By contrast, quantitative iFOBT is a stool-based test for colon 200 cancer screening and is available worldwide (Yen & Hsu 2019). Unlike Crohn’s disease, UC 201 involves the superficial colonic wall; therefore, mucosal hemorrhage may be used as a surrogate 202 marker for predicting mucosal damage (Kuriyama et al. 2010). 203 187 disease relapse or subsequent development of complications(Peyrin-Biroulet et al. 2015). 188 Colonoscopy has been the gold standard for the evaluation of mucosal status; however, it is 189 invasive and less acceptable by patients compared with non-invasive tests such as blood and 190 stool tests. In the present study, 25% of the patients with an endoscopic Mayo score of 3 for 191 severe colonic inflammation reported no rectal bleeding and/or had normal or near-normal stool 192 frequencies. Therefore, relying on patient-reported symptoms may underestimate the severity of 193 colonic inflammation. 194 Calprotectin is a calcium-binding protein, which is mainly found in neutrophils (Wei 2016). FC 195 has been correlated with mucosal inflammation and has been used as a surrogate biomarker for 196 evaluating IBD activity in Western countries (Kim et al. 2020; Lin et al. 2015; Takashima et al. 197 2015). Although non-invasive, FC is not readily available in Asia and it costs the same as or 198 more than colonoscopy in Taiwan (Lin et al. Manuscript to be reviewed 2015; Wei et al. 2017); therefore, its use in our 199 clinical practice has been limited. By contrast, quantitative iFOBT is a stool-based test for colon 200 cancer screening and is available worldwide (Yen & Hsu 2019). Unlike Crohn’s disease, UC 201 involves the superficial colonic wall; therefore, mucosal hemorrhage may be used as a surrogate 202 marker for predicting mucosal damage (Kuriyama et al. 2010). PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 205 and FC for mucosal healing in patients with IBD, particularly with UC. Further, in Asia, the 206 interest in the use of iFOBT as a test for predicting mucosal damage among patients with UC has 207 been increasing (Naganuma et al. 2019; Nakarai et al. 2013; Ryu et al. 2016; Ryu et al. 2019; Shi 208 et al. 2017; Takashima et al. 2015). Using different cutoff levels and test kits, iFOBT was 209 reported to predict endoscopy mucosal healing with 58%–94.9% sensitivity and 38.3%–100% 210 specificity. Data on the optimal cutoff levels of these stool-based tests for predicting EMH has 211 varied among different studies due to the different testing kits used. 212 213 Although the UC treatment guidelines in Taiwan have recommended FC as a biomarker for 214 detecting colonic inflammation and for evaluating disease activity (Wei et al. 2017), this test is 215 not widely available and is not reimbursed by the national insurance in Taiwan. Few studies have 216 compared the performances of iFOBT and FC (Table 5), and the results are conflicting. 217 Takashima et al. (Takashima et al. 2015) conducted the first study on the use of FC and iFOBT 218 to predict the mucosal status of 92 patients with UC. They found that both iFOBT and FC 219 efficiently predicted mucosal healing in UC, but compared with FC, iFOBT appeared to be more 220 sensitive in predicting CMH. Ryu et al. (Ryu et al. 2019) from Korea found a more accurate 221 prediction of endoscopic activity with FC than with iFOBT. Kim et al. (Kim et al. 2018), also 222 from Korea, found that compared with iFOBT, FC was better in predicting CMH but with a 209 reported to predict endoscopy mucosal healing with 58%–94.9% sensitivity and 38.3%–100% 210 specificity. Data on the optimal cutoff levels of these stool-based tests for predicting EMH has 211 varied among different studies due to the different testing kits used. 212 213 Although the UC treatment guidelines in Taiwan have recommended FC as a biomarker for 214 detecting colonic inflammation and for evaluating disease activity (Wei et al. 2017), this test is 215 not widely available and is not reimbursed by the national insurance in Taiwan. Few studies have 216 compared the performances of iFOBT and FC (Table 5), and the results are conflicting. 217 Takashima et al. (Takashima et al. Manuscript to be reviewed 223 similar performance with regard to predicting EMH (Kim et al. 2018; Kim et al. 2020). To the 224 best of our knowledge, the present study is the first from Taiwan to compare the relative 225 predictive values of iFOBT and FC. Our findings are consistent with those of a recent Japanese 226 nationwide cohort study (Naganuma et al. 2020), which suggested similar predictive values of 227 iFOBT and FC for the mucosal status of patients with UC. Given the low cost and similar 228 predictive value of FC, iFOBT may be advantageous as a first-line monitoring tool for patients 229 with UC in Taiwan. 230 231 There are several limitations to this study. First, our study had a single-center retrospective 232 design; therefore, the results require validation by further large-scale studies outside Taiwan. 233 Endoscopic findings were retrospectively reviewed and may have been biased due to the fact that 234 different endoscopists were involved. Second, we only performed one iFOBT and FC test before 235 colonoscopy owing to the high cost of FC. Therefore, we were not able to investigate and 236 compare the potential diagnostic relevance of iFOBT and FC in predicting treatment response or 237 disease relapse after remission. The combined use of both fecal tests is promising, and Japanese 238 researchers have shown promising results with regard to predicting disease relapse (Naganuma et 239 al. 2020; Nakarai et al. 2018). Third, colonic polyps or cancer was not identified in any of our 240 patients during colonoscopy. Theoretically, iFOBT cannot differentiate between UC and 223 similar performance with regard to predicting EMH (Kim et al. 2018; Kim et al. 2020). To the 224 best of our knowledge, the present study is the first from Taiwan to compare the relative 225 predictive values of iFOBT and FC. Our findings are consistent with those of a recent Japanese 226 nationwide cohort study (Naganuma et al. 2020), which suggested similar predictive values of 227 iFOBT and FC for the mucosal status of patients with UC. Given the low cost and similar 228 predictive value of FC, iFOBT may be advantageous as a first-line monitoring tool for patients 229 with UC in Taiwan. 230 231 There are several limitations to this study. First, our study had a single-center retrospective 232 design; therefore, the results require validation by further large-scale studies outside Taiwan. Manuscript to be reviewed 2015) conducted the first study on the use of FC and iFOBT 218 to predict the mucosal status of 92 patients with UC. They found that both iFOBT and FC 219 efficiently predicted mucosal healing in UC, but compared with FC, iFOBT appeared to be more 220 sensitive in predicting CMH. Ryu et al. (Ryu et al. 2019) from Korea found a more accurate 221 prediction of endoscopic activity with FC than with iFOBT. Kim et al. (Kim et al. 2018), also 222 from Korea, found that compared with iFOBT, FC was better in predicting CMH but with a 211 varied among different studies due to the different testing kits used. PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 233 Endoscopic findings were retrospectively reviewed and may have been biased due to the fact that 234 different endoscopists were involved. Second, we only performed one iFOBT and FC test before 235 colonoscopy owing to the high cost of FC. Therefore, we were not able to investigate and 236 compare the potential diagnostic relevance of iFOBT and FC in predicting treatment response or 237 disease relapse after remission. The combined use of both fecal tests is promising, and Japanese 238 researchers have shown promising results with regard to predicting disease relapse (Naganuma et 239 al. 2020; Nakarai et al. 2018). Third, colonic polyps or cancer was not identified in any of our 240 patients during colonoscopy. Theoretically, iFOBT cannot differentiate between UC and PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Fecal immunochemical test for predicting mucosal 263 healing in ulcerative colitis patients: A systematic review and meta-analysis. J 264 Gastroenterol Hepatol 33:990-997. 10.1111/jgh.14121 265 Freeman K, Willis BH, Fraser H, Taylor-Phillips S, and Clarke A. 2019. Faecal calprotectin to 266 detect inflammatory bowel disease: a systematic review and exploratory meta-analysis 267 of test accuracy. BMJ Open 9:e027428. 10.1136/bmjopen-2018-027428 268 Jackson BD, and De Cruz P. 2019. Quality of Care in Patients With Inflammatory Bowel Disease. 269 Inflamm Bowel Dis 25:479-489. 10.1093/ibd/izy276 261 10.1016/j.jfma.2019.12.005 262 Dai C, Jiang M, Sun MJ, and Cao Q. 2018. Fecal immunochemical test for predicting mucosal 263 healing in ulcerative colitis patients: A systematic review and meta-analysis. J 264 Gastroenterol Hepatol 33:990-997. 10.1111/jgh.14121 265 Freeman K, Willis BH, Fraser H, Taylor-Phillips S, and Clarke A. 2019. Faecal calprotectin to 266 detect inflammatory bowel disease: a systematic review and exploratory meta-analysis 267 of test accuracy. BMJ Open 9:e027428. 10.1136/bmjopen-2018-027428 268 Jackson BD, and De Cruz P. 2019. Quality of Care in Patients With Inflammatory Bowel Disease. 269 Inflamm Bowel Dis 25:479-489. 10.1093/ibd/izy276 270 Jung YS. 2020. Trends in Healthcare Costs for Inflammatory Bowel Disease in South Korea. Gut 271 Liver 14:3-4. 10.5009/gnl19374 268 Jackson BD, and De Cruz P. 2019. Quality of Care in Patients With Inflammatory Bowel Disease. 269 Inflamm Bowel Dis 25:479-489. 10.1093/ibd/izy276 270 Jung YS. 2020. Trends in Healthcare Costs for Inflammatory Bowel Disease in South Korea. Gut 271 Liver 14:3-4. 10.5009/gnl19374 270 Jung YS. 2020. Trends in Healthcare Costs for Inflammatory Bowel Disease in South Korea. Gut 271 Liver 14:3-4. 10.5009/gnl19374 272 Kim DJ, Jeoun YM, Lee DW, Koo JS, and Lee SW. 2018. Usefulness of fecal immunochemical test 273 and fecal calprotectin for detection of active ulcerative colitis. Intest Res 16:563-570. 274 10.5217/ir.2018.00020 272 Kim DJ, Jeoun YM, Lee DW, Koo JS, and Lee SW. 2018. Usefulness of fecal immunochemical test 273 and fecal calprotectin for detection of active ulcerative colitis. Intest Res 16:563-570. 274 10.5217/ir.2018.00020 275 Kim ES, Lee HS, Kim SK, Kim EY, Jang BI, Kim KO, Yang CH, Lee YJ, Crohn's, and Colitis Association 276 in D-G. 2020. Fecal calprotectin is more accurate than fecal immunochemical test for 277 predicting mucosal healing in quiescent ulcerative colitis: a prospective multicenter 278 study. Scand J Gastroenterol:1-6. 10.1080/00365521.2020.1714716 279 Kuriyama M, Kato J, Takemoto K, Hiraoka S, Okada H, and Yamamoto K. 2010. Manuscript to be reviewed 241 colorectal neoplasms. Importantly, iFOBT should not replace surveillance colonoscopy; we 242 suggest its use as a monitoring tool to assess disease activity rather than for cancer surveillance. 243 244 Conclusions 245 246 The increased prevalence of UC in Taiwan has raised the need for a practical tool to monitor 247 disease activity. Our study found similar predictive values of iFOBT and FC for both CMH and 248 EMH. Therefore, iFOBT might be useful as a first-line non-invasive tool in clinical practice to 249 evaluate disease severity and may assist in clinical decision making. 250 251 Acknowledgments 252 253 The authors received funding for this study from Changhua Christian Hospital (106-IRP-CCH- 254 030 and 108-CCHIRP-018). 255 256 References 257 258 Chang CW, Tu CH, Chou JW, Huang TY, Hsu WH, Wang YP, Chen CC, Chung CS, Lin CP, Lin WC, 259 Tai CM, Wang HY, and Chen MJ. 2020. Endoscopic management of strictures in patients 241 colorectal neoplasms. Importantly, iFOBT should not replace surveillance colonoscopy; we 242 suggest its use as a monitoring tool to assess disease activity rather than for cancer surveillance. 243 244 Conclusions 245 246 The increased prevalence of UC in Taiwan has raised the need for a practical tool to monitor 247 disease activity. Our study found similar predictive values of iFOBT and FC for both CMH and 248 EMH. Therefore, iFOBT might be useful as a first-line non-invasive tool in clinical practice to 249 evaluate disease severity and may assist in clinical decision making. 250 251 Acknowledgments 252 253 The authors received funding for this study from Changhua Christian Hospital (106-IRP-CCH- 258 Chang CW, Tu CH, Chou JW, Huang TY, Hsu WH, Wang YP, Chen CC, Chung CS, Lin CP, Lin WC, 259 Tai CM, Wang HY, and Chen MJ. 2020. Endoscopic management of strictures in patients Manuscript to be reviewed 260 with Crohn's disease - A multi-center experience in Taiwan. J Formos Med Assoc. 261 10.1016/j.jfma.2019.12.005 262 Dai C, Jiang M, Sun MJ, and Cao Q. 2018. Fecal immunochemical test for predicting mucosal 263 healing in ulcerative colitis patients: A systematic review and meta-analysis. J 264 Gastroenterol Hepatol 33:990-997. 10.1111/jgh.14121 265 Freeman K, Willis BH, Fraser H, Taylor-Phillips S, and Clarke A. 2019. Faecal calprotectin to 266 detect inflammatory bowel disease: a systematic review and exploratory meta-analysis 267 of test accuracy. BMJ Open 9:e027428. 10.1136/bmjopen-2018-027428 268 Jackson BD, and De Cruz P. 2019. Quality of Care in Patients With Inflammatory Bowel Disease. 269 Inflamm Bowel Dis 25:479-489. 10.1093/ibd/izy276 270 Jung YS. 2020. Trends in Healthcare Costs for Inflammatory Bowel Disease in South Korea. Gut 271 Liver 14:3-4. 10.5009/gnl19374 272 Kim DJ, Jeoun YM, Lee DW, Koo JS, and Lee SW. 2018. Usefulness of fecal immunochemical test 273 and fecal calprotectin for detection of active ulcerative colitis. 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Significance of Conducting 2 Types of Fecal Tests in Patients with 298 Ulcerative Colitis. Clin Gastroenterol Hepatol. 10.1016/j.cgh.2019.07.054 299 Naganuma M, Kobayashi T, Nasuno M, Motoya S, Kato S, Matsuoka K, Hokari R, Watanabe C, 300 Sakamoto H, Yamamoto H, Sasaki M, Watanabe K, Iijima H, Endo Y, Ichikawa H, Ozeki K, 301 Tanida S, Ueno N, Fujiya M, Sako M, Takeuchi K, Sugimoto S, Abe T, Hibi T, Suzuki Y, and 302 Kanai T. 2020. Significance of Conducting 2 Types of Fecal Tests in Patients With 303 Ulcerative Colitis. Clin Gastroenterol Hepatol 18:1102-1111.e1105. 304 10.1016/j.cgh.2019.07.054 305 Nakarai A, Hiraoka S, Takahashi S, Inaba T, Higashi R, Mizuno M, Takashima S, Inokuchi T, 306 Sugihara Y, Takahara M, Harada K, Kato J, and Okada H. 2018. Simultaneous 307 Measurements of Faecal Calprotectin and the Faecal Immunochemical Test in Quiescent 308 Ulcerative Colitis Patients Can Stratify Risk of Relapse. 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Fecal calprotectin 284 correlated with endoscopic remission for Asian inflammatory bowel disease patients. 285 World J Gastroenterol 21:13566-13573. 10.3748/wjg.v21.i48.13566 282 Lin WC, Wong JM, Tung CC, Lin CP, Chou JW, Wang HY, Shieh MJ, Chang CH, Liu HH, Wei SC, and 283 Taiwan Society of Inflammatory Bowel Disease Multicenter S. 2015. Fecal calprotectin 284 correlated with endoscopic remission for Asian inflammatory bowel disease patients. 285 World J Gastroenterol 21:13566-13573. 10.3748/wjg.v21.i48.13566 286 Ma C, Lumb R, Walker EV, Foshaug RR, Dang TT, Verma S, Huang VW, Kroeker KI, Wong K, 287 Dieleman LA, Fedorak RN, and Halloran BP. 2017. Noninvasive Fecal Immunochemical 288 Testing and Fecal Calprotectin Predict Mucosal Healing in Inflammatory Bowel Disease: 289 A Prospective Cohort Study. Inflamm Bowel Dis 23:1643-1649. 290 10.1097/MIB.0000000000001173 291 Motaganahalli S, Beswick L, Con D, and van Langenberg DR. 2019. Faecal calprotectin delivers 292 on convenience, cost reduction and clinical decision-making in inflammatory bowel 293 disease: a real-world cohort study. Intern Med J 49:94-100. 10.1111/imj.14027 294 Naganuma M, Kobayashi T, Nasuno M, Motoya S, Kato S, Matsuoka K, Hokari R, Watanabe C, 295 Sakamoto H, Yamamoto H, Sasaki M, Watanabe K, Iijima H, Endo Y, Ichikawa H, Ozeki K, 294 Naganuma M, Kobayashi T, Nasuno M, Motoya S, Kato S, Matsuoka K, Hokari R, Watanabe C, 295 Sakamoto H, Yamamoto H, Sasaki M, Watanabe K, Iijima H, Endo Y, Ichikawa H, Ozeki K, PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 332 Endoscopic and Histological Healing in Ulcerative Colitis: A Prospective Study Based on 333 Validated Histological Scores. J Crohns Colitis 11:1071-1077. 10.1093/ecco-jcc/jjx088 334 Takashima S, Kato J, Hiraoka S, Nakarai A, Takei D, Inokuchi T, Sugihara Y, Takahara M, Harada 335 K, Okada H, Tanaka T, and Yamamoto K. 2015. Evaluation of Mucosal Healing in 336 Ulcerative Colitis by Fecal Calprotectin Vs. Fecal Immunochemical Test. Am J 337 Gastroenterol 110:873-880. 10.1038/ajg.2015.66 338 Wei SC. 2016. Could fecal calprotectin enter mainstream use for diagnosing and monitoring 339 inflammatory bowel disease? Intest Res 14:293-294. 10.5217/ir.2016.14.4.293 340 Wei SC, Chang TA, Chao TH, Chen JS, Chou JW, Chou YH, Chuang CH, Hsu WH, Huang TY, Hsu 341 TC, Lin CC, Lin HH, Lin JK, Lin WC, Ni YH, Shieh MJ, Shih IL, Shun CT, Tsang YM, Wang CY, 342 Wang HY, Weng MT, Wu DC, Wu WC, Yen HH, and Wong JM. 2017. Management of 343 ulcerative colitis in Taiwan: consensus guideline of the Taiwan Society of Inflammatory 344 Bowel Disease. Intest Res 15:266-284. 10.5217/ir.2017.15.3.266 345 Wei SC, Lin MH, Tung CC, Weng MT, Kuo JS, Shieh MJ, Wang CY, Ho WC, Wong JM, and Chen 346 PC. 2013. A nationwide population-based study of the inflammatory bowel diseases 347 between 1998 and 2008 in Taiwan. BMC Gastroenterol 13:166. 10.1186/1471-230X-13- 348 166 349 Yen HH, Chang CW, Chou JW, and Wei SC. 2017. Balloon-Assisted Enteroscopy and Capsule 350 Endoscopy in Suspected Small Bowel Crohn's Disease. Clin Endosc 50:417-423. 351 10.5946/ce.2017.142 352 Yen HH, and Hsu YC. 2019. Changing from two- to one-operator colonoscopy insertion 353 technique is feasible with similar quality outcomes. JGH Open 3:159-162. 354 10.1002/jgh3.12124 355 Yen HH, Weng MT, Tung CC, Wang YT, Chang YT, Chang CH, Shieh MJ, Wong JM, and Wei SC. 356 2019. Epidemiological trend in inflammatory bowel disease in Taiwan from 2001 to 357 2015: a nationwide populationbased study. Intest Res 17:54-62. 10.5217/ir.2018.00096 358 332 Endoscopic and Histological Healing in Ulcerative Colitis: A Prospective Study Based on 333 Validated Histological Scores. 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Assessment of disease 325 activity by fecal immunochemical test in ulcerative colitis. World J Gastroenterol 326 22:10617-10624. 10.3748/wjg.v22.i48.10617 324 Ryu DG, Kim HW, Park SB, Kang DH, Choi CW, Kim SJ, and Nam HS. 2016. Assessment of disease 325 activity by fecal immunochemical test in ulcerative colitis. World J Gastroenterol 326 22:10617-10624. 10.3748/wjg.v22.i48.10617 327 Ryu DG, Kim HW, Park SB, Kang DH, Choi CW, Kim SJ, and Nam HS. 2019. Clinical implications of 328 fecal calprotectin and fecal immunochemical test on mucosal status in patients with 329 ulcerative colitis. Medicine (Baltimore) 98:e17080. 10.1097/MD.0000000000017080 330 Shi HY, Chan FKL, Chan AWH, Higashimori A, Kyaw M, Ching JYL, Luk AKC, Wong SH, Wu JCY, 331 Sung JJY, and Ng SC. 2017. Accuracy of Faecal Immunochemical Test to Predict 327 Ryu DG, Kim HW, Park SB, Kang DH, Choi CW, Kim SJ, and Nam HS. 2019. Clinical implications of 328 fecal calprotectin and fecal immunochemical test on mucosal status in patients with 329 ulcerative colitis. Medicine (Baltimore) 98:e17080. 10.1097/MD.0000000000017080 330 Shi HY, Chan FKL, Chan AWH, Higashimori A, Kyaw M, Ching JYL, Luk AKC, Wong SH, Wu JCY, 331 Sung JJY, and Ng SC. 2017. Accuracy of Faecal Immunochemical Test to Predict PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 10.1038/ajg.2015.66 345 Wei SC, Lin MH, Tung CC, Weng MT, Kuo JS, Shieh MJ, Wang CY, Ho WC, Wong JM, and Chen 346 PC. 2013. A nationwide population-based study of the inflammatory bowel diseases 347 between 1998 and 2008 in Taiwan. BMC Gastroenterol 13:166. 10.1186/1471-230X-13- 348 166 349 Yen HH, Chang CW, Chou JW, and Wei SC. 2017. Balloon-Assisted Enteroscopy and Capsule 350 Endoscopy in Suspected Small Bowel Crohn's Disease. Clin Endosc 50:417-423. 351 10.5946/ce.2017.142 352 Yen HH, and Hsu YC. 2019. Changing from two- to one-operator colonoscopy insertion 353 technique is feasible with similar quality outcomes. JGH Open 3:159-162. 354 10.1002/jgh3.12124 355 Yen HH, Weng MT, Tung CC, Wang YT, Chang YT, Chang CH, Shieh MJ, Wong JM, and Wei SC. 356 2019. Epidemiological trend in inflammatory bowel disease in Taiwan from 2001 to 357 2015: a nationwide populationbased study. Intest Res 17:54-62. 10.5217/ir.2018.00096 358 359 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Table 1(on next page) Demographic data of the patients PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Table 1. Demographic data of the patients Clinical variable Sex (M/F) 31/19 Age, years (mean, 95% CI) 46 (39 ~ 52.4) Disease duration (median, IQR) 3.5 (2–6) Disease distribution (E1/E2/E3) 13/20/17 (26%/40%/34%) Smoking status (nonsmoker/current smoker/ever-smoker) 34/4/12 (68%/8%/24%) Current medication - steroid (Y/N) 17/33 (34%/66%) - oral 5-ASA (Y/N) 47/3 (94%/6%) - rectal 5-ASA (Y/N) 27/23 (54%/47%) - immune modulator (Y/N) 12/38 (24%/76%) - biologic agent (Y/N) 1/49 (2%/98%) WBC count, 103/μL (median, IQR) 6.5 (5.6–7.4) Hemoglobin level, g/dL (median, IQR) 14.15 (13.26–14.6) Platelet count,103/μL (median, IQR) 261 (229.83–295.95) NLR (median, IQR) 2.26 (1.84–2.74) CRP level, mg/dL (median, IQR) 0.13 (0.09 –0.24) ESR, mm/h (median, IQR) 10 (8–13.8) Endoscopy Mayo score (0/1/2/3) 15/16/8/11(30%/32%/16%/22%)) iFOBT, ng/ml (median, IQR) 44.5 (7–101.7) FC,μg/g (median,IQR) 135.88 (83.06–651.6) 1 Table 1. Demographic data of the patients PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 2 5-ASA, 5-aminosalicylic acid; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; 3 iFOBT, immune fecal occult blood test; WBC, white blood cell; FC, fecal calprotectin PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 1 Table 2. Comparison of patients with and without complete mucosal healing CMH (+) (n = 15) CMH (−) (n = 35) P-value Age (mean, SD) 48.13 (15.17) 45.71 (12.51) 0.560 WBC count (median, IQR) 5.7 (5.4 – 8.6) 6.7 (5.4 – 8.2) 0.8489 Hb (median, IQR) 16.7 (13.8–15.3) 17 (11.4–14.7) 0.0284 Platelet (median, IQR) 233 (192–265) 287 (220–350)) 0.0466 iFOBT, ng/mL (median, IQR) 7 (7–22.5) 121 (8.5–400) 0.0003 FC, μg/g (median, IQR) 59.95 (12.88–110.5) 555 (79.96–1687.34) 0.0028 Disease distribution (E1/E2/E3) 3/7/5 10/13/12 0.7643 Mayo score stool frequency (0/1/2/3) 4/9/2/0 7/13/10/5 0.0694 Mayo score rectal bleeding (0/1/2/3) 12/3/0/0 17/8/8/2 0.0162 Physician rating of disease activity (0/1/2/3) 9/6/0/0 7/20/7/1 0.0032 1 Table 2. Comparison of patients with and without complete mucosal healing IQR) Manuscript to be reviewed Manuscript to be reviewed Table 2(on next page) Table 2(on next page) Table 2(on next page) Comparison of patients with and without complete mucosal healing PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed hemoglobin; iFOBT, immune fecal occult blood test; WBC, white blood cell (0/1/2/3) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed – steroids (%) 20% 40% 0.1756 – oral 5-ASA (%) 93.3% 94.3% 0.8976 – rectal 5-ASA (%) 60% 51.4% 0.5812 – immune modulator (%) 20% 25.7% 0.6678 - biologic agent (%) 6.7% 0% 0.1266 2 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal healing;; Hb, 3 hemoglobin; iFOBT, immune fecal occult blood test; WBC, white blood cell (%) - biologic agent (%) 6.7% 0% 0.1266 2 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal healing;; Hb, 2 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal healing;; Hb, 3 hemoglobin; iFOBT, immune fecal occult blood test; WBC, white blood cell 3 hemoglobin; iFOBT, immune fecal occult blood test; WBC, white blood cell 3 hemoglobin; iFOBT, immune fecal occult blood test; WBC, white blood cell PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 4 4 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 1 Table 3. Comparison of patients with and without endoscopic mucosal healing EMH (+) (n = 31) EMH (−) (n = 19) P-value Age (mean, SD) 47.16 (15.26) 45.26 (9.38) 0.628 WBC count (median, IQR) 6.1 (5.1 –7.7) 7.4 (5.5–8.7) 0.2630 Hb (median, IQR) 14.2 (13.4–15.1) 12.7 (9.9–14.7) 0.0324 Platelet (median, IQR) 246 (207–281) 319 (249–403) 0.0115 iFOBT, ng/mL (median, IQR) 7 (7–29.5) 283 (129.3–400) <0.0001 FC, μg/g (median, IQR) 71.2 (39.1–222.8) 912 (448.8–1800) 0.0002 Disease distribution (E1/E2/E3) 8/14/9 5/6/8 0.5665 Mayo score stool frequency (0/1/2/3) 11/16/3/1 0/6/9/4 0.0001 Mayo score rectal bleeding (0/1/2/3) 23/6/2/0 6/5/6/2 0.0079 Physician rating of disease activity (0/1/2/3) 15/15/1/0 1/11/6/1 0.0015 Manuscript to be r 1 Table 3. Comparison of patients with and without endoscopic mucosal healing EMH (+) (n = 31) EMH (−) (n = 19) P-value Age (mean, SD) 47.16 (15.26) 45.26 (9.38) 0.628 WBC count (median, IQR) 6.1 (5.1 –7.7) 7.4 (5.5–8.7) 0.2630 Hb (median, IQR) 14.2 (13.4–15.1) 12.7 (9.9–14.7) 0.0324 Platelet (median, IQR) 246 (207–281) 319 (249–403) 0.0115 iFOBT, ng/mL (median, IQR) 7 (7–29.5) 283 (129.3–400) <0.0001 FC, μg/g (median, IQR) 71.2 (39.1–222.8) 912 (448.8–1800) 0.0002 Disease distribution (E1/E2/E3) 8/14/9 5/6/8 0.5665 Mayo score stool frequency (0/1/2/3) 11/16/3/1 0/6/9/4 0.0001 Mayo score rectal bleeding (0/1/2/3) 23/6/2/0 6/5/6/2 0.0079 Physician rating of disease activity (0/1/2/3) 15/15/1/0 1/11/6/1 0.0015 1 Table 3. Comparison of patients with and without endoscopic mucosal healing Manuscript to be reviewed Manuscript to be reviewed Table 3(on next page) Table 3(on next page) Comparison of patients with and without endoscopic mucosal healing PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed (0/1/2/3) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal Manuscript to be reviewed – steroids (%) 22.6% 52.6% 0.0311 – oral 5-ASA (%) 93.55 94.7% 0.8650 – rectal 5-ASA (%) 54.8% 52.6% 0.8804 – immune modulator (%) 19.4% 31.6% 0.3308 - biologic agent (%) 3.2% 0% 0.4337 2 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal healing;; EMH, 3 endoscopic mucosal healing;; Hb, hemoglobin; iFOBT, immune fecal occult blood test; WBC, 4 white blood cell (%) 2 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal healing;; EMH, 3 endoscopic mucosal healing;; Hb, hemoglobin; iFOBT, immune fecal occult blood test; WBC, 4 white blood cell 2 5-ASA, 5-aminosalicylic acid; BMI, body mass index; CMH, complete mucosal healing;; EMH, 3 endoscopic mucosal healing;; Hb, hemoglobin; iFOBT, immune fecal occult blood test; WBC, 4 white blood cell PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Table 4(on next page) Table 4(on next page) Table 4(on next page) Table 4(on next page) Correlations of endoscopy activity with iFOBT, FC, ESR, and CRP PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed Table 5(on next page) Manuscript to be reviewed 1 Table 4. Correlations of endoscopy activity with iFOBT, FC, ESR, and CRP Mayo Endoscopy Score 0.708 0.449 0.548 0.497 iFOBT 0.708, p<0.0001 0.481 0.568 0.316 ESR 0.449, p= 0.0012 0.481, p=0.0005 0.312 0.607 FC 0.548, p< 0.0001 0.568, p<0.0001 0.312, p=0.0291 0.317 CRP 0.497, p=0.0003 0.316, p=0.0271 0.607, p<0.0001 0.317, p=0.0266 Mayo Endoscopy Score iFOBT ESR FC CRP Spearman rank correlation coefficient. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; iFOBT, immune fecal occult blood test; FC, fecal calprotectin 1 Table 4. Correlations of endoscopy activity with iFOBT, FC, ESR, and CRP Table 4. Correlations of endoscopy activity with iFOBT, FC, ESR, and CRP Mayo Endoscopy Score 0.708 0.449 0.548 0.497 iFOBT 0.708, p<0.0001 0.481 0.568 0.316 ESR 0.449, p= 0.0012 0.481, p=0.0005 0.312 0.607 FC 0.548, p< 0.0001 0.568, p<0.0001 0.312, p=0.0291 0.317 CRP 0.497, p=0.0003 0.316, p=0.0271 0.607, p<0.0001 0.317, p=0.0266 Mayo Endoscopy Score iFOBT ESR FC CRP Spearman rank correlation coefficient. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; iFOBT, immune fecal occult blood test; FC, fecal calprotectin Table 4. Correlations of endoscopy activity with iFOBT, FC, ESR, and CRP Spearman rank correlation coefficient. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; iFOBT, immune fecal occult blood test; FC, fecal calprotectin Spearman rank correlation coefficient. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; iFOBT, immune fecal occult blood test; FC, fecal calprotectin 2 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Table 5(on next page) Table 5(on next page) Table 5(on next page) Literature comparing the use of FC and iFOBT for the prediction Mayo endoscopic mucosal healing among patients with UC PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 1 Table 5. Literature comparing the use of FC and iFOBT for the prediction Mayo endoscopic 2 mucosal healing among patients with UC Author, Region Case Number, Study Type, Description of Results Year Yen, Taiwan 50, R, S No difference in AUC of FC (cutoff level, 156μg/g) vs. iFOBT (cutoff level, 30 ng/mL) for MES = 0 vs. MES ≥ 1 (0.769 vs. 0.813, P = 0.5581) No difference in AUC of FC (cutoff level, 156μg/g) vs. iFOBT (cutoff level, 43 ng/mL) for MES≤1 vs. MES≥2 (0.812 vs. 0.906, P = 0.1207) Present Study Naganuma, Japan 429, P,M No difference in AUC of FC (cutoff level, 146.0 mg/kg) vs. iFOBT (cutoff level, 77.0 ng/mL) for MES = 0 vs. MES ≥ 1 (0.7774 vs. 0.8085, P = 0.394) No difference in AUC of FC (cutoff level, 277.0 mg/kg) vs. iFOBT (cutoff level, 201.0 ng/mL) for MES ≤ 1 vs. MES≥2 (0.8166 vs. 0.8353, P = 0.394) 2020[17] erature comparing the use of FC and iFOBT for the prediction Mayo endoscopic erature comparing the use of FC and iFOBT for the prediction Mayo endoscopic PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) P: prospective; S: single-center study; M: multicenter study Manuscript to be reviewed Kim, Korea 127, P, M AUC of FC (cutoff level, 70 μg/g) > iFOBT (cutoff level, 0 ng/mL) for MES = 0 vs. MES ≥ 1 (0.858 vs. 0.707, P = 0.0009) No difference in AUC of FC (cutoff level, 200 μg/g) vs. iFOBT (cutoff level, 60 ng/mL) for MES≤1 vs. MES≥2 (0.82 vs. 0.813, P = 0.089) 2020[16] Ryu, Korea 128, P, S AUC of FC (cutoff level, 170 μg/g) > iFOBT (cut-off value,100 ng/mL )for MES = 0 vs. MES ≥ 1 (0.847 vs. 0.757, P < 0.0001) AUC of FC (cutoff level, 170 μg/g)> IFOBT (cut-off value,100 ng/mL) for MES≤1 vs. MES≥2 (0.863 vs. 0.765, P < 0.0001) 2019[18] Kim, Korea 68,R,S No difference in AUC of FC vs. iFOBT for MES ≤ 1 vs. MES≥2 (0.727 vs. 0.717, P = 0.8643) 2018[20] Takashima, Japan 92,P,S No difference in AUC of FC (cut-off value, 200 μ g/g) vs. iFOBT (cut-off value, 75 ng/ml) for MES = 0 vs. MES ≥ 1 (0.83 vs. 0.82, P = 0.394) No difference in AUC of FC (cut-off value, 369 μ g/g) vs. iFOBT (cut-off value, 280 ng/ml) for MES≤1 vs. MES≥2 (0.80 vs. 0.79, P = 0.394) 2015[19] PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Manuscript to be reviewed 3 3 PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Figure 1 Figure 1 Correlation of complete Mayo score with iFOBT (A, Left) and FC (B, Right) (A, Left) Spearman's coeﬃcient of rank correlation =0.737, P < 0.0001 (B, Right) Correlation of FC with a complete Mayo Score (A, Left) Spearman's coeﬃcient of rank correlation =0.737, P < 0.0001 (B, Right) Correlation of FC with a complete Mayo Score PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Figure 2 Figure 2. ROC curve analysis for the use of iFOBT to assess complete mucosal healing (A, Left) and endoscopic mucosal healing (B, Right). (A, Left ) Using an iFOBT criterion of ≤30 ng/mL (95% CI ≤27 to ≤52), the sensitivity is 93.33%, and the speciﬁcity is 71.43% for predicting complete mucosal healing. Dot line: 95% Conﬁdence interval of ROC curve. (B, Right) Using an iFOBT criterion of ≤43 ng/mL (95% CI ≤28 to ≤88), the sensitivity is 80.65%, and the speciﬁcity is 100% for predicting endoscopic mucosal healing. Dot line: 95% Conﬁdence interval of ROC curve. PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Manuscript to be reviewed Figure 3 Figure 3. ROC curve analysis for the use of FC to assess complete mucosal healing (A, Left) and endoscopic mucosal healing (B, Right). (A, Left) Using an FC criterion of ≤156 μg/g (95% CI ≤100 to ≤1800), the sensitivity is 86.67%, and the speciﬁcity is 62.86% for predicting complete mucosal healing. Dot line: 95% Conﬁdence interval of ROC curve. (B, Right) Using an FC criterion of ≤156 μg/g (95% CI ≤63.7 to ≤638.9), the sensitivity is 74.19% and the speciﬁcity is 84.21% for predicting endoscopic mucosal healing.Dot line: 95% Conﬁdence interval of ROC curve. PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) Figure 4 Figure 4. ROC curve comparing iFOBT and FC for predicting complete mucosal healing (A, Left) and endoscopic mucosal healing (B, Right). A, Left) Pairwise comparison of ROC curves: iFOBT vs. FC, p = 0.5581 . (B, Right) Pairwise omparison of ROC curves: iFOBT vs. FC, p = 0.1207. PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020) (A, Left) Pairwise comparison of ROC curves: iFOBT vs. FC, p = 0.5581 . (B, Right) Pairwise comparison of ROC curves: iFOBT vs. FC, p = 0.1207. PeerJ reviewing PDF \| (2020:05:48530:1:1:NEW 18 Jun 2020)
https://openalex.org/W3195019176	https://ejournal.unibabwi.ac.id/index.php/BIOSENSE/article/download/1421/954	Indonesian	null	PENGARUH EKSTRAK ETANOL DAUN UBI JALAR UNGU (Ipomoea batatas L.) TERHADAP LEUKOSIT DAN LIMFOSIT MENCIT JANTAN BALB/C YANG DI INDUKSI VAKSIN HEPATITIS B	Jurnal Biosense	2,021	cc-by-sa	3,894	PENGARUH EKSTRAK ETANOL DAUN UBI JALAR UNGU (Ipomoea batatas L.) TERHADAP LEUKOSIT DAN LIMFOSIT MENCIT JANTAN BALB/C YANG DI INDUKSI VAKSIN HEPATITIS B Devi Kurnia1), Desi Sagita2), Siti Hamidatul Aliyah1), 1 Program Studi Farmasi, Sekolah Tinggi Ilmu Kesehatan Harapan Ibu, Jambi, Indonesi 2Program Studi Farmasi, Universitas Adiwangsa Jambi, Indonesia Email: sitihamidatula@stikes-hi.ac.id Devi Kurnia1), Desi Sagita2), Siti Hamidatul Aliyah1), 1 Program Studi Farmasi, Sekolah Tinggi Ilmu Kesehatan Harapan Ibu, Jambi, Indonesia 2Program Studi Farmasi, Universitas Adiwangsa Jambi, Indonesia Email: sitihamidatula@stikes-hi.ac.id Abstrak Latar Belakang: Daun ubi jalar ungu (Ipomoea batatas L.) yang telah diketahui mempunyai aktifitas imunomodulator, yang mengandung beberapa senyawa kimia, diantaranya vitamin, mineral, flavonoid (antosianin) khususnya zat besi ditemukan dalam proporsi yang lebih tinggi dalam daun. Oleh karena itu, perlu dilakukan penelitian terhadap aktifitas imunomodulator ekstrak daun ubi jalar ungu terhadap respon imun pada mencit Balb/c yang diinduksi dengan vaksin hepatitis B p p y g g p Metode: Penginduksian dilakukan di hari ke 0 dan hari ke 15. Mencit dengan bobot ± 20 g yang telah dibagi kedalam kelompok kontrol normal, kelompok I, II, III, IV dan V (masing-masing 5 ekor), berturut-turut diberi peroral dengan ekstrak daun ubi jalar ungu (Ipomoea batatas L.) (dosis 100 mg/kg BB, 200 mg/kg BB, 300 mg/kg BB, 400 mg/kg BB dan 500 mg/kg BB), kontrol normal (Na CMC 0,5 %). Dengan volume pemberian 1 % BB mencit, selama dilakukan penelitian (20 hari). Pengambilan darah dilakukan melalui vena ekor (vena lateralis), untuk menghitung jumlah sel leukosit (hari ke 21) dan pengambilan limfa untuk menghitung jumlah sel limfosit (hari ke 21). Hasil: Berdasarkan penelitian bahwa ekstrak daun ubi jalar ungu (ipomoea batatas L.), terutama dosis 500 mg/kg BB memiliki aktifitas munomodulator dapat meningkatkan jumlah sel limfosit. Kesimpulan: Ekstrak daun ubi jalar ungu memiliki aktivitas terhadap peningkatan jumlah sel limfosit terutama dosis 500 mg/kg BB yang di induksi oleh vaksin hepatitis B yang dapat menstimulasi respon imun. BIOSENSE Vol. 04 No. 1, Juni 2021 BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Abstract Background: Purple sweet potato leaves (Ipomoea batatas L.) which has been known as immunomodulatory activity, which contains chemical compounds, such as vitamins, minerals, flavonoids (anthocyanin), especially iron found in a higher proportion in the leaves. Therefore, it is necessary to study the immunomodulatory activity of the leaf extract of purple sweet potato (Ipomoea batatas L.) on the immune response in Balb/c mice who induced by hepatitis B vaccines. 59 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Method: Inducing performed at day 0 and day 15. Mice with weights ± 20 g, which has been divided into normal control group, I, II, III, IV and V (each 5 mice), respectively were given orally by the leaf extract of purple sweet potato (Ipomoea batatas L.) (doses 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg and 500 mg / kg), normal controls (CMC Na 0,5%). With the volume of giving 1% of body weight in mice, performed during the study (20th days). Blood sampling is done via the tail vein (laeralis vein), to calculate the number of leukocytes (21st days) and lymph decision to count lymphocytes (21st days). Results: The results that the leaf extract of purple sweet potato (ipomoea batatas L.), especially a dose of 500 mg / kg as immunomodulatory activity can increase the number of lymphocytes proliferation cell. Conclusion: Purple sweet potato leaf extract has activity against the increase in the number of lymphocytes cell especially on dose 500 mg/kg BW who induced by hepatitis B vaccine that can stimulated an immune response. Keywords: Purple sweet potato leaf, Imunomodulatory, Hepatitis B Vaccines, Leukocytes, Lymphocyte Keywords: Purple sweet potato leaf, Imunomodulatory, Hepatitis B Vaccines, Leukocytes, Lymphocyte 1. PENDAHULUAN Daun ubi jalar ungu (Ipomoea batatas L.) mengandung sejumlah besar protein, dan tinggi asam amino. Semua bagian dari ubi jalar ungu (Ipomoea batatas L.) yang kaya serat makanan. Selain itu daun ubi jalar ungu (Ipomoea batatas L.) juga mengandung vitamin seperti β-karoten, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin C, vitamin E dan vitamin K (Milind & Monika, 2015). Kandungan mineral khususnya zat besi ditemukan dalam proporsi yang lebih tinggi dalam daun. Selain vitamin, daun ubi jalar ungu (Ipomoea Batatas L.) juga mengandung zink, (Ishiguro et al., 2004; Yoshimoto et al., 2003), antosianin seperti karotenoid (β-karoten, dan leutin), flavonoid seperti antosianin (cyanidin) tilirosida, astragalin, rhamnositrin, rhamnetin, dan kamferol (Islam, 2008; Milind & Monika, 2015). Daun ubi jalar ungu memiliki beberapa efek terapi karena kandungan senyawa cyanidin (golongan antosianin) diantaranya sebagai imunomodulator, anti bakteri (Islam, 2006), aprodisiak, anstringen, pencahar, penambah energi, fungisida (Milind & Monika, 2015), anti mikroba (Islam, 2008), anti mutagen (Hanieh et al., 2012; Yoshimoto et al., 2003). Selain itu, mengkonsumsi daun ubi jalar ungu (Ipomoea 60 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 batatas L.) dapat memodulasi berbagai fungsi kekebalan tubuh termasuk meningkatkan proliferasi responsivitas PBMC sekresi sitokin IL-4 dan aktifitas litik sel NK (Chen et al., 2005). Berdasarkan uraian diatas bahwa perlunya dilakukan penelitian pada daun ubi jalar ungu (Ipomoea batatas L.) memiliki aktifitas imunostimulan apakah dapat meningkatkan respon imun yaitu terhadap sel leukosit dan meningkatnya jumlah sel limfosit dan bobot limfa pada mencit jantan Balb/c. 2.1 Bahan dan Alat Bahan yang digunakan dalam penelitian ini adalah sampel segar daun ubi jalar ungu (Ipomoea batatas L.) yang diperoleh dari desa Bukit Suban Sarolangun. Etanol 96 %, mencit jantan Balb/c, alkohol 70 %, Vaksin hepatitis B (Engerix B®), pewarna giemsa, Na EDTA, serbuk Mg, asam asetat anhidrat, asam sulfat pekat, kloroform, aquades, reagen Turk, FeCl3 1 % , dapar fosfat pH 7,4, dan Na CMC. Alat-alat yang digunakan adalah vakum rotari evaporator (Eyela®), erlemeyer, beaker gelas, tabung reaksi, mikroskop (Olympus®), botol vial, kapas, aluminium foil, serbet, timbangan analitik (Shimadzu®), kaca objek, batang pengaduk, pinset, haemocytometer, tisu, gunting bedah, spet oral 1 mL, syringe, pipet tetes, pipet pencampur, mikro pipet (Eppendorf®). 2.2 Pembuatan Sampel p Daun segar ubi jalar ungu 2 kg diekstraksi menggunakan etanol 96% kemudian diuapkan secara vakum sehingga diperoleh ekstrak kental sebanyak 135,7802 g. 2.4 Penghitungan Jumlah Sel Leukosit Cuplikan darah dihisap menggunakan pipet pencampur leukosit sampai angka 1,0, ujungnya dibersihkan dengan kertas isap atau tisu. Segeralah isaplah larutan Turk menggunakan pipet yang sama sampai angka 11. Lepaskan alat penghisap dari pipet pencampur. Ujung pipet dipegang dengan ibu jari dan ujung yang satunya dengan telunjuk. Kemudian kocoklah selama 2 menit. Buanglah 2-3 tetesan pertama, baru tetes-tetes berikutnya digunakan untuk penghitungan. Siapkan haemocytometernya. Ujung pipet ditempelkan pada tepi gelas penutup dengan sendirinya cairan dalam pipet akan masuk mengisi bilik hitung terlihat jelas. Hitunglah semua leukosit yang tedapat di dalam keempat bujur sangkar pojok. Jumlah sel leukosit dihitung berdasarkan rumus : 25 x L 2.3 Perlakuan Hewan Uji T t l 30 k it j Total 30 ekor mencit jantan galur Balb/c dengan berat 20 g dibagi menjadi 5 kelompok, masing-masing kelompok terdiri dari 5 ekor. Larutan uji diberikan selama 20 hari secara peroral. Ekstrak yang diberikan peroral dibedakan menjadi 5 dosis berbeda. Kelompok kontrol hewan uji hanya diberi suspensi Na CMC 0,5 %. 61 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Kelompok perlakuan adalah sebagai berikut: Kelompok 1: kelompok kontrol; kelompok 2: diberi ekstrak daun ubi jalar ungu dosis 100 mg/kg BB;kelompok 3: diberi ekstrak daun ubi jalar ungu dosis sebanyak 200 mg/kg BB; kelompok 4: diberi ekstrak daun ubi jalar ungu dosis 300 mg/kg BB; kelompok 5: diberi ekstrak daun ubi jalar ungu dosis 400 mg/kg BB; kelompok 6: diberi ekstrak daun ubi jalar ungu dosis 500 mg/kg BB. Pemberian vaksin dilakukan secara i.p sebanyak 2 kali pada hari ke-0 (setelah 7 hari aklimasi) dan hari ke-15. Volume vaksin yang diberikan berdasarkan konversi dosis manusia pada mencit yaitu 2,6 µL/20 g BB. Kemudian dilakukan pengenceran vaksin menggunakan menggunakan aquades hingga volume 26 µL. 2.6 Perhitungan Limfosit Limfa Timbang 50 mg limpa. Kemudian limpa dihancurkan menggunakan syringe. Kemudian disuspensikan ke dalam 3 mL larutan dapar fosfat pH 7,4. Suspensi limpa diambil sebanyak 20 μL dan diteteskan di atas kaca objek, biarkan mengering. Selanjutnya dilakukan fiksasi dengan metanol selama dua menit, kemudian dibilas dengan aquadest dan dikering anginkan. Untuk pewarnaan menggunakan pewarna Giemsa sebanyak 5 tetes giemsa diencerkan dengan aquadest 1:10. Biarkan selama 5 menit, kemudian bilas dengan aquadest, kemudian dikeringanginkan. Preparat yang telah diwarnai, dihitung jumlah limfositnya dibawah mikroskop dengan perbesaran 100x. Persentase kenaikan sel limfosit dihitung dengan rumus: 2.5 Penentuan Bobot Limfa Relatif Mencit yang diberi perlakuan pada masing-masing kelompok dianestesi Dietil eter, kemudian mencit dibedah dan diambil organ limfanya, timbang dan tentukan bobot relatif limfa berdasarkan rumus: Bobot Limfa x 100% Bobot Mencit Bobot Limfa x 100% Bobot Mencit 62 iskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 2.7 Analisis Data Jumlah sel leukosit, Jumlah sel limfosit dan berat limfa mencit yang diberi perlakuan terhadap berat limfa mencit kontrol. Masing-masing data tersebut dikalkulasikan kemudian dianalisa secara statistik dengan metode analisis one way (ANOVA). Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] 3. HASIL DAN PEMBAHASAN Hasil identifikasi tumbuhan dilakukan di Herbarium Universitas Andalas Jurusan Biologi FMIPA. Terhadap herba yang didapatkan dari Bukit Suban, menyatakan bahwa sampel yang digunakan adalah tumbuhan daun ubi jalar ungu (Ipomoea batatas L.) dengan family Convolvulaceae. Daun ubi jalar ungu (Ipomoea batatas L.) mengandung sejumlah besar vitamin, zat besi, zink, dan flavonoid (antosianin) ditemukan dalam proporsi yang lebih tinggi dalam daun. Hasil skrining fitokimia menunjukkan bahwa flavonoid, saponin, fenol dan tanin memberikan hasil yang positif (Tabel 1). Hal ini terjadi karena senyawa flavonoid bersifat polar sehingga senyawa polar dapat ditarik oleh pelarut polar. Sedangkan Alkaloid memberikan hasil 63 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 ISSN : 2622 - 6286 BIOSENSE Vol. 04 No. 1, Juni 2021 negatif, karena Alkaloid merupakan senyawa non polar sehingga tidak dapat ditarik oleh pelarut polar. Tabel 1. Skrining Fitokimia Ekstrak Daun Ubi Jalar Ungu Golongan Senyawa Hasil Flavonoid Positif Fenol Positif Tanin Positif Steroid Negatif Alkaloid Negatif Saponin Positif Tabel 1. Skrining Fitokimia Ekstrak Daun Ubi Jalar Ungu Pengujian imunomodulator ekstrak daun ubi jalar ungu (Ipomoea batatas L.) yaitu dengan melihat jumlah leukosit, berat limfa dan jumlah sel limfosit. Hewan uji yang digunakan adalah mencit jantan galur Balb/c dengan range berat badan 20- 34gram sebanyak 30 ekor. Mencit yang digunakan sebagai hewan uji adalah galur Balb/c dipilih karena galur ini sensitif dan umum digunakan untuk uji imunologi, karena mencit ini menunjukkan Th2 sebagai respon kekebalan tubuh. Mencit yang sudah diaklimasi selama 7 hari kemudian diberi vaksin hepatitis B. Tujuan pemberian vaksin untuk merangsang respon imun primer. Kemudian pada pemberian vaksin kedua akan memunculkan respon imun sekunder. Pada saat inilah mencit dikorbankan untuk diambil organ limfanya dan penghitungan jumlah sel limfosit. Penginduksian vaksin Hepatitis B dapat merangsang respon imun sehingga sel limfosit akan berproliferasi dan berdiferensiasi menjadi sel limfosit matang yang dapat mensekresikan antibodi dan dapat merangsang antibodi (immunoglobulin) terutama immunoglobulin M (IgM) dan immunoglobulin G (IgG). Dimana, fungsi utama IgM adalah menghasilkan respon primer terhadap antigen sedangkan IgG berfungsi mengopsonisai virus atau bakteri sehingga memudahkannya untuk difagositosis (Wahab & Julia, 2002). Bisa juga menggunakan vaksin yang lain karena sama-sama untuk merangsang respon imun sekunder, sehingga jika ada pajanan kedua antibodi akan memberikan respon yang lebih kuat dan memudahkannya untuk 64 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 BIOSENSE Vol. 04 No. 3. HASIL DAN PEMBAHASAN 1, Juni 2021 ISSN : 2622 - 6286 mengeliminasi pathogen (Khusnawati et al., 2016). Rute pemberian vaksin secara i.p (intra peritoneal) agar vaksin dapat diabsorpsi dengan baik. Pemberian vaksin dilakukan pada hari ke-0 (hari ke 8 setelah aklimasi) dan hari ke-15. Wahab & Julia (2002) melaporkan bahwa pada pemberian vaksin pertama, akan membangkitkan respon imun primer (pajanan pertama, IgM), kemudian pemberian vaksin kedua akan muncul respon imun sekunder (pajanan kedua) yang lebih kuat. Permenkes no 42 tahun (2013) menyatakan bahwa agar tidak menggunakan alat suntik dan vaksin berulang, untuk menghindarkan terjadinya penyebaran penyakit. Hasil perhitungan sel leukosit pada darah mencit putih jantan setelah diinduksi vaksin hepatitis B dan diberi ekstrak etanol daun ubi jalar ungu (Ipomoea batatas L.) terdiri atas kelompok kontrol Na CMC rata-rata 6080 sel/mm3, kelompok 1 dosis 100 mg.kg BB rata-rata 5850 sel/mm3, kelompok 2 dosis 200 mg/kg BB rata-rata 7250 sel/mm3, kelompok 3 dosis 300 mg/kg BB rata-rata 9030 sel/mm3, kelompok 4 dosis 400 mg/kg BB rata-rata 10360 sel/mm3, dan kelompok 5 dosis 500 mg/kg BB rata- rata 12135 sel/mm3 (Gambar 1). Meningkatnya sel darah putih terutama pada dosis 500 mg/kg BB menunjukkan bahwa perlakuan pemberian ekstrak daun ubi jalar ungu (Ipomoea batatas L.) pada berbagai dosis berbeda nyata terhadap jumlah sel darah putih (leukosit) kontrol Na CMC. Dalam penelitian ini sesuai dengan Aldi, dkk., (2016). Leukosit meningkat setelah diberi ekstrak etanol ubi jalar ungu (Ipomoea batatas L.) yang mengandung senyawa antosianin, saponin dan tanin. Ashland (2013) menyatakan bahwa diietil eter merupakan anestesi yang umum digunakan untuk operasi bedah, sifat mengiritasinya rendah dan bersifat revelsibel serta tidak memiliki efek mutagenik. Sehingga cukup aman bagi peneliti. 65 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Gambar 1. Grafik jumlah sel leukosit mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu (Ipomoea batatas L.) Gambar 1. Grafik jumlah sel leukosit mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu (Ipomoea batatas L.) Hasil bobot relatif limfa mencit putih jantan setelah diinduksi vaksin hepatitis B dan diberi ekstrak etanol daun ubi jalar ungu (Ipomoea batatas L.) kelompok kontrol Na CMC berat relatif limfa rata-rata 0,4124 %. Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] 3. HASIL DAN PEMBAHASAN kelompok 1 dosis 100 mg/kg BB rata- rata berat relatif limfa adalah 0,4072 %, kelompok 2 dosis 200 mg/kg BB rata-rata 0,43 %, kelompok 3 dosis 300 mg/kg BB rata-rata 0,5798 %, kelompok 4 dosis 400 mg/kg BB rata-rata 0,6324 %, kelompok 5 dosis 500 mg/kg BB memiliki persentase bobot relatif limfa rata-rata 0,9438 % (Gambar 2). Gambar 2. Grafik Berat relatif limfa mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu Gambar 2. Grafik Berat relatif limfa mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] 66 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Jumlah sel limfosit limfa mencit putih jantan jantan setelah diinduksi vaksin hepatitis B dan diberi ekstrak etanol daun ubi jalar ungu (Ipomoea batatas L.). Kelompok kontrol Na CMC jumlah sel limfosit rata-rata 80200 sel/µL, Kelompok 1 dosis 100 mg/kg BB rata-rata 82000 sel/µL, Kelompok 2 dosis 200 mg/kg BB rata- rata 85500 sel/µL, kelompok 3 dosis 300 mg/kg BB rata-rata 89600 sel/µL, kelompok 4 dosis 400 mg/kg BB rata-rata 94800 sel/ µL, dan kelompok 5 dosis 500 mg/kg BB memiliki jumlah sel limfosit rata-rata 101800 sel/µL (Gambar 3). Gambar 3. Grafik Sel Limfosit dan Persentase Kenaikan Sel LImfosit mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu (Ipomoea batatas L.) Gambar 3. Grafik Sel Limfosit dan Persentase Kenaikan Sel LImfosit mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu (Ipomoea batatas L.) Dari data dapat dilihat bahwa kenaikan bobot relatif limfa juga diikuti dengan kenaikan sel limfosit pada limfa, Wahab dan Julia (2002) menyatakan bahwa sel limfosit ini sangat berperan dalam proses respon imun spesifik, dan membunuh sel yang terinfeksi virus. Aldi & Suhatri (2015) melaporkan bahwa peningkatan jumlah sel limfosit dan bobot relatif limfa mengindikasikan adanya peningkatan respon imun spesifik. Dimana sel limfosit terdiri dari sel limfosit B dan sel limfosit T. Sel limfosit B ini akan mengalami proliferasi dan differensiasi. Sehingga terjadi pembesaran pada limfa, membentuk sel plasma dan sel memori. Sel plasma inilah yang membentuk antibodi yang terbentuk setelah kontak dengan antigen. 67 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] 3. HASIL DAN PEMBAHASAN 04 No. 1, Juni 2021 BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Jumlah sel limfosit limfa mencit putih jantan jantan setelah diinduksi vaksin hepatitis B dan diberi ekstrak etanol daun ubi jalar ungu (Ipomoea batatas L.). Kelompok 100 mg/kg BB persentase kenaikan sel limfosit rata-rata 102,24 %, Kelompok 200 mg/kg BB rata-rata 106,6 %, Kelompok 300 mg/kg BB rata-rata 111,716 %, Kelompok 400 mg/kg rata-rata 118,262 %, Kelompok 500 mg/kg BB rata-rata 126,926 % (Gambar 4). Gambar 4. Grafik Persentase kenaikan sel limfosit mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu (Ipomoea batatas L.) Gambar 4. Grafik Persentase kenaikan sel limfosit mencit putih jantan Balb/c setelah diinduksi vaksin hepatitis B dan diberi ekstrak daun ubi jalar ungu (Ipomoea batatas L.) Dari hasil uji statistik one way ANOVA menunjukkan bahwa pada analisa sel limfosit limfa terhadap kontrol Na CMC, nilai pvalue < 0,05 menunjukkan bahwa terdapat perbedaan yang bermakna antara kontrol Na CMC dengan kelompok perlakuan. Sedangkan pada sel leukosit dan berat limfa memiliki nilai p value > 0,05 tidak ada perbedaan antara kelompok. Sehingga dapat dikatakan secara statistik ekstrak daun ubi jalar ungu (Ipomoea batatas L.) hanya dapat memberikan pengaruh terhadap sel limfosit hewan uji. Penelitian ini merujuk pada beberapa penelitian, dimana beberapa metabolit sekunder yang mendukung sebagai imunostimulan, yaitu senyawa antosianin, saponin dari golongan flavonoid Mekanisme flavonoid sebagai imunostimulan adalah dengan cara menstimulasi limfosit agar memproduksi interleukin (IL)-2 dan interferon (INF)- 68 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 γ. Flavonoid dalam ekstrak daun ubi jalar ungu (Ipomoea batatas L.) juga dapat memodulasi berbagai fungsi kekebalan tubuh termasuk meningkatkan proliferasi responsivitas PBMC sekresi sitokin IL-4 dan aktifitas litik sel NK (Aldi et al., 2016; Aldi & Suhatri, 2015; Chen et al., 2005; Hanieh et al., 2012; Plohmann et al., 1997). γ. Flavonoid dalam ekstrak daun ubi jalar ungu (Ipomoea batatas L.) juga dapat memodulasi berbagai fungsi kekebalan tubuh termasuk meningkatkan proliferasi responsivitas PBMC sekresi sitokin IL-4 dan aktifitas litik sel NK (Aldi et al., 2016; Aldi & Suhatri, 2015; Chen et al., 2005; Hanieh et al., 2012; Plohmann et al., 1997). Peneliti sebelumnya telah menemukan senyawa saponin yang terkandung dalam ekstrak daun ubi jalar ungu (Ipomoea batatas L.) juga memiliki aktifitas imunostimulan. 3. HASIL DAN PEMBAHASAN Mekanismenya adalah menginduksi produksi sitokin seperti interleukin dan interferon yang dapat memediasi efek imunostimulan. Saponin tidak hanya memiliki efek stimulasi pada komponen imunitas spesifik, tetapi juga mempengaruhi respon imun non-spesifik seperti pada inflamasi dan proliferasi monosit (Cabral de Oliveira et al., 2001; Delmas et al., 2000; Haridas et al., 2004; Yui et al., 2001). 4.1 Kesimpulan Dari penelitian ini dapat disimpulkan bahwa pemberian ekstrak etanol daun ubi jalar ungu (Ipomoea batatas L.) dapat meningkatkan jumlah sel limfosit pada mencit jantan Balb/c terutama pada dosis 500 mg/kg BB. 4.2 Saran Hasil penelitian akan lebih maksimal jika dilakukan uji hematologi lengkap. Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] 5. REFERENSI Aldi, Y., D, D., Florina, T., & Friardi, D. (2016). Activity and Capacity Test of Macrophage Peritoneal Cell and Number Leukocyte of Ethanol Extract Purple Sweet Potato Peel (Ipomoea batatas (L.) Lam. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 7(178), 178–186. Aldi, Y., & Suhatri, S. (2015). Aktivitas Ekstrak Etanol Biji Jintan Hitam (Nigella sativa Linn.) terhadap Titer Antibodi dan Jumlah Sel Leukosit pada Mencit Putih Jantan. Scientia : Jurnal Farmasi Dan Kesehatan, 1(1). https://doi.org/10.36434/scientia.v1i1.14 bral de Oliveira, A. C., Perez, A. C., Merino, G., Prieto, J. G., & Alvarez, A. I 69 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 (2001). Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 130(3). https://doi.org/10.1016/S1532- 0456(01)00262-9 (2001). Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 130(3). https://doi.org/10.1016/S1532- 0456(01)00262-9 Chen, C. M., Li, S. C., Lin, Y. L., Hsu, C. Y., Shieh, M. J., & Liu, J. F. (2005). Consumption of purple sweet potato leaves modulates human immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production. World Journal of Gastroenterology, 11(37). https://doi.org/10.3748/wjg.v11.i37.5777 Delmas, F., Di Giorgio, C., Elias, R., Gasquet, M., Azas, N., Mshvildadze, V., Dekanosidze, G., Kemertelidze, E., & Timon-David, P. (2000). Antileishmanial activity of three saponins isolated from ivy, α- hederin, β-hederin and hederacolchiside A1, as compared to their action on mammalian cells cultured in vitro. Planta Medica, 66(4). https://doi.org/10.1055/s-2000-8541 Hanieh, H., Narabara, K., Tanaka, Y., Gu, Z., Abe, A., & Kondo, Y. (2012). Immunomodulatory effects of Alliums and Ipomoea batata extracts on lymphocytes and macrophages functions in White Leghorn chickens: In vitro study. Animal Science Journal, 83(1). https://doi.org/10.1111/j.1740- 0929.2011.00918.x Haridas, V., Hanausek, M., Nishimura, G., Soehnge, H., Gaikwad, A., Narog, M., Spears, E., Zoltaszek, R., Walaszek, Z., & Gutterman, J. U. (2004). Triterpenoid electrophiles (avicins) activate the innate stress response by redox regulation of a gene battery. Journal of Clinical Investigation, 113(1). https://doi.org/10.1172/JCI200418699 Ishiguro, K., Toyama, J., Islam, M. S., Yoshimoto, M., Kumagai, T., Kai, Y., Nakazawa, Y., & Yamakawa, O. (2004). Suioh, a new sweetpotato cultivar for utilization in vegetable greens. Acta Horticulturae, 637. https://doi.org/10.17660/ActaHortic.2004.637.42 Islam, S. (2006). Sweetpotato (Ipomoea batatas L.) leaf: Its potential effect on human health and nutrition. 5. REFERENSI In Journal of Food Science (Vol. 71, Issue 2). https://doi.org/10.1111/j.1365-2621.2006.tb08912.x Islam, S. (2008). Antimicrobial activities of Ipomoea batatas (L.) leaf. Journal of Food, Agriculture and Environment, 6(1). Khusnawati, N. N., Pramono, S., & Sasmito, E. (2016). Effect of 50% ethanolic extract of pegagan herb (centella asiatica (l.) Urban) on cell proliferation of lymphocytes in balb/c male mice induced by hepatitis b vaccine. Effect of 50% ethanolic extract of pegagan herb (centella asiatica (l.) Urban) on cell proliferation of lymphocytes in balb/c male mice induced by hepatitis b vaccine, 20(3), 164–169. https://doi.org/10.22146/tradmedj.8861 70 Tuliskan judul Artikel lengkap [Times New Roman, 9, Normal] BIOSENSE Vol. 04 No. 1, Juni 2021 ISSN : 2622 - 6286 Milind, P., & Monika. (2015). Sweet Potato As A Super-Food. International Journal of Research in Ayurveda and Pharmacy, 6(4). https://doi.org/10.7897/2277- 4343.064104 Plohmann, B., Bader, G., Hiller, K., & Franz, G. (1997). Immunomodulatory and antitumor effects of triterpenoid saponins. Pharmazie, 52(12). Yoshimoto, M., Okuno, S., Islam, M. S., Kurata, R. A., & Yamakawa, O. (2003). 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https://openalex.org/W3157266870	https://www.researchsquare.com/article/rs-434947/latest.pdf	English	null	Lag Between COVID-19 Cases And Subsequent Deaths	Research Square (Research Square)	2,021	cc-by	2,066	Lag Between COVID-19 Cases And Subsequent Deaths Sara Donovan University of Nebraska Medical Center https://orcid.org/0000-0002-7358-716X David Brett-Major University of Nebraska Medical Center James V. Lawler (  james.lawler@unmc.edu ) University of Nebraska Medical Center https://orcid.org/0000-0003-3736-8485 Research article Keywords: COVID-19, epidemiology, health policy, community health, communicable disease Posted Date: May 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-434947/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License DOI: https://doi.org/10.21203/rs.3.rs-434947/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/7 Background Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), originated in Wuhan, China in late 2019, and was declared a pandemic by the World Health Organization on March 11, 2020 [1]. As of April 5, 2021, over 131 million cases had been confirmed in 223 countries, areas, or territories, and over 2.8 million virus-associated deaths had been reported [2]. In the face of persistent consequences from the health emergency and absence of widespread vaccination, public health authorities have employed a variety of non-pharmaceutical interventions (NPIs) to control community spread of COVID-19 [3]. While effective when fully employed, these NPIs can have secondary economic and social consequences, often receiving push-back from political leaders and the public. In gauging the appropriate level of NPI response, many have focused on health system data such as active hospitalizations and deaths, without appreciating their temporal relationship [4, 5]. By reacting to deaths or hospitalizations in isolation, or expecting both to be high at the same time, actions of health systems and jurisdictions may come weeks behind changes in true disease burden that presage resultant morbidity and mortality and societal consequence. To investigate the nature of this potential relationship, we investigated the time lag between changes in COVID-19 epidemic case curves and deaths in 16 U.S. states and three countries. Abstract Background: Observed COVID-19 cases and deaths are frequently used as independent factors to justify the use or de-escalation of non-pharmaceutical interventions. To investigate and describe the temporal relationship between changes in epidemic curves for COVID-19 cases and deaths, a longitudinal analysis of these metrics was performed for 16 states in the United States and three countries. Results: The analysis performed demonstrates a considerable, consistent lag between a surge in COVID- 19 cases and the subsequent rise in attributable deaths, with approximately a month-long lag observed. Conclusions: The time lag identified must be incorporated in health emergency decision making in order to avoid the pre-mature de-escalation of disease mitigation measures. Methods Data from the COVID Tracking Project [6] and the European Centre for Disease Prevention and Control [7] were used to identify surges in COVID-19 cases and subsequent virus-associated deaths. To minimize the impacts of variability in reporting, a surge was defined as a two-fold or greater increase in the seven-day average case or death count in a two-month period. The nadir of the seven-day average preceding the surge in cases or deaths was determined, and the inflection point was defined as the date that marked a 10 percent increase in the seven-day average of cases or deaths above the nadir. If the nadir was zero, the inflection point was defined as the next date when a seven-day average was greater than zero. When Page 2/7 Page 2/7 surges in both cases and deaths were observed, the lag between them was calculated as the number of days between each of their inflection points (Fig. 1). surges in both cases and deaths were observed, the lag between them was calculated as the number of days between each of their inflection points (Fig. 1). After surveying U.S. rates across the 50 states, we identified those that experienced a discrete surge in cases between June 1, 2020 and August 1, 2020. Sixteen states in the United States (Georgia, Idaho, Iowa, Kansas, Minnesota, Mississippi, Missouri, Montana, Nevada, New Mexico, Ohio, Oklahoma, South Dakota, Virginia, West Virginia, and Wisconsin) were analyzed, along with three countries (France, Netherlands, and the United Kingdom) incorporated for comparison. Surges occurring prior to June 1, 2020 were not included, as laboratory supply shortages in the winter and spring of 2020 hampered COVID-19 diagnostic testing [8]. Results The lag between the surge in COVID-19 cases and the subsequent surge in deaths ranged from 14 to 55 days, with the shortest lag observed in New Mexico and the longest lag occurring in the United Kingdom (Table 1 and Fig. 2). The median time lags were 27 and 50 days, and the means were 29 and 43 days, with standard deviations of 10 and 14 days for states and countries, respectively. Analyzed together, the median time lag was 27 days, and the mean was 31 days, with a standard deviation of 12 days. Page 3/7 Table 1 Table 1 Table 1 Inflection Dates for Cases and Deaths and Time Lag Location Inflection Date for Cases Inflection Date for Deaths Lag (Days) New Mexico June 20, 2020 July 4, 2020 14 Iowa June 21, 2020 July 8, 2020 17 Nevada June 4, 2020 June 26, 2020 22 Idaho June 16, 2020 July 9, 2020 23 Oklahoma June 2, 2020 June 25, 2020 23 Virginia July 4, 2020 July 29, 2020 25 Missouri June 15, 2020 July 11, 2020 26 Montana June 12, 2020 July 9, 2020 27 Wisconsin June 21, 2020 July 18, 2020 27 Kansas June 15, 2020 July 13, 2020 28 Mississippi June 7, 2020 July 7, 2020 30 Ohio June 18, 2020 July 18, 2020 30 Georgia June 4, 2020 July 10, 2020 36 Minnesota June 18, 2020 July 29, 2020 41 West Virginia June 14, 2020 July 27, 2020 43 South Dakota July 18, 2020 September 10, 2020 54 Netherlands July 13, 2020 August 5, 2020 23 France July 21, 2020 September 9, 2020 50 United Kingdom July 22, 2020 September 15, 2020 55 Inflection Dates for Cases and Deaths and Time Lag Consent for Publication: Not applicable Availability of data and materials: The datasets analyzed during the current study are available in the COVID Tracking Project repository, https://covidtracking.com/data/download and the European Centre for Disease Prevention and Control repository, https://www.ecdc.europa.eu/en/covid-19/data Competing Interests: The authors declare that they have no competing interests Authors’ Contributions: JVL conceived the study and designed the analyses. DMB participated in study design. SD performed the analyses. All authors contributed to the writing of the manuscript. Acknowledgments: Not applicable Acknowledgments: Not applicable Acknowledgments: Not applicable Conclusions Political jurisdictions must rely on NPIs to mitigate the impact of COVID-19 even as vaccine uptake increases; for many regions of the world, this may encompass the next year or more, as well as the prospect of subsequent waves of disease. If political and health system leaders better understand the time lag between changes in community incidence and subsequent documented deaths, they may be better prepared to make more timely decisions, responding more effectively to community epidemics. Further research should explore the trend identified in this analysis, as well as temporal relationships among other commonly used metrics such as hospitalizations. Discussion Our analysis demonstrates a considerable, consistent lag between a surge in COVID-19 cases and the subsequent rise in attributable deaths. Based upon this review, deaths as a metric of community epidemic status reflect transmission events occurring in a community more than a month prior, on average. While hospitalizations ostensibly would have a shorter lag-time and may represent a more responsive metric, accurate daily curves on real-time hospitalizations are not as readily available, can be impacted by level of hospital stress, and may not capture particularly vulnerable populations counted among deaths such as long-term care facilities. We anticipate that hospitalization data would lag case incidence data, and additional research into this is warranted. Page 4/7 Page 4/7 Limitations of our analysis may include an underestimation of COVID-19 cases and deaths—both those directly and indirectly related. There is variability between jurisdictions in laboratory test availability, performance, and reporting, as well as how such results are captured. Although testing was more widely accessible in the summer of 2020 compared to the winter and spring, test availability in the United States was still unable to meet the overwhelming demand [9]. As widespread community transmission occurred, scarcity of supplies amalgamated with backlogs at laboratories responsible for processing tests [9] causing reporting shortfalls and delays that contribute to an underestimation of disease incidence. Death certification errors and delays in ascertainment as COVID-19 related are known to impact mortality statistics [10, 11]. The COVID-19 pandemic has highlighted limitations within the reporting process, as deaths attributable to COVID-19 have been assigned to other causes [12]. Declarations Ethics Approval and Consent to Participate: Not applicable Consent for Publication: Not applicable References Page 5/7 Page 5/7 Page 5/7 1. World Health Organization (2020) Rolling updates on coronavirus disease (COVID-19). In: World Health Organization. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as- they-happen. Accessed 30 Oct 2020 2. World Health Organization (2020) Coronavirus disease (COVID-19) pandemic. In: World Health Organization. https://www.who.int/emergencies/diseases/novel-coronavirus-2019. Accessed 5 Apr 2021 2. World Health Organization (2020) Coronavirus disease (COVID-19) pandemic. In: World Health Organization. https://www.who.int/emergencies/diseases/novel-coronavirus-2019. Accessed 5 Apr 2021 3. Brauner JM, Mindermann S, Sharma M, et al (2020) Inferring the effectiveness of government interventions against COVID-19. Science. https://doi.org/10.1126/science.abd9338 3. Brauner JM, Mindermann S, Sharma M, et al (2020) Inferring the effectiveness of government interventions against COVID-19. Science. https://doi.org/10.1126/science.abd9338 4. Gov. Ricketts announces phased public health restrictions tied to coronavirus hospitalization rate [media release]. In: Nebraska Department of Health and Human Services. http://dhhs.ne.gov/Pages/Gov-Ricketts-Announces-Phased-Public-Health-Restrictions-Tied-to- Coronavirus-Hospitalization-Rate.aspx. Accessed 19 Jan 2021 4. Gov. Ricketts announces phased public health restrictions tied to coronavirus hospitalization rate [media release]. In: Nebraska Department of Health and Human Services. http://dhhs.ne.gov/Pages/Gov-Ricketts-Announces-Phased-Public-Health-Restrictions-Tied-to- Coronavirus-Hospitalization-Rate.aspx. Accessed 19 Jan 2021 4. Gov. Ricketts announces phased public health restrictions tied to coronavirus hospitalization rate [media release]. In: Nebraska Department of Health and Human Services. 5. Testa CC, Krieger N, Chen JT, Hanage W (2020) Visualizing the lagged connection between COVID-19 cases and deaths in the United States: An animation using per capita state-level data (January 22, 2020 – July 8, 2020). Harvard Center for Population and Development Studies 5. Testa CC, Krieger N, Chen JT, Hanage W (2020) Visualizing the lagged connection between COVID-19 cases and deaths in the United States: An animation using per capita state-level data (January 22, 2020 – July 8, 2020). Harvard Center for Population and Development Studies 6. The COVID Tracking Project at The Atlantic (2020) Our data: Data download. In: The COVID Tracking Project. https://covidtracking.com/data/download. Accessed 15 Nov 2020 6. The COVID Tracking Project at The Atlantic (2020) Our data: Data download. In: The COVID Tracking Project. https://covidtracking.com/data/download. Accessed 15 Nov 2020 7. European Centre for Disease Prevention and Control (2020) COVID-19 data. In: European Centre for Disease Prevention and Control. https://www.ecdc.europa.eu/en/covid-19/data. Accessed 15 Nov 2020 7. European Centre for Disease Prevention and Control (2020) COVID-19 data. In: European Centre for Disease Prevention and Control. https://www.ecdc.europa.eu/en/covid-19/data. Accessed 15 Nov 2020 8. Schneider EC (2020) Failing the test: The tragic data gap undermining the U.S. pandemic response. NEJM 383:299–302 8. Schneider EC (2020) Failing the test: The tragic data gap undermining the U.S. pandemic response. References NEJM 383:299–302 9. Mervosh S, Fernandez M (2020) Months into virus crisis, U.S. cities still lack testing capacity. The New York Times 10. McGivern L, Shulman L, Carney JK, Shapiro S, Bundock E (2017) Death certification errors and the effect on mortality statistics. Public Health Rep 132:669–675 10. McGivern L, Shulman L, Carney JK, Shapiro S, Bundock E (2017) Death certification errors and the effect on mortality statistics. Public Health Rep 132:669–675 11. Pritt BS, Hardin NJ, Richmond JA, Shapiro SL (2005) Death certification errors at an academic institution. Arch Pathol Lab Med 129:1476–1479 11. Pritt BS, Hardin NJ, Richmond JA, Shapiro SL (2005) Death certification errors at an academic institution. Arch Pathol Lab Med 129:1476–1479 12. Gill JR, DeJoseph ME (2020) The importance of proper death certification during the COVID-19 pandemic. JAMA 324:27–28 12. Gill JR, DeJoseph ME (2020) The importance of proper death certification during the COVID-19 pandemic. JAMA 324:27–28 Figures Page 6/7 Figure 1 The Determination of Inflection Points and Calculation of Time Lag The Determination of Inflection Points and Calculation of Time Lag The Determination of Inflection Points and Calculation of Time Lag Figure 2 Time Lag for the 19 Locations Analyzed Figure 2 Time Lag for the 19 Locations Analyzed Time Lag for the 19 Locations Analyzed Time Lag for the 19 Locations Analyzed Page 7/7
https://openalex.org/W4385397817	https://jurnal.medanresourcecenter.org/index.php/PED/article/download/1049/847	Indonesian	null	Analisis Faktor Penyebab Minat Kuliah pada Perguruan Tinggi Islam	Pedagogika	2,023	cc-by	3,009	ABSTRACT In the pursuit of educational development, the youth of Pakamban Laok Village in Pragaan Subdistrict show a strong preference for pursuing higher education at Islamic institutions affiliated with Islamic boarding schools. This growing interest among the youth of Pakamban Laok serves as a foundation for self-development in the realm of education. This study aims to identify and gather data on the internal and external factors influencing the inclination to study at the Islamic College of Pakamban Laok Village in Pragaan District, Sumenep Regency. Employing a qualitative descriptive research method, data and information were collected and subsequently analyzed to address the objectives of this study. The findings reveal that internal factors, such as motivation, aspirations, and hopes, significantly influence the interest in learning at the Youth Islamic Boarding School in Pakamban Laok Village, Pragaan Sumenep District. External factors, including family, friends, school, and religious practices, also play a significant role in shaping the interest in learning among adolescents attending Madrasah Ibtidaiyah Pakamban Laok Village, Pragaan Sumenep District. Analisis Faktor Penyebab Minat Kuliah pada Perguruan Tinggi Islam Latifah, Institut Dirosat Islamiyah Al-Amien, Indonesia Ahmadi, Institut Dirosat Islamiyah Al-Amien, Indonesia ARTICLE HISTORY Received Revised Accepted Published 23/06/2023 30/06/2023 10/07/2023 13/07/2023 KEYWORDS Youth education; Islamic higher education; factors influencing interest. CORRESPONDENCE AUTHOR ifalatifah4300@gmail com PEDAGOGIKA: JURNAL ILMU-ILMU KEPENDIDIKAN https://doi.org/10.57251/ped.v3i1.1049 Vol. 3, No. 1, 2023 \| 145-149 g g p y This is an Open Access article distributed under the terms of the Creative Common Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited This is an Open Access article distributed under the terms of the Creative Common Attribution License (https://creativecommons.org/licenses/by/4.0 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. g g p y This is an Open Access article distributed under the terms of the Creative Common Attribution License (https://creativecommons.or which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PEDAGOGIKA: JURNAL ILMU-ILMU KEPENDIDIKAN https://doi.org/10.57251/ped.v3i1.1049 © 2023 The Author(s). Pedagogika: Jurnal Ilmu-Ilmu Kependidikan. ISSN: 2808-9502. Published by Medan Resource Center This is an Open Access article distributed under the terms of the Creative Common Attribution License (https://creativecommons.org/licenses/ © 2023 The Author(s). Pedagogika: Jurnal Ilmu-Ilmu Kependidikan. ISSN: 2808-9502. Published by Medan Resource Center This is an Open Access article distributed under the terms of the Creative Common Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ( ) g g p y This is an Open Access article distributed under the terms of the Creative Common Attribution License (https://creativecommons.org/licenses/by/4.0/), © 2023 The Author(s). Pedagogika: Jurnal Ilmu-Ilmu Kependidikan. ISSN: 2808-9502. Published by Medan Resource Center METODE Penelitian ini menggunakan pendekatan kualitatif deskriptif. Menurut penelitian kualitatif merupakan penelitian yang tidak menggunakan analisis kuantifikasi (Moleong, 2012). Penelitian kualitatif bermaksud memahami fenomena yang dialami subjek penelitian, misalnya perilaku, persepsi, motivasi, tindakan dan sebagainya. Penelitian ini dilakukan dengan cara deskripsi dalam bentuk kata dan bahasa dengan menggunakan berbagai metode alamiah. Adapun jenis penelitian ini adalah studi kasus, yang mana ditunjukkan untuk memahami permasalahan- permasalahan dari sudut pandang atau perspektif partisipan. Jadi dalam penelitian ini mengungkap tentang apa faktor penyebab minat kuliah pada Perguruan Tinggi Islam. Penelitian ini menggunakan pendekatan kualitatif deskriptif. Menurut penelitian kualitatif merupakan penelitian yang tidak menggunakan analisis kuantifikasi (Moleong, 2012). Penelitian kualitatif bermaksud memahami fenomena yang dialami subjek penelitian, misalnya perilaku, persepsi, motivasi, tindakan dan sebagainya. Penelitian ini dilakukan dengan cara deskripsi dalam bentuk kata dan bahasa dengan menggunakan berbagai metode alamiah. Adapun jenis penelitian ini adalah studi kasus, yang mana ditunjukkan untuk memahami permasalahan- permasalahan dari sudut pandang atau perspektif partisipan. Jadi dalam penelitian ini mengungkap tentang apa faktor penyebab minat kuliah pada Perguruan Tinggi Islam. Sumber data ditentukan peneliti dengan menggunakan Purposive Sampling, yaitu memilih informan yang telah mengetahui dengan pasti untuk dapat menjawab pertanyaan-pertanyaan dari peneliti. Sumber data dalam penelitian ini adalah 5 keluarga, yang terdiri dari anak (pemuda desa Pakamban Laok yang melanjutkan kuliah ke perguruan tinggi Islam), ayah, ibu dan kakaknya. Prosedur pengumpulan data dalam penelitian ini menggunakan teknik wawancara, observasi dan dokumentasi. Dalam melakukan wawancara, peneliti menggunakan pedoman wawancara terstruktur, agar pertanyaan-pertanyaan yang diajukan kepada subjek penelitian dapat terarah dan fokus pada data atau informasi yang diperlukan oleh peneliti terkait faktor penyebab minat kuliah pada Perguruan Tinggi Islam. Observasi yang digunakan dalam penelitian ini adalah jenis observasi non partisipan, di mana peneliti hanya melakukan pengamatan biasa. Peneliti menggunakan dokumentasi sebagai salah satu data atau bukti untuk mengetahui faktor dari penyebab minat kuliah pada perguruan tinggi Islam di Desa Pakamban Laok. Analisis data dalam penelitian ini adalah mengacu pada konsep Miles and Huberman yaitu: 1) reduksi data, pada penelitian ini, reduksi data dimulai ketika peneliti melakukan penelitian di lokasi penelitian sewaktu proses pengumpulan data mengenai faktor penyebab minat kuliah pada Perguruan Tinggi Islam pemuda desa Pakamban Laok Kecamatan Pragaan Sumenep. Kemudian peneliti membuat catatan-catatan dari hasil penelitian. Hasil penelitian ini berupa hasil wawancara dan hasil observasi. kemudian, peneliti membuat ringkasan sesuai dengan hasil penelitian; 2) penyajian data, pada penyajian data ini, peneliti menyusun hasil penelitian yang menggambarkan tentang . PENDAHULUAN Setiap orang di Indonesia berhak mendapatkan pendidikan dan diharapkan dapat terus berkembang di dalamnya karena pendidikan sangat penting bagi kehidupan manusia. Pendidikan secara umum mempunyai arti suatu proses kehidupan dalam mengembangkan diri tiap individu untuk bisa hidup dan untuk melangsungkan kehidupan. Sehingga untuk menjadi orang yang terdidik itu sangat penting. Manusia mengenyam pendidikan untuk menjadi orang yang berkontribusi bagi negara, nusa, dan bangsa. Lingkungan keluarga (pendidikan informal), lingkungan sekolah (pendidikan formal), dan lingkungan masyarakat merupakan lingkungan pendidikan awal yang diperoleh manusia (pendidikan nonformal) (Alpian et al, 2019). Pendidikan adalah usaha sadar dan terencana untuk menciptakan lingkungan belajar dan proses pembelajaran agar peserta didik dapat secara aktif mengembangkan potensi dirinya untuk memiliki kekuatan spiritual keagamaan, pengendalian diri, kepribadian, kecerdasan, akhlak mulia, dan keterampilan yang dibutuhkan bagi dirinya, masyarakat, bangsa dan negara. Gagasan ini tertuang dalam Undang-Undang Republik Indonesia Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional (Hidayat and Abdillah, 2019). Pengertian pendidikan dalam arti yang paling sederhana dan seluas-luasnya adalah usaha manusia untuk menumbuhkan dan mengembangkan potensi-potensi intrinsik, baik jasmani maupun rohani, sesuai dengan cita-cita dan standar masyarakat. Agar manusia dapat menggunakan ilmu dan bakatnya untuk kepentingan masyarakat, lingkungan dan negara, pendidikan juga bertujuan untuk menanamkan rasa keimanan dan ketakwaan kepada Allah SWT. Sebagaimana Allah SWT berfirman dalam surat At-Taubah ayat 122 yang berbunyi: Artinya: “Dan tidak sepatutnya bagi mukmin itu pergi semuanya (ke medan perang). Mengapa tidak pergi dari tiap- tiap golongan di antara mereka beberapa orang untuk memperdalam pengetahuan mereka tentang agama dan untuk memberi peringatan kepada kaumnya apabila mereka telah kembali kepadanya, supaya mereka itu dapat menjaga dirinya” (QS. At-Taubah: 122) (Zaim, 2019). Mengingat sulitnya mendapatkan pekerjaan di tengah persaingan masyarakat luas. Perguruan tinggi sangatlah penting supaya memiliki kemampuan dan keterampilan yang cukup sebagai bekal untuk menjadi tenaga kerja. Lebih bermutunya sebuah pekerjaan apabila mengikuti pendidikan di perguruan tinggi terlebih dahulu, karena di dalam 145 146 \| Ahmadi & Latifah perguruan tinggi tidak hanya mementingkan teori saja melainkan juga praktik. Kemudian, akan lebih mudah untuk mendapatkan pekerjaan dengan posisi yang lebih baik dan layak. perguruan tinggi tidak hanya mementingkan teori saja melainkan juga praktik. Kemudian, akan lebih mudah untuk mendapatkan pekerjaan dengan posisi yang lebih baik dan layak. Meskipun, mengingat realitas perguruan tinggi saat ini, hanya sedikit orang yang benar-benar menginginkannya. Hal ini disebabkan oleh menurunnya minat belajar dan kurangnya keyakinan akan kemampuan untuk maju secara pribadi melalui perguruan tinggi. PENDAHULUAN Namun, tidak demikian dengan para pemuda yang secara khusus berada di Desa Pakamban Laok Kecamatan Pragaan Kabupaten Sumenep. Desa Pakamban Laok merupakan desa yang mayoritas masyarakatnya memiliki mata pencaharian sangat beragam. Seperti bertani, nelayan, buruh bangunan dan sebagainya. hal tersebut mereka lakukan untuk memenuhi kebutuhan hidup sehari-hari. Para pemuda di desa Pakamban Laok memiliki kesadaran yang tinggi akan pentingnya pendidikan perguruan tinggi. Sebagaimana di Desa Pakamban Laok Kecamatan Pragaan Kabupaten Sumenep, di sana terdapat lulusan MA atau SMA pada tahun 2020 sebanyak 28 orang. Melanjutkan pada jenjang perguruan tinggi Islam hanya 9 orang. Lulusan pada tahun 2021 dari jumlah lulusan 23 orang. Yang melanjutkan ke perguruan tinggi hanya sebanyak 7 orang. Berdasarkan latar belakang di atas, peneliti tertarik untuk meneliti apa yang menarik kemauan para pemuda Desa Pakamban Laok lebih memilih Perguruan Tinggi Islam. Faktor Internal yang Mempengaruhi Minat Kuliah pada Perguruan Tinggi Islam Faktor internal yang mempengaruhi minat kuliah pada Perguruan Tinggi Islam yaitu: Pertama, Motivasi. Motivasi merupakan suatu hal yang timbul dari dalam diri seseorang untuk melakukan sesuatu sesuai keinginan individu itu sendiri. Sehingga, ada hal yang akan dicapai dari aktivitas/kegiatan tersebut. Motif merupakan suatu dorongan atau suatu kehendak yang mendasari munculnya suatu tingkah laku. Dengan kata lain motivasi dapat diartikan sebagai suatu kekuatan atau tenaga pendorong untuk melakukan sesuatu hal atau menampilkan sesuatu perilaku tertentu (Gunarsa, 2008). Dorongan dari dalam individu itu sendiri mengakibatkan timbulnya minat untuk melakukan aktivitas atau tindakan tertentu untuk memenuhinya. Misalnya, dorongan untuk belajar dan menimbulkan minat untuk belajar (Tafonao, 2018). Dalam perkembangannya, motivasi diartikan suatu kondisi internal (fisik ataupun mental, fitrah maupun perolehan) yang merangsang perilaku, menentukan jenis dan orientasinya, dan mengantarkannya untuk mencapai tujuan-tujuan tertentu yang dapat memuaskan salah satu aspek dari kehidupan manusia (Az-Za’balawi, 2007). Motivasi biasanya akan menghasilkan kualitas pencapaian yang tinggi, pencapaian tugas tepat waktu, memenuhi tantangan dan mendapatkan penyempurnaan dalam tugas. Hal ini dapat terjadi karena adanya dorongan dari dalam diri pemuda tersebut untuk mengukur batas kemampuannya atau bahkan untuk mencapai suatu target yang ditetapkan untuk dirinya sendiri. Pemuda desa Pakamban Laok melanjutkan pendidikannya ke Perguruan Tinggi Islam tentu karena adanya motivasi atau karena adanya kebutuhan yang harus dipenuhi dalam kehidupannya. Kebutuhan tersebut mengacu pada teori Abraham Maslow tentang motivasi atau hierarki kebutuhan manusia, di mana ada lima kebutuhan manusia menurut teori Abraham Maslow. Adapun kelima kebutuhan pemuda desa Pakamban Laok tersebut adalah sebagai berikut: a) Kebutuhan fisik (Physiological Needs). Kebutuhan fisik di sini merupakan kebutuhan yang paling mendasar dan paling mendominasi kebutuhan pemuda desa Pakamban Laok. Kebuthan ini bersifat biologis seperti oksigen, makanan, air dan sebagainya; b) Kebutuhan akan rasa aman (Safety Needs). Setelah kebutuhan fisiologis terpenuhi, maka pemuda desa Pakamban Laok akan cenderung mencari rasa aman, bisa berupa kebutuhan akan perlindugan, kebebasan dari rasa takut, kekacauan dan sebagainya. Kebutuhan ini bertujuan untuk mengembangkan hidup pemuda desa Pakamban Laok supaya menjadi lebih baik; c) Kebutuhan akan kepemilikan dan cinta (The Belongingness and Love Needs). Setelah kebutuhan fisik dan rasa aman terpenuhi, pemuda desa Pakamban Laok akan cenderung mencari cinta orang lain selain dari keluarganya sendiri, misalnya pemuda desa Pakamban Laok tersebut ingin dicintai oleh pasangannya supaya bisa dimengerti dan dipahami oleh orang lain; d) Kebutuhan untuk dihargai (The Esteem Needs). METODE faktor penyebab minat kuliah pada Perguruan Tinggi Islam. Dan seluruh data yang disajikan oleh peneliti berbentuk tulisan peneliti, kemudian peneliti menyajikan data yang diperoleh dengan menyelesaikan berdasarkan kategori yang berkaitan dengan fokus penelitian; 3) penarikan kesimpulan, terkait penarikan kesimpulan, peneliti menyimpulkan hasil penelitian yang telah diseleksi sesuai dengan kategori yang berkaitan dengan fokus penelitian. Penyajian laporan penelitian dikonsultasikan dengan dosen pembimbing supaya memperoleh hasil yang sistematis dan mudah untuk dipahami (Sugiyono, 2017). Pedagogika: Jurnal Ilmu-Ilmu Kependidikan \| 147 Pedagogika: Jurnal Ilmu-Ilmu Kependidikan \| 147 HASIL DAN PEMBAHASAN Faktor Internal yang Mempengaruhi Minat Kuliah pada Perguruan Tinggi Islam Setelah ketiga kebutuhan di atas terpenuhi, maka sudah menjadi naluri pemuda desa Pakamban Laok untuk bisa dihargai oleh sesama bahkan oleh masyarakat; e) Kebutuhan aktualisasi diri (Self Actualization). Kebutuhan inilah yang menjadi puncak tertinggi pencapaian pemuda desa Pakamban Laok setelah kebutuhan-kebutuhan di atas terpenuhi. Pencapaian aktualisasi diri ini berdampak pada kondisi psikologi yang meninggi pula seperti perubahan persepsi, dan motivasi untuk selalu tumbuh dan berkembang (Trygu, 2021) Kedua, Cita-cita merupakan suatu tolak ukur dan bentuk nyata yang timbul dari dalam diri setiap individu. Serta, Cita-cita merupakan tujuan akhir dari keinginan yang berkesinambungan. Cita-cita adalah keinginan/kehendak yang selalu ada di dalam pikiran, tujuan yang sempurna (yang akan dicapai atau dilaksanakan), cita-cita adalah keinginan, harapan, atau tujuan yang selalu ada dalam pikiran (Carsel, 2020). Jadi dalam perkembangannya cita-cita bisa dikatakan padanan kata dari ideologi. cita-cita bersifat tetap dan harus dicapai, sehingga cita-cita yang bersifat tetap tersebut merupakan dasar, pandangan atau paham (Pramono, 2020). Ketiga, Harapan merupakan suatu hal yang abstrak namun akan menjadi hal yang nyata jika sudah tercapai. Harapan adalah asa atau bentuk dasar dari kepercayaan akan sesuatu yang diinginkan akan didapatkan, atau suatu kejadian akan berbuah kebaikan di waktu yang akan datang (Angggraeni, 2019). Jadi dengan kata lain, Harapan 148 \| Ahmadi & Latifah adalah suatu keinginan yang diharapkan oleh seseorang untuk menjadi kenyataan (Syamsu, 2018). Faktor internal yang paling berpengaruh dalam melanjutkan pendidikan ke Perguruan Tinggi Islam adalah motivasi. Faktor Eksternal yang Mempengaruhi Minat Kuliah pada Perguruan Tinggi Islam Faktor eksternal yang mempengaruhi minat kuliah pada Perguruan Tinggi Islam yaitu: Pertama, Keluarga merupakan lingkungan sosial terkecil yang ada di tatanan masyarakat. Keluarga juga dapat membentuk karakter siswa, baik dalam perilaku dan prestasi belajar. Jadi, dukungan keluarga sangat dibutuhkan terutama dalam kegiatan belajar siswa. Keluarga sangat berpengaruh terhadap gaya belajar peserta didik. Karena mereka akan menerima pengaruh dari orang-orang terdekat (keluarga) seperti cara orang tua mendidik, hubungan yang baik antara anggota keluarga, suasana rumah dan keadaan ekonomi keluarga (Supuwiningsih, 2021). Serta adanya dukungan yang kuat dari keluarga dapat meningkatkan minat siswa (Rosyada, Kartikasari, and Annisa, 2021). Kedua, Teman adalah individu yang selalu bersama dalam proses kegiatan belajar secara formal dan informal. Teman, mempunyai peran penting dalam dunia pendidikan. Pengaruh-pengaruh dari teman bergaul siswa lebih cepat masuk dalam jiwa anak jika teman bergaulnya, baik akan berpengaruh baik terhadap diri siswa, begitu juga sebaliknya. Jika teman bergaulnya jelek pasti memengaruhi sifat yang jelek pada diri siswa. Sepatutnya orang tua memperhatikan pergaulan anak-anaknya, jangan sampai anaknya berteman dengan anak yang memiliki tingkah laku yang tidak diharapkan (Sutrisno, 2021). Karena Teman atau sahabat dapat menyebabkan timbulnya minat siswa dalam belajar (Trygu, 2021). Ketiga, sekolah sebagai sarana untuk menuntut ilmu. Jika sarana pelengkap dalam proses belajar siswa tidak mendukung seperti suasana dan keadaan gedung sekolah tidak mendukung maka akan berakibat pada terhambatnya semangat dalam belajar siswa. Sekolah dapat menjadi faktor penghambat timbulnya minat siswa jika: sarana dan prasana sekolah kurang memadai, hubungan antar peserta didik yang kurang baik, kurangnya bimbingan yang maksimal dari guru, lokasi gedung sekolah yang buruk seperti dekat pasar (Syam et al, 2021). Keempat, Doa, sebagai umat Islam yang mempunyai keimanan tentunya tidak boleh mengesampingkan peranan doa. Doa menurut istilah yaitu permohonan atau permintaan kepada Allah SWT sebagai penguasa alam semesta (Muratika, 2020). Dengan demikian, doa merupakan hubungan pribadi manusia dengan Tuhannya. Melalui doa Tuhan dapat memberikan segalanya, salah satunya timbul minat dalam belajar (Trygu, 2021). Adapun faktor eksternal yang paling berpengaruh dalam melanjutkan pendidikan ke Perguruan Tinggi Islam adalah berasal dari keluarga (ayah dan ibu). KESIMPULAN Faktor internal yang mempengaruhi minat kuliah pada Perguruan Tinggi Islam adalah motivasi, seperti ingin memperdalam pengetahuan tentang Islam dan ingin mendapat ilmu pengetahuan yang berdasar pada ajaran Islam. Cita-cita, seperti mempunyai keinginan untuk menjadi guru karena ingin menjadi orang yang bermanfaat bagi orang lain. Harapan, agar bisa seperti bapaknya menjadi seorang guru dan menjadi penerus sekaligus kebanggaan keluarga. Dan faktor eksternal yang mempengaruhi minat kuliah pada Perguruan Tinggi Islam antara lain: Keluarga, seperti: support/dukungan dari orang tua kepada anak yang dapat menumbuhkan minat untuk belajar. Teman, seperti: teman merupakan partner dalam pembelajaran. Sekolah, seperti: sekolah merupakan salah satu sarana pelengkap dan penunjang dalam tercapainya tujuan KBM. Doa, seperti : ikhtiar tanpa doa kurang lengkap. Alpian, Y., Anggraeni, S. W., Wiharti, U., & Soleha, N. M. (2019). Pentingnya Pendidikan Bagi Manusia. Jurnal Buana Pengabdian, 1(1), 66-72. https://doi.org/10.36805/jurnalbuanapengabdian.v1i1.581 Angggraeni, S. (2019). Menggali Potensi, Menggapai Mimpi. Depok: Diandra Kreatif. Az-Za’balawi, M. S. M. (2007). Pendidikan Remaja Antara Islam dan Ilmu Jiwa. Jakarta: Gema Insani Press. Carsel, H. R., & Syamsunie. (2020). Budaya Akademik dan Kemahasiswaan. Ponorogo: Uwais Inspirasi Indonesia dan Reativ. G S D (2008) P ik l i Ol h P t i (2 d d ) J k t PT BPK G M li Gunarsa, S. D. (2008). Psikologi Olahraga Prestasi (2nd ed.). Jakarta: PT BPK Gunung Mulia. Syamsu, R. F. (2018). Anatomi Rindu 2. Gowa: Jariah Publishing Intermedia. REFERENSI Pedagogika: Jurnal Ilmu-Ilmu Kependidikan \| 14 149 Hidayat, R., & Abdillah. (2019). Ilmu Pendidikan: Konsep, Teori Dan Aplikasinya. Medan: Lembaga Peduli Pengembangan Pendidikan Indonesia (LPPPI). Moleong, L. J. (2012). Metodologi Penelitian Kualitatif (Edisi Revisi). Bandung: PT Remaja Ro Muratika. (2020). Jadikan Allah Sebagai Sandaran Motivasi Hidup Dalam Perspektif Islam Berdasarkan Filosofi Kehidupan. Riau: Dot Plus. Pramono, B. (2020). Sosiologi Hukum. Surabaya: Scopindo Media Pustaka. mono, B. (2020). Sosiologi Hukum. Surabaya: Scopindo Media Pustaka. Rosyada, A., Kartikasari, C. D. P., & Annisa, D. H. (2021). Nalar Kritis Mahasiswa (Kumpulan Mahasiswa FTIK). Salatiga: Academia Publication. Sugiyono. (2017). Metode Penelitian & Pengembangan. Bandung: Alfabeta. Sugiyono. (2017). Metode Penelitian & Pengembangan. Bandung: Alfabeta. Supuwiningsih, N. N. (2021). E-Learning Untuk Pembelajaran Abad 21 Dalam Menghadapi Era Revolusi Industri 4.0. Bandung: Media Sains Indonesia. Supuwiningsih, N. N. (2021). E-Learning Untuk Pembelajaran Abad 21 Dalam Menghadapi Era Revolusi Industri 4.0. Bandung: Media Sains Indonesia. Sutrisno. (2021). Meningkatkan Minat Dan Hasil Belajar TIK Materi Tipologi Jaringan Dengan Media Pembelajaran. Malang: Ahlimedia Press. Sutrisno. (2021). Meningkatkan Minat Dan Hasil Belajar TIK Materi Tipologi Jaringan Dengan Media Pembelajaran. Malang: Ahlimedia Press. Syam, S., Cecep, Fahmi, A. I., Chamidah, D., Damayanti, W. K., Saputro, A. N. C., Halim, N. M., Herlina, E. S., & Haris, A. (2021). Pengantar Ilmu Pendidikan. Yayasan Kita Menulis. Syam, S., Cecep, Fahmi, A. I., Chamidah, D., Damayanti, W. K., Saputro, A. N. C., Halim, N. M., Herlina, E. S., & Haris, A. (2021). Pengantar Ilmu Pendidikan. Yayasan Kita Menulis. Tafonao, T. (2018). Peranan Media Pembelajaran Dalam Meningkatkan Minat Belajar Mahasiswa. Jurnal Komunikasi Pendidikan, 2(2), 112. Tafonao, T. (2018). Peranan Media Pembelajaran Dalam Meningkatkan Minat Belajar Mahasiswa. Jurnal Komunikasi Pendidikan, 2(2), 112. Trygu. (2021). Teori Motivasi Abraham H. Maslow dan Implikasinya dalam Belajar Matematika. Guepedia. Zaim, M. (2019). Tujuan Pendidikan Islam Perspektif Al-Quran Dan Hadis (Isu Dan Strategi Pengembangan Pendidikan Islam). Jurnal Muslim Heritage, 4(2), 240. Zaim, M. (2019). Tujuan Pendidikan Islam Perspektif Al-Quran Dan Hadis (Isu Dan Strategi Pengembangan Pendidikan Islam). Jurnal Muslim Heritage, 4(2), 240. Ó Ahmadi, Latifah, 2023
https://openalex.org/W2891488608	https://fundaj.emnuvens.com.br/CIC/article/download/1734/1426	Portuguese	null	ESTUDO COMPARATIVO ENTRE A HISTÓRIA DA CONTABILIDADE TRADICIONAL E A SUA NOVA HISTÓRIA	Ciência & Trópico	2,018	cc-by	12,687	Estudio comparativo entre la historia de la contabilidad tradicional y su nueva historia Estudio comparativo entre la historia de la contabilidad tradicional y su nueva historia Paulo Schmidt 1 Júlia de Medeiros Gass 2 Paulo Schmidt 1 Júlia de Medeiros Gass 2 p g 2 Pós-graduada em Gestão Empresarial – Instituto Federal do Rio Grande do Sul; Graduada em Ciências Contábeis – Universidade Federal do Rio Grande do Sul - UFRGS; Graduada em Administração de Empresas – Rede Metodista do Sul - IPA. juliamgass@yahoo.com.br. 1 Doutor em Controladoria pela Universidade de São Paulo; professor titular do Departamento de Ciên cias Contábeis e Atuariais da Universidade Federal do Rio Grande do Sul. pschmidt@ufrgs.br. 1 Doutor em Controladoria pela Universidade de São Paulo; professor titular do Departamento de Ciên cias Contábeis e Atuariais da Universidade Federal do Rio Grande do Sul. pschmidt@ufrgs.br. 2 Pós-graduada em Gestão Empresarial – Instituto Federal do Rio Grande do Sul; Graduada em Ciências Contábeis – Universidade Federal do Rio Grande do Sul - UFRGS; Graduada em Administração de Empresas – Rede Metodista do Sul - IPA. juliamgass@yahoo.com.br. ABSTRACT From the beginning of its existence, Accounting has followed the evolutionary steps of society. Initially everything was recorded precariously, but from the onset of writing and numbers, accounting records became more developed and complex. This evolutionary process has led schools of accounting thinking to succeed each other through history, especially with traditional views, following the social and political precepts of their day. However, from 1929 a new historical perspective for accounting appeared with the emergence of the so-called New Accounting History or Critical History, led by a new chain of thinkers with ideas contrary to those presented by traditionalists. From this new moment, this study was developed with the purpose of presenting the characteristics that distinguish the New Accounting History (NAH) from the Traditional Accounting History (TAH). For this, a descriptive and documentary analysis was performed on NAH and TAH, applying a qualitative approach. The result indicates that there is no consensus on this evolutionary process and that there are still significant divergences between the proponents of each historical current. The main contributions of the study are related to the understanding that this new conception about history can significantly influence the focus of research on Accounting. KEYWORDS: History. Traditional accounting history. New accounting history. RESUMO Desde o início de sua existência, a Contabilidade seguiu os passos evolutivos da sociedade. Inicialmente tudo era registrado de modo precário, mas, a partir do surgimento da escrita e dos números, os registros contábeis tornaram-se mais desenvolvidos e complexos. Esse processo evolutivo fez com que escolas de pensamentos contábeis se sucedessem através da história, especialmente com visões tradicionais, seguindo os preceitos sociais e políticos de sua época. Porém, a partir de 1929, surgiu uma nova perspectiva histórica para a con tabilidade com o aparecimento da chamada Nova História da Contabilidade ou História Crítica, liderada por uma nova corrente de pensadores com ideias contrárias as apresentadas pelos tradicionalistas. A partir desse novo momen to, este estudo foi desenvolvido com o objetivo de apresentar quais as caracte rísticas que distinguem a Nova História da Contabilidade (NHC) da História Tradicional da Contabilidade (HTC). Para tanto, foi realizada uma análise descritiva e documental sobre a NHC e a HTC, aplicando-se uma abordagem qualitativa. O resultado indica que não existe consenso sobre esse processo evolutivo e que, ainda existem divergências significativas entre os defensores de cada corrente histórica. As principais contribuições do estudo estão relacio nadas com o entendimento de que essa nova concepção sobre a história pode influenciar significativamente o foco de pesquisas sobre Contabilidade. PALAVRAS-CHAVE: História. História tradicional da contabilidade. Nova história da contabilidade. 71 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história RESUMEN Desde el comienzo de su existencia, la Contabilidad siguió los pasos evolutivos de la sociedad. Al principio todo era registrado precariamente, pero desde la aparición de la escritura y números, registros de contabilidad se convirtió en más desarrollado y complejo. Este proceso evolutivo ha hecho escuelas de pen samientos de contabilidad tuvisteis éxito a lo largo de la historia, especialmen te con vistas al tradicionales, siguiendo los preceptos sociales y políticos de su tiempo. Sin embargo, en 1929, surgió una nueva perspectiva histórica para la contabilidad con la aparición de la llamada Nueva Historia de la Contabili dad o Historia Crítica, dirigido por una nueva corriente de pensadores con ideas que compiten con las presentadas por tradicionalistas. A partir de este momento nuevo, este estudio fue desarrollado con el objetivo de presentar cuáles son las características que distinguen a la Nueva Historia de la Contabi lidad (NHC) de la Historia Tradicional de la Contabilidad (HTC). Para eso, un análisis descriptivo y documental fue realizado sobre la NHC y la THC, aplicándose un enfoque cualitativo. El resultado indica que no existe consenso sobre este proceso evolutivo y que hay todavía diferencias entre los defensores 72 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass de cada corriente histórica. Las principales aportaciones del estudio se relacio nan con la comprensión de este nuevo concepto en la historia puedan afectar significativamente el enfoque de las investigaciones en la contabilidad. PALABRAS CLAVE: Historia. Historia Tradicional de la Contabilidad. Nueva Historia de la Contabilidad. PALABRAS CLAVE: Historia. Historia Tradicional de la Contabilidad. Nueva Historia de la Contabilidad. Data de submissão: 03/04/2018 Data de aceite: 17/07/2018 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 2 FUNDAMENTOS TEÓRICOS SOBRE HISTÓRIA O objetivo desta seção é apresentar o conceito e a caracterização da história como um ramo do conhecimento humano, da história tradicional, assim como da nova história. Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Esse processo evolutivo de uma visão tradicional para uma nova visão da Contabilidade foi denominado por Carnegie (2014) como a transição da História da Contabilidade Tradicional (HCT) para a Nova História da Con tabilidade (NHC). O surgimento da chamada Nova História da Contabilidade, conforme Es teve (1997), pode ser considerado um dos acontecimentos mais importantes dos últimos anos na Contabilidade. Assim, essa nova visão baseou sua pesquisa em vá rias disciplinas sociais para fazer uma “história total”, ou seja, uma história única, mas tendo a influência de outras áreas. Ela considera que “tudo tem um passado que, em princípio, pode ser reconstruído com o resto do passado” (Burke, 1992). O estudo da NHC tem despertado o interesse de vários pesquisadores do mundo, objetivando entender pontos como o significado dessa transição, seus principais conceitos, quais são as implicações para o estudo da História da Contabilidade, dentre outros. Dentre os pesquisadores, podem ser destacados Carnegie (2014), Bertalan e Napier (2013), Carmona, Ezzamel e Gutiérrez (2004) e Miller, Hopper e Laughlin (1991). A partir desses e de outros estudos contábeis com abordagem evolutiva da perspectiva tradicional para a nova história da contabilidade, esse trabalho busca contribuir para o entendimento da seguinte questão: quais as principais diferenças conceituais entre o estudo da História Tradicional da Contabilida de e da Nova História da Contabilidade? Esse artigo inicia com a apresentação do intento que será desenvolvi do, seguido dos estudos relacionados com os principais conceitos de História Tradicional e da Nova História da Contabilidade, dos procedimentos meto dológico utilizados na pesquisa, e encerra com a apresentação das principais diferenças conceituais entre os dois enfoques históricos de análise da Contabi lidade e as considerações finais. 1 INTRODUÇÃO O tema História representa um dos principais tópicos nos atuais estu dos no campo das Ciências Sociais, apesar de serem escassos os estudos histó ricos voltados para a área da Contabilidade, especialmente no Brasil. Como defendem Padilha e Borenstein (2005, p. 576): “é possível fazer sentido no mundo sem abordar a questão da história”? Apesar dos estudos históricos serem mais raros do que em outras áreas de interesse contábil, no mundo existem periódicos que publicam exclusiva mente estudos históricos de Contabilidade, como é o caso da norte-americana The Accounting Historians Journal, que iniciou suas publicações em 1974, da inglesa Accounting History, que passou a publicar em 1996, da espanhola De Computis – Revista Española de Historia de la Contabilidad, que começou suas publicações em 2004 e da inglesa Accounting History Review, que desde 1990 passou a publicar artigos em história da contabilidade (SCHMIDT e SANTOS, 2017). No Brasil, mesmo não havendo nenhuma revista que se dedique exclu sivamente à publicação de artigos de história da contabilidade, existem alguns pesquisadores que publicam nessa área, como é o caso de Silva (2017), Sch midt e Santos (2017), Miranda et al. (2013), Peleias et al. (2007), Iudícibus, Martins e Carvalho (2005), Ricardino Filho e Martins (2003), dentre outros. As pesquisas na área da histórica, como defende Barros (2011), têm se tornado mais complexas nos últimos anos, especialmente, considerando que o próprio conhecimento científico tem buscado mais especialidades nos processos de pesquisa. Essa evolução nas pesquisas históricas, também repercutiu diretamente no próprio estudo da história da contabilidade, principalmente a partir do sur gimento da chamada Nova História. Como destaca Burke (1992), as pesquisas histórias passam a direcionar seus focos para toda atividade humana, não ape nas para os grandes homens e líderes mundiais, mas para as opiniões das pessoas comuns, fazendo com que a história passasse a ser explicada por outras fontes e não apenas por documentos tradicionais, onde o próprio historiador passou a entender a dificuldade de explicar como os fatos realmente aconteceram. 73 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 2.1 HISTÓRIA COMO UM RAMO DO CONHECIMENTO HUMANO Segundo Maia et al (2011, p. 139) a história “que significa em grego “ver”, “começou com um relato, a narração daquele que pode ouvir e vêm mantendo este aspecto de “história testemunho” no processo de seu desenvol vimento. Os autores ainda mencionam que a história “é constituída pela expe 74 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass riência humana vivida integral e socialmente, numa constante contradição de ideias, necessidades e aspirações que se manifestam num movimento de “fazer, desfazer e refazer” (MAIA et al, 2011, p. 139). Dessa maneira, “a história é ciência e não arte, consistindo a tarefa do historiador não em evocar ou reviver o passado [...] mas sim em narrar/des crever os acontecimentos deste passado tal como eles realmente se passaram” (FALCON, 1997, p. 66). Corroborando com o autor, Maia et al citam que: A constituição de bibliotecas e arquivos e elaboração de méto dos de crítica científica vêm conferindo a história, desde o sé culo XVII, o “status” de ciência. O fato de não “existir história sem erudição” vem sendo questionada no século XX a partir da crítica de noção de fato histórico, que é definido como objeto que não é acabado, pois resulta da construção do historiador (MAIA et al 2011 p 139) Dessa maneira, “a história é ciência e não arte, consistindo a tarefa do historiador não em evocar ou reviver o passado [...] mas sim em narrar/des crever os acontecimentos deste passado tal como eles realmente se passaram” (FALCON, 1997, p. 66). Corroborando com o autor, Maia et al citam que: A constituição de bibliotecas e arquivos e elaboração de méto A partir da história, tem-se a historiografia que se trata de um ramo da ciência que estuda a evolução no desenvolvimento histórico global. A ideia é da história como estudo do homem no tempo por meio da redefinição de conceitos fundamentais como documento, fato histórico e tempo (Costa et al, 2010). Os autores citam ainda, as principais características da historiografia: a) discurso histórico construído em função de uma epistemolo gia empirista; b) análises de curtos períodos temporais; c) foco no âmbito político e na atribuição de relevância aos eventos conjunturais e personagens específicos (história baseada em ideias e ações de alguns poucos agentes históricos individuais) (COSTA et al, 2010, p. 293). Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Assim, as maiores dificuldades para os novos historiadores são as que estão relacionados às fontes e aos métodos: “quando os historiadores come çaram a fazer novos tipos de perguntas sobre o passado, para escolher novos objetos de pesquisa, tiveram de buscar novos tipos de fontes, para suplementar os documentos oficiais” (BURKE, 1992, p. 25). Esse novo grupo de pesquisadores tem estudado ideias contrárias as clássicas, dissociando-se das escolas tradicionais, o que gerou a nova histó ria. “O movimento de mudança surgiu a partir de uma percepção difundida da inadequação do paradigma tradicional. Esta percepção da inadequação só pode ser compreendida se olharmos além do âmbito do historiador, para as mudanças no mundo mais amplo” (Burke, 1992, p.7).i Portanto identificar as visões provenientes do debate entre as perspecti vas da história tradicional e da história nova pode contribuir para: “a) melhor compreensão dos fenômenos administrativos; b) formação de pesquisadores mais conscientes de seus caminhos de pesquisa e c) fortalecimento da interdis ciplinaridade por meio da criação de vínculos mais profundos entre as áreas” (Costa et al, 2010, p. 290). 2.1 HISTÓRIA COMO UM RAMO DO CONHECIMENTO HUMANO Durante o começo do século XX “manteve-se quase inalterada a hege monia da historiografia política tradicional, começando o seu declínio somen te a partir dos anos 1930 com a história social e a partir dos anos 1970 com a nova história” (Costa et al, 2010, p. 293). No século XX, a historiografia renovada promove um profun do questionamento tanto da noção de fato histórico (não mais um objeto dado e acabado e sim construção do historiador) quanto da noção de documento (noção ampliada e não mais considerado algo objetivo e inocente). A própria ausência de documentos passa a ser significativa, pois elucida que estes são sempre produtos de uma sociedade que os fabricou segundo relações de dominação e poder (COSTA et al, 2010, p. 294). No século XX, a historiografia renovada promove um profun do questionamento tanto da noção de fato histórico (não mais um objeto dado e acabado e sim construção do historiador) quanto da noção de documento (noção ampliada e não mais considerado algo objetivo e inocente). A própria ausência de documentos passa a ser significativa, pois elucida que estes são sempre produtos de uma sociedade que os fabricou segundo relações de dominação e poder (COSTA et al, 2010, p. 294). Esse movimento em direção a um novo tipo de história foi iniciado e conduzido por Marc Bloch e Lucien Febvre, em 1929, com a publicação do primeiro número da revista dos Annales d’Histoire Économique et Sociale. Foi o início da disciplina de história às temáticas e métodos das outras ciências humanas por meio da formulação de novos problemas, novos métodos e novas abordagens da pesquisa histórica (CASTRO, 1997). 75 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 2.2 HISTÓRIA TRADICIONAL Segundo Le Goff (1992, p. 9), a história começou como uma história relato ou como testemunho, assim como “a narração daquele que pode dizer ‘eu vi, senti’”. Dessa forma a ciência histórica passou a priorizar a reunião de documentos escritos, transformando-os em testemunhos. A visão tradicional dedicou seus esforços para compartilhar a história com a “história oficial”, como uma história cujo objeto principal de investigação era os fatos políticos. Sua atenção foi direcionada exclusivamente por instituições como o Estado, a Igreja e a Guerra, o que resultou em histórias centradas nas façanhas dos “Grandes Homens (aqueles que verdadeiramente fazem história), tratando-os como eventos únicos (às vezes predestinados) que dão novas orienta ções para a vida de uma nação ou de uma sociedade” (MARTÍN, 2006, p. 189). A história tradicional é caracterizada como: A história tradicional é caracterizada como: Uma narrativa dos acontecimentos políticos e militares, apre sentada como a história dos grandes feitos de grandes homens e legitimada por uma visão centralizada e institucionalizada do poder, a história política tradicional foi definindo e disse minando seus objetos, princípios e métodos mesmo diante de tentativas de contestação a esse tipo de narrativa (COSTA et al, 2010, p. 296). Dessa forma, essa visão não se vinculou à vida cotidiana dos homens, importando apenas com os grandes fatos e os grandes personagens, uma história conclusa aos grandes feitos e às grandes narrativas, o que era um 76 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass Paulo Schmidt / Júlia de Medeiros Gass movimento quase que inerte (CLEMENTE, 2011). Assim, perdurou du rante muito tempo como “objeto por excelência na produção histórica” (FALCON, 1997, p. 65). movimento quase que inerte (CLEMENTE, 2011). Assim, perdurou du rante muito tempo como “objeto por excelência na produção histórica” (FALCON, 1997, p. 65). Esta abordagem considera que a história podia ser vista como uma série de decisões econômicas racionais e que tinha como função o ensinamento por meio de exemplos reais e ilustres, o que levaram ao progresso que foi feito até hoje. Vale destacar que esta visão é a predominante da história, em que consi dera uma série de eventos encadeados até o fim (MARTÍN, 2006). i O paradigma tradicional pregava que a disciplina devia dizer respeito es sencialmente à política. “História é política passada; política é história presen te”, nas palavras de Sir John Seeley (BURKE, 1992). 2.2 HISTÓRIA TRADICIONAL Desse modo, essa dimen são política era vista a partir do Estado e dedicava às grandes homens, batalhas e guerras, considerando predominantemente aqueles documentos oficiais. Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 2.3 NOVA HISTÓRIA Durante o século XX surgiram novas concepções do conhecimento his tórico tradicional, repercutindo em diversos outros caminhos para a pesquisa histórica, através de diferentes abordagens e problemas. “Os autores dessas no vas concepções, mesmo que não comunguem de uma homogeneidade de ideias e posicionamentos perante a História, alinham-se a uma corrente historiográ fica que passou a denominar-se Nova História” (MAIA et al, 2011, p. 139). Este novo paradigma “possibilitou historicizar novos temas que antes não eram pesquisados e novas metodologias e técnicas para a análise das fontes concorrendo para o melhor desempenho da investigação”. [...] Assim, a histó ria também pode ser revista ganhando novas respostas historiográficas e outros temas podem ser investigados “provando que tudo e todos têm uma história a ser desvendada e reconhecida” (MAIA et al, 2011, p. 140). Essa visão também tem sido chamada de história vista de baixo, pois se trata de uma história relacionada com o movimento, às mudanças econô micas e sociais, com a sociedade, e entre os outros; assim sem desvalorizar os “grandes feitos” que não deixam de ser importantes. Esta pretensão da história total requer fontes documentais que não sejam apenas oficiais, mas produzidas por setores que “não tinham história” e, portanto, tendem a investigar história oral, estatística, etc. (MARTÍN, 2006). Assim, a história vista de baixo considera as opiniões das pessoas co muns com suas experiências da mudança social e da história da vida cotidiana, o que difere da história tradicional, que vê os fatos “de cima” concentrados nos grandes feitos, nos grandes homens e na elite. Conforme Maia et al (2011, p. 139) a nova história “tem uma tradição própria, a dos fundadores da revista Annales d’histoire économique et sociale, 77 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história editada a partir de 1929, e faz uma crítica à noção de fato histórico, como se houvesse uma “realidade histórica acabada que se entregaria por si só ao histo riador”. Sobre os Annales, os autores mencionam: Os primeiros historiadores dos Annales são vistos como respon sáveis por uma nova concepção historiográfica, pela ampliação da noção de “fonte histórica”, pela valorização de uma “história estrutural” em oposição à “história factual”, por uma prática interdisciplinar e por uma série de outras contribuições (MAIA et al, 2011; p. 139). Já Burke (1992) cita: Já Burke (1992) cita: Para muitas pessoas, a nova história está associada à Lucien Febvre e a Marc Bloch, que fundaram arevista Annales em 1929 para divulgar sua abordagem, e na geração seguinte, a Fernand Braudel. Na verdade,seria difícil negar a importância do movimento para a renovação da história liderado por esses homens (BURKE, 1992, p. 5). Associada à chamada École des Annales, “La nouvelle histoire”, expres são francesa de A Nova História, é o título de uma coleção de ensaios do fran cês Jacques Le Goff. Apesar de não ter uma definição exata, este movimento é a união daqueles que se opõem as ideias tradicionais. É a história escrita como uma reação contra o “paradigma” clássico (BURKE, 1992). “É por isso difícil apresentar mais que uma descrição vaga, caracterizando a nova história como história total (histoire totale) ou história estrutural. [...] Em outras palavras, definir a nova história em termos do que ela não é, daquilo a que se opõem seus estudiosos” (BURKE, 1992, p. 2). Poderíamos também chamar este paradigma de a visão do senso co mum da história, não para enaltecê-lo, mas para assinalar que ele tem sido com frequência — com muita frequência — considerado a maneira de se fazer história, ao invés de ser percebido como uma dentre várias abordagens possíveis do passado (BURKE, 1992, p. 2).i Os novos historiadores estão enfrentando problemas de definição por que eles “estão avançando em território não familiar. Como normalmente fa zem os exploradores de outras culturas, eles começam com uma espécie de imagem negativa daquilo que estão procurando” (Burke, 1992, p. 6). Entre tanto, outros obstáculos surgiram: Os maiores problemas para os novos historiadores, no entanto, são certamente aqueles das fontes e dos métodos. Já foi sugeri do que quando os historiadores começaram a fazer novos tipos de perguntas sobre o passado, para escolher novos objetos de pesquisa, tiveram de buscar novos tipos de fontes, para suple mentar os documentos oficiais. Alguns se voltaram para a his tória oral; outros à evidência das imagens; outros à estatística (BURKE, 1992, p. 8). 78 Ci & Tróp. Já Burke (1992) cita: Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass Este novo paradigma faz com que o pesquisador não utilize apenas um simples modelo de análise, mas sim, que ele busque uma conexão entre a teoria e a filosofia, para agregar valor positivo à sua pesquisa, além de trazer inúmeras possibilidades de vantagens, como uma melhor interpretação e compreensão dos significados e sentidos dos eventos e contribuir para o desenvolvimento e construção do conhecimento histórico (MAIA et al, 2011). Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 3 PROCEDIMENTOS METODOLÓGICOS A pesquisa realizada neste estudo foi classificada quanto aos seguintes aspectos: pela forma de abordagem do problema; de acordo com seus objeti vos; e com base nos procedimentos técnicos utilizados. Quanto à forma de abordagem do problema, a pesquisa é qualitativa. Tal pesquisa de acordo com o Minayo (1994, p. 21), “responde a questões muito particulares. Ela se preocupa nas ciências sociais, com um nível de qua lidade que não pode ser quantificado”. Já Lima (2008, p. 40) destaca que a “abordagem qualitativa pressupõe a investigação de aspectos sociologicamente construídos e que, por isso, não são facilmente mensuráveis”. Bauer e Gaskell (2012, p. 24) também demonstram a importância da pesquisa qualitativa: A mensuração dos fatos sociais depende da categorização do mundo social. As atividades sociais devem ser distinguidas an tes que qualquer frequência ou percentual possa ser atribuído a qualquer distinção. É necessário ter uma noção das distin ções qualitativas entre categorias sociais, antes que se possam medir quantas pessoas pertencem a uma ou outra categoria (BAUER e GASKELL, 2012, p. 24). Ou seja, a pesquisa foi classificada nesse aspecto, pois os dados que fo ram analisados, não eram numéricos, mas sim de diferentes abordagens, o que possibilitou ao pesquisador informações mais aprofundadas e maior liberdade na condução dos documentos a fim de obter melhores respostas, aprofundan do os pontos que se considerarem mais importantes para o cumprimento dos objetivos do estudo. De acordo com objetivos propostos, a pesquisa é descritiva. Este tipo de pesquisa tem como finalidade descrever características de uma determinada população ou fenômeno ou estabelecer relações entre variáveis (GIL, 2009). Para Cervo e Bervian (1996, p. 49) “a pesquisa descritiva procura descobrir, com a precisão possível, a frequência com que um fenômeno ocorre, sua rela ção e conexão, com os outros, sua natureza e características, correlacionando fatos ou fenômenos sem manipulá-los”. A pesquisa em questão teve o objetivo de descrever e analisar as carac terísticas que distinguem a NHC da HTC. Assim, quanto aos procedimentos técnicos utilizados, o estudo classificou-se como pesquisa documental. 79 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história A estratégia de Pesquisa Documental é característica dos estudos que utilizam documentos como fonte de dados, informações e evidências. 4 A TRANSIÇÃO DA HISTÓRIA TRADICIONAL DA CONTABILIDADE PARA A NOVA HISTÓRIA DA CONTABILIDADE Esta seção apresenta a História Tradicional da Contabilidade, a Nova História da Contabilidade e as diferenças entre elas. 3 PROCEDIMENTOS METODOLÓGICOS [...] a pesquisa documental emprega fontes primá rias, assim considerados os materiais compilados pelo próprio autor do trabalho, que ainda não foram objeto de análise, ou que ainda podem ser reelaborados de acordo com os propósitos da pesquisa (MARTINS e THEÓPHILO, 2009, p. 55). Foi utilizado este tipo de procedimento, pois foi realizada a pesquisa em documentos e artigos publicados como fonte de dados, informações e evidên cias. Dessa forma, o plano de coleta foi investigar documentos, livros e artigos publicados referentes ao assunto em questão. A análise de dados foi realizada através de uma análise de conteúdo. Já Bardin (1977, p. 42 apud VERGARA, 2006, p. 16) a define como: Um conjunto de técnicas de análise das comunicações visando obter, por procedimentos sistemáticos e objetivos de descrição do conteúdo das mensagens, indicadores (quantitativos ou não) que permitam a inferência de conhecimentos relativos às condições de produção/recepção (variáveis inferidas) destas mensagens. Para Vergara (2006), a análise de conteúdo é uma técnica de tratamen to de dados, que busca identificar o que foi dito acerca de um determinado assunto e também pode ser utilizada tanto para fins exploratórios (de des coberta), quanto para fins de verificação para confirmar ou não hipóteses e suposições preestabelecidas. 4.1 HISTÓRIA TRADICIONAL DA CONTABILIDADE A História Tradicional apresenta uma dimensão técnica da contabi lidade, que a prioriza como um sistema de mensuração e comunicação de transações econômicas para chegar a representações verificáveis próximas à realidade. Por um longo período, essa visão técnica foi privilegiada com carac terísticas de neutralidade e objetividade e utilidade para a tomada de decisões (GOMES, 2008). Como citado por Morgan e Willmott (1993, p. 8), “nesta visão, a con tabilidade é incontestável porque é simplesmente medir ou relatar a realida de”. Assim, cabe ressaltar que na contabilidade tradicional o lado social não foi 80 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass explorado, entretanto, não quer dizer que ele tenha sido ignorado, o fato é que não houve a mistura dos dois, da técnica e do social, ou não foi devidamente levado em consideração (GOMES, 2008). “Ciente da importância do contexto e do meio ambiente, era natural para que os historiadores tradicionais da contabilidade julguem os registros contábeis históricos em termos de sua capacidade de fornecer informações úteis para a tomada de decisões” (CARNEGIE e NAPIER, 1996, p.15). A história da contabilidade seria pouco mais do que uma história de progresso em que a contabilidade evolui em resposta aos desenvolvimentos técnicos de um determinado período. A contabilidade é relativa e progressiva. Os fenômenos que formam seu objeto estão em constante mudança. Os métodos mais antigos tornam-se menos efetivos em condições alteradas, ideias anteriores tornam-se irrelevantes diante de novos pro blemas. Assim, as condições circundantes geram novas ideias e estimulam os engenhosos para aconselhar novos métodos. E, como tal, ideias e métodos são bem sucedidos, eles, por sua vez, começam a modificar as condições circundantes (LITTLE TON, 1966, p. 361). A contabilidade é relativa e progressiva. Os fenômenos que formam seu objeto estão em constante mudança. Os métodos mais antigos tornam-se menos efetivos em condições alteradas, ideias anteriores tornam-se irrelevantes diante de novos pro blemas. Assim, as condições circundantes geram novas ideias e estimulam os engenhosos para aconselhar novos métodos. E, como tal, ideias e métodos são bem sucedidos, eles, por sua vez, começam a modificar as condições circundantes (LITTLE TON, 1966, p. 361). Assim, percebe-se nas escolas dos pensamentos contábeis que o pro gresso contábil está relacionado com os fatos ocorridos na economia. Con forme Canabarro (2008, p. Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Ao pensar na História da Contabilidade, surgem as escolas contábeis, em que se deve entender como se deu o surgimento e o contexto na época em que estavam inseridas. Percebe-se que elas estão relacionadas com os aconte cimentos sociais, econômicos e políticos que ocorreram no mundo, e dessa maneira muitas delas, sofreram grandes influências desses fatos, o que conse quentemente, fez com que se tornassem parte da história tradicional. A história da contabilidade é tão antiga quanto à própria história da civilização. “Está ligada às primeiras manifestações humanas da necessidade social de proteção à posse e de perpetuação e interpretação dos fatos ocorridos com o objeto material de que o homem sempre dispôs para alcançar os fins propostos” (ZANLUCA e ZANLUCA, 2017, p. 01). Não se sabe exatamente quando começaram os primeiros registros con tábeis, mas se tem conhecimento que na época dos sumérios já havia alguns indícios, através de registros primitivos de pagamentos, em placas de barro. Com o passar do tempo, diversos outros povos como egípcios, romanos e entre outros, também tiveram a sua forma de anotar suas transações. Alguns historiadores relatam que os primeiros sinais da exis tência das contas datam de aproximadamente 4.000 A. C.. Entretanto, antes disto, o homem primitivo, ao inventariar as primeiras ferramentas e instrumentos de caça e pesca disponí vel, ao contar seus rebanhos, já estava praticando uma forma rudimentar de contabilidade (COTRIN et al., 2012, p. 45). No início do século XIV, com as grandes navegações, consequente do aumento do comércio que ocorria na Europa, veio junto à peste bubônica. Co nhecida como peste negra, essa infecção matou mais de um terço da população em curto espaço de tempo. Devido a ela, a mão de obra começou a tornar-se escassa, pois já não havia quem trabalhasse. Aproveitando a situação, muitos trabalhadores começaram a cobrar mais por seus trabalhos, o que fez muitos deixarem o campo atrás de salários maiores. Os efeitos dessa doença e outros fa tores resultaram no declínio do sistema feudal (SCHMIDT e SANTOS, 2006). Com o passar do tempo, toda a instabilidade em que a Europa passou durante o século XIV, virou um novo período, que foi marcado por expansão e crescimento. 4.1 HISTÓRIA TRADICIONAL DA CONTABILIDADE 41) “as escolas históricas possuem características próprias, o que influenciou diretamente na concepção de historiografia, pois esta muda de acordo com cada uma”. O autor ainda cita que elas representam a maneira “como os historiadores pensam, como formulam seus trabalhos de pesquisa e como constroem o próprio conhecimento histórico” (CANABAR RO, 2008, p. 41). A Contabilidade é tão antiga quanto a história da civilização humana. A sua evolução está diretamente relacionada e ligada com a história política e econômica do mundo, caracterizando-se assim, como uma visão tradicional. (SÁ, 1997). A História da Contabilidade divide-se, pois, em nítidos perío dos, cada um servindo de base para demarcar as evoluções, em bora a maior parte de nosso saber se tenha situado, apenas, no campo dos registros e demonstrações, em razão do imenso va lor que a humanidade atribuiu à escrita, como arte, e ao poder da memória gravada que ela ofereceu como fonte de evidência, controle, base de decisão e prova. A escrituração contábil nas ceu antes mesmo que a escrita comum aparecesse, ou seja, o registro da riqueza antecedeu aos demais, como comprovam os estudos realizados sobre a questão, na antiga Suméria. A His tória da Contabilidade, pois, percorre milênios, participando das diversas modificações sobre o uso da riqueza e dos recursos sobre os meios de registro (SÁ, 1997, p. 11). 81 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história O sistema feudal entrou em crise e os trabalhadores já não tinham mais interesses em voltar para os campos, pois estavam em busca de condições e salários melhores, fazendo com que as cidades começassem a crescer, e junto com ela, o comércio. Assim o trabalho escravo cedia lugar ao trabalho assalaria do, alterando-se as relações de trabalho. Esse processo gerou a acumulação de capital, tornando os registros mais complexos (SCHMIDT e SANTOS, 2006). A partir desse momento, o comércio começa a se desenvolver devido a necessidade de tentar atender a toda nova população que estava se desenvol vendo e ao interesse dos comerciantes em lucrar mais. Em razão das intensas 82 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass vendas e trocas que estavam acontecendo, cresce ainda mais as relações econô micas, surgindo pequenos e grandes comerciários, que sentem a necessidade de fazer registros e anotações sobre as combinações feitas entre eles. Foi pensando no futuro, devida a preocupação em ter que saber de to dos seus bens, rendimentos, produção e oportunidades de crescimento de seus negócios, que levou o homem a fazer os seus primeiros registros, e assim, mais tarde surgindo à necessidade do controle (COTRIN et al., 2012). “À medida que o homem começava a possuir maior quantida de de valores, preocupava-lhe saber quanto poderiam render e qual a forma mais simples de aumentar as suas posses; tais informações não eram de fácil memorização quando já em maior volume, requerendo registros (ZANLUCA e ZANLU CA, 2017, p. 01). “À medida que o homem começava a possuir maior quantida de de valores, preocupava-lhe saber quanto poderiam render e qual a forma mais simples de aumentar as suas posses; tais informações não eram de fácil memorização quando já em maior volume, requerendo registros (ZANLUCA e ZANLU CA, 2017, p. 01). Em virtude dessa maior preocupação com as anotações, começam a aparecer os processos contábeis, com o surgimento da primeira escola, a Con tista, no século XV. Esse pensamento tinha como base o sistema de contas, que tinha a preocupação em registrar dívidas a receber ou pagar, ou seja, o haver e dever. Como tudo era muito simples, apenas as combinações feitas entre os comerciantes e compradores, que era o sistema que eles precisavam na época, eram registradas. Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Juntamente com esse crescimento que a Europa teve, houve intensi ficação das navegações marítimas, onde foram se descobrindo novas rotas e novos comércios. Com esses avanços, os comércios foram se desenvolvendo até chegar à Espanha e Portugal, que se tornaram os novos centros comerciais. Esse novo foco fez com que as cidades italianas começassem a decair e entrar em recessão econômica, pois já não era mais de grande interesse, como antes (SCHMIDT e SANTOS, 2006). Após o período, de imenso crescimento eco nômico, político e social, as cidades italianas, começam a entrar numa época de declínio e juntamente com ela, a contabilidade também vive esse período de estagnação, que foi de 1494 e durou até 1840, com o surgimento da obra “La Contabilità Applicatta alle Amministrazioni Private e Pubbliche” de Fran cesco Villa (SCHMIDT, 2000). O termo “Era da Estagnação”, para Hendriksen e Van Breda (1999, p. 45) não é muito apropriado, pois “esse período se iniciou como uma era de descobrimento e encerrou-se como uma era de revolução”. Na verdade, durante esse momento, não surgiram outros pensadores, porém foi a época da consolidação contábil, com a divulgação do sistema das partidas dobradas pelo mundo inteiro. p Depois de anos de estagnação, a Inglaterra, passa por um período de crescimento, fruto de uma série de fatos, como o melhoramento da saúde e nutrição da população, devido a boas colheitas e baixo preço do alimento. Esse desenvolvimento fez com que a população aumentasse, e em razão disso, sur gisse a necessidade de novos trabalhos para atender aquela demanda crescente. O resultado disso foi o aumento das fábricas e consequentemente, o aumento da produção. Iniciava a era da Revolução Industrial. Diante desses novos fatos que vinham acontecendo, foi crescendo a necessidade de uma contabilidade que englobasse outros requisitos, além do crédito e do débito. Havia a vontade de obter mais informações sobre o que era produzido e os seus custos, ou seja, de um sistema que abrangesse mais dados e que fosse mais complexa (SCH MIDT e SANTOS, 2006). Da necessidade de uma contabilidade mais completa, em 1840, ini cia a contabilidade do mundo científico, que dura até hoje, com o surgindo da escola lombarda ou administrativa, com Francesco Villa. Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Esse sistema é reflexo ainda de um sistema muito primitivo de relações econômicas, onde a escrituração era muito fragmentada e pouca, pois não havia tanta necessidade já que eram poucas as relações existentes, não precisando de uma contabilidade mais complexa, pois ainda o comércio estava no início (SCHMIDT e SANTOS, 2006). A introdução da técnica contábil nos negócios privados foi uma contribuição de comerciante italianos do sec. XII. Quan do apareceu o Tratactus de Computis et Scripturis (Contabi lidade das Partidas Dobradas) de Frei Luca Pacioli, publicado em 1494, enfatizando que a teoria contábil do débito e do cré dito corresponde a teoria dos números positivos e negativos, obra que contribuiu para inserir a contabilidade entre os ramos do conhecimento humano. Pacioli apesar de ser considerado o pai da contabilidade, não foi o criador das partidas dobradas, o método já era utilizado na Itália, principalmente na Toscana, desde o século XIV (COTRIN et al., 2012, p. 47). Luca Pacioli enfatizou que “a teoria contábil do débito e do crédito corresponde à teoria dos números positivos e negativos, obra que contribuiu para inserir a contabilidade entre os ramos do conhecimento humano” (ZAN LUCA e ZANLUCA, 2017, p. 02). Segundo Schmidt e Santos (2006, p. 34), Pacioli incluiu esse método no Summa, “por achar bastante necessário ao mercado, que deve saber regis trar corretamente suas contas, a partida de crédito e de débito e seus corres pondentes e também determinar os lucros e as perdas de todo o negócio”. 83 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Foi uma escola científica, que acreditava que contabilidade não deveria ficar restrita a estrutu ração, mas ser voltada para o controle da gestão também. Esses interesses dos autores da época se deram em função ao crescente número de fábricas e aos adventos econômicos da Revolução Industrial, que trouxe uma contabilidade mais complexa e preocupação maior com a administração dos negócios. O período científico foi aquele em que as doutrinas se agigan taram determinadas, não só em buscar a delimitação de um ob jeto verdadeiro de estudos para a Contabilidade, mas também, 84 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass especialmente de buscar conhecer a substância gerida pelo ser humano no sentido da satisfação de suas necessidades materiais (SÁ, 1997, p. 55). Hendriksen e Van Breda (1999, p. 46) consideram este período o se gundo importante avanço da contabilidade, pois foi: Marcado por grandes transformações, uma vez que os de tentores do poder, ou seja, os empresários da época começa ram a fazer grandes investimentos e, por outro lado, houve a necessidade de contratação de mão-de-obra para trabalhar por mais tempo nas empresas e não por um período deter minado. Os pensadores da lombarda, influenciados pelo contexto da época, queriam melhorar as informações a serem passadas para os proprietários das fábricas, aliando a contabilidade com a administração, visando a produtivida de com a máxima qualidade. Francesco Villa, uns dos principais autores, surge com os princípios econômicos gerais, mostrando a preocupação com a gestão das organizações e defendendo a ideia de que para um novo empreendimento, precisavam-se analisar vários procedimentos e fazer cálculos (SCHMIDT e SANTOS, 2006). Francesco Villa deu início ao grande progresso da Ciência Contábil: Extrapolou os conceitos tradicionais de Contabilidade, segun do os quais escrituração e guarda livros poderiam ser feitas por qualquer pessoa inteligente. Para ele, a Contabilidade implica va conhecer a natureza, os detalhes, as normas, as leis e as prá ticas que regem a matéria administrada, ou seja, o patrimônio (ZANLUCA e ZANLUCA, 2017, p. 08). Extrapolou os conceitos tradicionais de Contabilidade, segun do os quais escrituração e guarda livros poderiam ser feitas por qualquer pessoa inteligente. Para ele, a Contabilidade implica va conhecer a natureza, os detalhes, as normas, as leis e as prá ticas que regem a matéria administrada, ou seja, o patrimônio (ZANLUCA e ZANLUCA, 2017, p. 08). Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Nas próximas décadas não houve fatos marcantes na história, pois a Revolução Industrial ocorria na Inglaterra e os outros países estavam na fase de desenvolvimento e industrialização. Como ainda era início desse novo perío do, a sociedade alemã, francesa e outras, estavam ainda sem muita evolução, começando um pouco mais adiante, no desenrolar da Revolução. Nesse mo mento a contabilidade, apresentou escolas como a personalista, que era mais focada com a ideia geral de personalizar as contas, ou seja, as contas deveriam ser abertas por pessoas envolvidas, como pessoa física ou jurídica (SCHMIDT e SANTOS, 2006). Sá (1997, p. 70) afirma que “o personalismo foi uma corrente que se ligou aos conceitos jurídicos, pessoais, mas, com sérios desenvolvimentos, também, com a administração”. Essa escola foi fundada na segunda metade do século XIX e teve muitos seguidores, como: Francesco Marchi, Giuseppe Cerboni e Giovanni Rossi (SCHMIDT, 2000). A teoria personalista teve extraordinário impulso com o traba lho desenvolvido por Cerboni, que teve grande interesse pelo aspecto jurídico das relações entre o proprietário e a entidade, 85 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história aspecto esse não abordado no trabalho de Marchi. Marchi foi o iniciador e Cerboni, o construtor da teoria personalista (SCH MIDT, 2000, p. 61). aspecto esse não abordado no trabalho de Marchi. Marchi foi o iniciador e Cerboni, o construtor da teoria personalista (SCH MIDT, 2000, p. 61). Por volta de 1860, o mundo começa numa nova era, a Segunda Revo lução Industrial, onde países como Alemanha, França, Rússia, Itália e EUA começam a se industrializarem, além disso, estavam também em busca de no vos territórios e de capitais para financiamento de suas indústrias. Nessa época surge a escola controlista, por volta de 1880 em Veneza, Itália, que tinham pensadores com que acreditava no controle da riqueza administrativa, tendo três fases: gestão, direção e controle (SCHMIDT e SANTOS, 2006). Como menciona Sá (1997, p. 80) “A escola personalista, Toscana, com grande força e prestígio, encontrou sua contestação ferrenha com o apareci mento da escola de Veneza (controlista)”. O surgimento do American Institut of Certield Public Ac countants foi de extrema importância no desenvolvimento da Contabilidade e dos princípios contábeis; várias associações empreenderam muitos esforços e grandes somas em pesquisas nos Estados Unidos. Havia uma total integração entre acadê micos e os já profissionais da Contabilidade (ZANLUCA e ZANLUCA, 2017, p. 09). Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Na Alemanha e Itália, a contabilidade começa a se desenvolver em tor no de 1919 e 1920, respectivamente, resultado de fatores como: o desenvol vimento industrial mais tardio de ambas e início das crises, consequência da Revolução Industrial, o que gerou a necessidade de uma contabilidade que trouxesse mais informações. Conforme isso, o desenvolvimento na Escola Ale mã, segundo Schmidt e Santos (2006, p. 123) “se deve, em parte, às crescentes necessidades dos usuários contábeis dos vários setores da contabilidade”. E a Escola Italiana, ou aziendal, tinha como ideia a de “mostrar que o fundamen to, era ter acrescido a parte cientifica da contabilidade, sendo, a organização, administração e o controle, contribuindo assim, à ciência administrativa e esquecia-se do campo da contabilidade ao levantamento patrimonial, só se lembrando das empresas” (MARQUES, 2010, p. 21). p p A partir de 1920, surge a escola americana. “Enquanto declinavam as escolas europeias, floresciam as escolas norte-americanas com suas teorias e práticas contábeis, favorecidas não apenas pelo apoio de uma ampla estrutura econômica e política, mas também pela pesquisa e trabalho sério dos órgãos associativos” (ZANLUCA e ZANLUCA, 2017, p. 08). “A teoria contábil avançou de acordo com as novas necessidades que se apresentaram, mas foi nos EUA que a contabilidade evoluiu para a prática” (COTRIN et al., 2012, p. 48). A escola norte americana apresentou a divisão da contabilidade em financeira e gerencial, sendo que teve destaque na área finan ceira com a padronização da contabilidade e com o surgimento de Associações. O surgimento do American Institut of Certield Public Ac countants foi de extrema importância no desenvolvimento da Contabilidade e dos princípios contábeis; várias associações empreenderam muitos esforços e grandes somas em pesquisas nos Estados Unidos. Havia uma total integração entre acadê micos e os já profissionais da Contabilidade (ZANLUCA e ZANLUCA, 2017, p. 09). 86 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass Conforme Schmidt e Santos (2006, p. 74) a “intensa participação das associações profissionais no desenvolvimento prático e teórico da disciplina”. As associações foram de extrema importância, pois foram elas que desenvolve ram a contabilidade, através de duas principais: American Accounting Associa tion (AAA) e Americam Association of Public Accountants (AICPA). Já a área gerencial foi importante, pois ajudou na gestão do capital obtido, em conse quência da industrialização, financiamentos e empréstimos, e para adequação das demonstrações contábeis. Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Essa escola foi resultado da necessidade da socie dade, tanto proprietários e quanto banqueiros, que estava precisando de uma contabilidade mais qualificada, padronizada e completa, além das associações, que tiveram inúmeras contribuições como, publicações, criação de revista, pro posta, regulamentações e entre outros (SCHMIDT e SANTOS, 2006). Nos inícios do século atual, com o surgimento das gigantescas corporações, aliado ao formidável desenvolvimento do merca do de capitais e ao extraordinário ritmo de desenvolvimento que os Estados Unidos da América experimentou e ainda expe rimenta, constitui um campo fértil para o avanço das teorias e práticas contábeis. Não é por acaso que atualmente o mundo possui inúmeras obras contábeis de origem norte-americanas que tem reflexos diretos nos países de economia (ZANLUCA e ZANLUCA, 2017, p. 09). Nos inícios do século atual, com o surgimento das gigantescas corporações, aliado ao formidável desenvolvimento do merca do de capitais e ao extraordinário ritmo de desenvolvimento que os Estados Unidos da América experimentou e ainda expe rimenta, constitui um campo fértil para o avanço das teorias e práticas contábeis. Não é por acaso que atualmente o mundo possui inúmeras obras contábeis de origem norte-americanas que tem reflexos diretos nos países de economia (ZANLUCA e ZANLUCA, 2017, p. 09). Assim, com a evolução da contabilidade, surge a Nova História da Contabilidade, formada por uma corrente de pensadores com novas concep ções e ideias. Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 4.2 NOVA HISTÓRIA DA CONTABILIDADE A NHC não constitui certamente uma escola ou corpo unitário de doutrina. Mas os seus partidários compartilham ideias e tendências comuns, que os distanciam da forma tradicional como a História da Contabilidade sempre tinha sido entendida (LIMA, 1999, p. 01). O aparecimento da “Nova História da Contabilidade”‚ sem dúvida o acontecimento mais importante surgido no seio desta disciplina nos últimos quinze anos e provavelmente causador de grande parte do novo interesse que ela desperta. Os seus impul sos geradores procedem da união entre o novo paradigma crítico radical da investigação contabilística, com a discussão metodo lógica e epistemológica que agita as águas das ciências históricas desde há cerca de duas dezenas de anos (LIMA, 1999, p. 01). O aparecimento da “Nova História da Contabilidade”‚ sem dúvida o acontecimento mais importante surgido no seio desta disciplina nos últimos quinze anos e provavelmente causador de grande parte do novo interesse que ela desperta. Os seus impul sos geradores procedem da união entre o novo paradigma crítico radical da investigação contabilística, com a discussão metodo lógica e epistemológica que agita as águas das ciências históricas desde há cerca de duas dezenas de anos (LIMA, 1999, p. 01). Este novo paradigma considera a ideia de que a contabilidade “não deve ser estudada unicamente como uma técnica, mas também como uma peça do contexto social e organizativo da sociedade” (ESTEVE, 1997, p. 2). Como argumenta Miller (1994, p. 20), “se quisermos entender plenamente como 87 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história surgiram formas particulares de contabilidade e por que isso lhes é concedido, temos que ir além dos limites da organização e examinar a prática social e institucional da contabilidade”. Chamada também de história “crítica” da contabilidade, ela “baseia seu estudo análise na interpretação das implicações mútuas com o contexto em que tais práticas são realizadas e não com em práticas contábeis isoladas” (MARTIN, 2006, p. 194). Durante anos, o estudo da contabilidade era relacionado apenas com a concepção de contabilidade como mera prática técnica, sendo explicada ape nas por razões econômicas, como no caso das escolas contábeis. No entanto, recentemente, a literatura assumiu um entendimento, em que ela também pode ser uma prática social, que ultrapassa a ideia anterior de ser uma única técnica neutra (GOMES, 2008). 4.2 NOVA HISTÓRIA DA CONTABILIDADE Miller (1994) menciona que há pelo menos três aspectos diferentes da contabilidade como prática social, que são complementares entre si, são elas: como uma tecnologia; a linguagem e os significados, que são os raciocínios in trínsecos e o domínio da contabilidade. A ênfase em tecnologia significa que a contabilidade é considerada como “uma forma de intervenção, um dispositivo para atuar sobre atividades, indivíduos e objetos de tal forma que o mundo possa ser transformado”. Já a racionalidade da contabilidade demonstra que as práticas contábeis são mais do que cálculos numéricos de custos, lucros, perdas e retornos; e “as práticas contábeis são dotadas de um significado que se estende para além da tarefa a que elas são aplicadas” (MILLER, 1994, p. 2-3). p q p p Embora a análise da contabilidade nas organizações continue a ser importante, é necessário “ultrapassar os limites da organização e examinar a prática social e institucional da contabilidade para poder entender como sur giram formas particulares de contabilidade e por que isso lhes é atribuído” (MILLER, 1994, p. 20). Os novos historiadores consideram a contabilidade mais como um fe nômeno cultural do que simplesmente como uma prática técnica e são estão mais predispostos a vê-la como um instrumento de poder e dominação do que como um conjunto de ideias e técnicas sem valor para implementar e monito rar contratos livremente (GOMES, 2008). Ao invés de ver a história da contabilidade como uma evolução natural das tecnologias administrativas, cada vez mais será vista como a formação de um determinado complexo de racionalida des e modos de intervenção entre muitos, um complexo que foi formado de diversos materiais e em relação a uma gama heterogê nea de questões e eventos (CARNEGIE; NAPIER, 1996, p. 7). Ao invés de ver a história da contabilidade como uma evolução natural das tecnologias administrativas, cada vez mais será vista como a formação de um determinado complexo de racionalida des e modos de intervenção entre muitos, um complexo que foi formado de diversos materiais e em relação a uma gama heterogê nea de questões e eventos (CARNEGIE; NAPIER, 1996, p. 7). Os esforços dos novos pesquisadores não estavam na busca de soluções para problemas específicos, mas sim em problematizar e desconstruir os desen volvimentos nas práticas contábeis, identificando seus efeitos sobre o funciona 88 Ci & Tróp. 4.2 NOVA HISTÓRIA DA CONTABILIDADE Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass mento organizacional e social, no entendimento de problemas, prioridades e soluções, entre outros (CARMONA, EZZAMEL e GUTIÉRREZ, 2004). Eles estão preocupados com “re-orientações, transformações e reversões que o tempo instarou, ao invés de procurar voltar no tempo para detectar uma continuidade ininterrupta que nos une ao nosso passado” (MILLER e NAPIER, 1993, p. 632). Chamada também de história “crítica” da contabilidade, ela ampliou as áreas nas quais a contabilidade é compreendida e trouxe novos atores para a aná lise, como o estado e as instituições. Essas contribuições podem ajudar a melho rar a compreensão das conexões entre as concepções de contabilidade adotadas por pesquisadores de contabilidade contemporânea e as escolas de pensamento adotadas por pesquisadores de contabilidade histórica (GOMES; 2008; p. 497). O novo paradigma fez com que os historiadores pensassem além das definições convencionais de contabilidade, de modo que uma visão muito mais abrangente foi desenvolvida para alcançar um maior conjunto de práti cas de quantificação, valorização e representação (CARMONA, EZZAMEL e GUTIÉRREZ, 2004). O novo paradigma fez com que os historiadores pensassem além das definições convencionais de contabilidade, de modo que uma visão muito mais abrangente foi desenvolvida para alcançar um maior conjunto de práti cas de quantificação, valorização e representação (CARMONA, EZZAMEL e GUTIÉRREZ, 2004). Dessa forma, estes novos estudos históricos podem ser uma importante fonte de informação para ajudar a melhorar a compreensão dos papéis da con tabilidade nas organizações e na sociedade, particularmente nos processos de mudança contábil que se tornaram importantes questões de pesquisa dentro da concepção de contabilidade como prática social (GOMES, 2008, p. 497). Os novos historiadores da contabilidade, conforme Carnegie e Napier (1996, p.7) “não são amplamente receptivos à abordagem tradicional de ex plicar modos específicos de contabilidade por referência exclusivamente a ra cionalidades econômicas”. Mas cabe ressaltar que os novos historiadores “são obrigados a usar os trabalhos dos historiadores tradicionais da contabilidade no desenvolvimento da literatura, em vez de ignorar essas contribuições e, portanto, reinventar o campo como resultado” (GOMES, 2008, p. 480). Dessa maneira, esta concepção social nas últimas décadas expandiu o domínio da contabilidade, aumentando o escopo limitado promovido pela história tradicional. Entretanto, conforme Burchell et al. (1994, p. 540): p p p história tradicional. Entretanto, conforme Burchell et al. (1994, p. Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Estudo comparativo entre a história da contabilidade tradicional e a sua nova história 4.3 DIFERENÇA ENTRE A HISTÓRIA TRADICIONAL E A NOVA HISTÓRIA A contabilidade apresenta duas diferentes dimensões: sendo por um lado, a Contabilidade Tradicional, em que os pesquisadores enfatizam uma visão mais técnica, observado ela como um conjunto de procedimentos que são utilizados para satisfazer os requisitos e necessidades de seus usuários. E por outro lado, a Nova Contabilidade, em que pesquisadores destacam a dimensão social e institucional, tentando entender e explicar os atributos da contabilida de, assim, como determinar os impactos sobre o funcionamento organizacio nal e social, requerendo uma maior compreensão de como a ela configura seu ambiente e de como, por sua vez, o meio ambiente a molda (GOMES, 2008). p Sendo assim, Gomes (2008) menciona a principal divergência entre as duas percepções: Os especialistas em contabilidade que se sentem confortáveis com a contabilidade apenas como uma prática técnica nor malmente ficariam confortáveis com as histórias contábeis tradicionais. Por outro lado, os especialistas em contabilida de que se sentem confortáveis com a noção de contabilidade como prática social normalmente ficariam confortáveis com novas histórias contábeis (GOMES, 2008, p. 481). Os especialistas em contabilidade que se sentem confortáveis com a contabilidade apenas como uma prática técnica nor malmente ficariam confortáveis com as histórias contábeis tradicionais. Por outro lado, os especialistas em contabilida de que se sentem confortáveis com a noção de contabilidade como prática social normalmente ficariam confortáveis com novas histórias contábeis (GOMES, 2008, p. 481). Entre alguns pontos de contraste entre a História Tradicional e a Nova História da Contabilidade são: no primeiro ponto, o paradigma tradicional, a história diz respeito essencialmente à política. E por outro lado, a nova histó ria começou a se interessar por virtualmente toda a atividade humana. Tudo tem uma história, ou seja, tudo tem um passado que pode em princípio ser reconstruído e relacionado ao restante do passado (BURKE, 1992). Outra diferença importante é em relação à visão, na qual a história tradi cional oferece uma visão de cima, no sentido de que tem sempre se concentrado nos grandes feitos dos grandes homens, estadistas, generais ou ocasionalmente eclesiásticos. Ao resto da humanidade foi destinado um papel secundário no drama da história. Entretanto, por outro lado, vários novos historiadores estão preocupados com a história vista de baixo, ou seja, com as opiniões das pessoas comuns e com sua experiência da mudança social (BURKE, 1992). 4.2 NOVA HISTÓRIA DA CONTABILIDADE 540): Como resultado, pouco se sabe de como as práticas técnicas de contabilidade estão ligadas ao social, de como as forças so ciais mais amplas podem afetar e mudar a contabilidade, e de como a própria contabilidade funciona no domínio do social, influenciando, além de apenas reagir para isso. Como resultado, pouco se sabe de como as práticas técnicas de contabilidade estão ligadas ao social, de como as forças so ciais mais amplas podem afetar e mudar a contabilidade, e de como a própria contabilidade funciona no domínio do social, influenciando, além de apenas reagir para isso. Assim, a nova história da contabilidade demonstrou o valor de atribuir papéis à prática contábil além da pura economia racional. Ou seja, de modo que não precisa mais considerar a contabilidade como um bem puramente econômico. Mas também não necessariamente excluindo, pois tais explicações do surgimento e do funcionamento das práticas contábeis ajudam a liberar o pensamento (MARTÍN, 2006). 89 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Procuramos identificar a importância da prática contábil para os comerciantes medievais ou mercantis do capitalismo nas cente, por exemplo; ou ao perguntar sobre o papel das mu lheres na formação e desenvolvimento da profissão contábil, o papel de outras formas de representação do que a dupla entra da, como a entrada única ou as diferentes formas contábeis das sociedades antigas (comprimidos, argila), quipus, entre outros (MARTÍN, 2006, p. 190). 4.3 DIFERENÇA ENTRE A HISTÓRIA TRADICIONAL E A NOVA HISTÓRIA p Para Martín (2006), a nova visão da história a partir de baixo, ocorre quando: Procuramos identificar a importância da prática contábil para os comerciantes medievais ou mercantis do capitalismo nas cente, por exemplo; ou ao perguntar sobre o papel das mu lheres na formação e desenvolvimento da profissão contábil, o papel de outras formas de representação do que a dupla entra da, como a entrada única ou as diferentes formas contábeis das sociedades antigas (comprimidos, argila), quipus, entre outros (MARTÍN, 2006, p. 190). 90 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass Outro ponto divergente é sobre o fato de que, no modelo tradicional, as fontes eram constituídas por documentos, registros oficiais que expressam o ponto de vista oficial. A Nova História, além de aumentar o conceito de fontes, faz críticas como em relação a tempo e fatos históricos e propõe uma história problematizada e não simplesmente automática (MAIA et al, 2011). Segundo o paradigma tradicional, a História é objetiva. A tarefa do his toriador é apresentar aos leitores os fatos, ou, a forma como eles aconteceram. Na nova história, os historiadores deslocam o “ideal da Voz da História para aquele da heteroglossia, definida como ‘vozes va riadas e opostas’”. A história nova reflete uma possibilidade para se “considerar mais seriamente as opiniões das pessoas comuns sobre seu próprio passado” do que comumente faziam os his toriadores na história tradicional (MAIA et al, 2011, p. 140). Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Diferente de Burke, Esteve (1997) menciona muito sinteticamente apenas duas características que distinguem as Histórias da Contabilidade: Diferente de Burke, Esteve (1997) menciona muito sinteticamente apenas duas características que distinguem as Histórias da Contabilidade: Em primeiro lugar a perda de convicção na possibilidade de se alcançar a objetividade, no estudo e descrição dos factos histó ricos, o que dá origem a que a interpretação dos factos ganhe a primazia sobre a apresentação dos mesmos. Em segundo lugar, o abandono de uma espécie de darwinismo histórico-contábil, que consiste em pensar a contabilidade como as restantes ati vidades humanas, sujeita a um processo mais ou menos linear de progresso contínuo. (ESTEVE, 1997, p. 2). Outro fato em que se diferem as duas histórias é referente ao debate sobre as origens versus gênese da contabilidade. Os pesquisadores tradicionais estão principalmente preocupados com as origens e a evolução, enquanto que os novos argumentam que essas áreas de interesse são problemáticas (CAR MONA, EZZAMEL e GUTIÉRREZ, 2004). Sobre a visão tradicional: O pesquisador normalmente procuraria identificar a “origem” das inovações na prática contábil e, portanto, a história é vis ta como uma coleção de eventos que podem ser organizados sistematicamente e cronologicamente, começando com o ‘primeiro’ evento e avançando gradualmente escala temporal (CARMONA, EZZAMEL e GUTIÉRREZ, 2004, p. 38). Já na nova abordagem, a busca pelas origens das inovações contábeis é considerada repleta de problemas e infrutíferas, pois não existe uma razão a priori porque um determinado evento (de fato, qualquer evento) deveria ter ocorrido em qualquer momento ou lugar (EZZAMEL et al, 1990, apud CARMONA, EZZAMEL e GUTIÉRREZ, 2004, p. 157). O histórico é visto como sendo muito mais do que simplesmente uma agregação de eventos. Os pesquisadores da NAH buscarão se concentrar em “gênese”, pelo que, ao invés de olhar para trás no tempo, tentando descobrir origens, o pesquisador pretende descobrir o cenário sociopolítico que causa ou dá origem a práticas contábeis específicas. (CARMONA, EZZAMEL e GUTIÉRREZ, 2004, p. 38). 4.3 DIFERENÇA ENTRE A HISTÓRIA TRADICIONAL E A NOVA HISTÓRIA De forma resumida, os seis pontos de diferença entre a antiga e a nova história, mencionados por Burke (1992), conforme quadro abaixo: 91 Quadro 1: Diferenças entre História Tradicional x Nova História Diferenças História Tradicional Nova História 1 História diz respeito à política; Toda atividade humana pode ser objeto da história; 2 Pensam na história como essencialmente uma narrativa dos acontecimentos; Está mais preocupada com a análise das estruturas; 3 Oferece uma visão de cima, no sentido de que tem sempre se concentrado nos grandes feitos dos grandes homens, estadistas, generais ou eclesiásticos; Estão preocupados com a história vista de baixo, com as opiniões das pessoas comuns e sua experiência de mudança social; 4 A história deveria ser baseada em documentos oficiais escritos; Expõe as limitações de tipo de documento, examinando maior variedade de evidências, atribuindo menor ênfase às fontes escritas e maior relevância ao uso da história oral, iconografia e vestígios arqueológicos; 5 Preocupa-se com as ações dos indivíduos; Considera relevantes movimen tos coletivos e individuais; 6 A história é objetiva, e a tarefa única do historiador é acrescentar aos seus leitores os fatos. Mostra a inevitabilidade da falta de isenção ao olhar sobre o passado. Fonte: Adaptado de Burke (1992, p. 2-4). Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Quadro 1: Diferenças entre História Tradicional x Nova História 91 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história Ao contrário da história clássica, a nova compreende várias abordagens heterogêneas para o estudo da história contábil: Uma proliferação de metodologias, um questionamento de noções recebidas, como progresso e evolução, um alargamen to do escopo, uma nova atenção à linguagem e racionalidades que dão significado às práticas contábeis e uma mudança de foco longe de personagens invariantes como o livro-deten tor e decisor para a preocupação com transformações mais amplas no conhecimento contábil (MILLER, HOPPER e LAUGHLIN, 1991, p. 395). 92 Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 Paulo Schmidt / Júlia de Medeiros Gass Entretanto, vale ressaltar que mesmo havendo divergências fundamen tais entre as duas concepções,histórico tradicional e novo, ambas puderam contribuir para enriquecer e aprofundar esse campo de pesquisa e compreen são da prática contábil (GOMES, 2008). Ci & Tróp. Recife, v.42, n.2, p.71-98, 2018 5 CONSIDERAÇÕES FINAIS A Contabilidade, assim como a sociedade, teve o seu desenvolvimento evoluindo gradual e lentamente, que continua até hoje. É possível afirmar que ambas estão relacionadas, na qual o crescimento da Contabilidade ocorreu de acordo com as necessidades humanas, políticas e econômicas. A história da Contabilidade perpassa pelas décadas. Percebe-se então, que o seu processo ao longo do tempo manteve-se sempre em constante evo lução. Assim, o objetivo deste estudo foi conhecer a Nova História da Conta bilidade e consequentemente apresentar quais as características que a distin guiram da História Tradicional da Contabilidade. Diferente da História Tradicional observa-se que uma nova corrente começou a tomar força, a da Nova História da Contabilidade, que apresentou uma visão contrária dos pensamentos clássicos, preocupando-se com o social e o institucional e não mais apenas com o econômico e político. Os autores dessas novas concepções, mesmo que não compartilhem de ideias e posicio namentos semelhantes perante a História, eles acreditam que a contabilidade deve ser vista como um todo, e não meramente de forma isolada. Dessa forma, analisando as principais escolas do pensamento contábil, percebe-se que desde a primeira a surgir, a Escola Contista, elas desenvolve ram de acordo com a necessidade que iria surgindo da sociedade. Nota-se que quando o mundo tinha um comércio primitivo, o processo contábil era muito rudimentar, apenas contas de debitar e creditar. Mas conforme ela foi evoluin do, foi cada vez mais se tornando complexas as informações que eram solicita das pelos usuários, o que foi gerando uma contabilidade mais completa. Entretanto, vale ressaltar que as escolas contábeis fazem parte da história da contabilidade tradicional, pois se preocupavam apenas em cumprir com as necessidades dos seus usuários, onde cada vez ficava mais forte a ideia de posse e propriedade. Assim, consequentemente, o homem passa a se preocupar em con servar e aumentar o patrimônio, com o objetivo de ver sua riqueza aumentar. Portanto, divergente em algumas características, destacam-se entre elas: a Nova História tem um pensamento mais abrangente sobre a contabilidade clássica, saindo da visão limitada que a anterior passava para um pensamento social e tendo uma visão de baixo, o que demonstra que ela não vê apenas os grandes feitos, as batalhas e a política, mas como também enxerga o homem como pessoa comum e como personagem desta história. Outro ponto relevan 93 Ci & Tróp. 5 CONSIDERAÇÕES FINAIS Recife, v.42, n.2, p.71-98, 2018 Estudo comparativo entre a história da contabilidade tradicional e a sua nova história te na nova dimensão são os vários tipos de documentos examinados, além da história oral como forma de compreender melhor o passado, e consequente mente, expor uma visão mais ampla. Apesar disso, percebe-se que ambas as visões tiveram sua importância. Contudo, apesar das discordâncias entre os pensamentos tradicionais e no vos, elas têm seu lugar na pesquisa de história e têm potencial para aumentar nosso conhecimento sobre o passado contábil. Por este motivo, deverá ter o seu passado sempre relembrado, pois sem este desenvolvimento não teria sido possível o novo, e para que os futuros profissionais possam entender de que forma a contabilidade iniciou e de que maneira os novos pensadores podem influenciar para trazer novas compreensões a essa ciência. Portanto, este estudo aqui realizado não se encerra. Pelo contrário, abre caminho para que sejam feitas novas pesquisas sobre o assunto, que ainda é pou co explorado, principalmente por pesquisadores brasileiros. Como sugestão para futuras pesquisas, pode-se colocar que estas se aprofundem mais em assuntos específicos ou pontuais na evolução da história, tanto pela tradicional quanto para a nova. 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https://openalex.org/W4315795536	https://jurnal.uns.ac.id/equilibrium/article/download/66334/39072	Indonesian	null	Peningkatan Kompetensi Siswa SMK Muhammadiyah 4 Surakarta mengenai Industri Baterai Lithium Ion	Equilibrium/Ekuilibrium	2,023	cc-by-sa	3,624	DOI: https://dx.doi.org/10.20961/equilibrium.v6i2.66334 Article History Received: 09-10-2022, Accepted: 03-01-2023, Published: 12-01-2023 Article History Received: 09-10-2022, Accepted: 03-01-2023, Published: 12-01-2023 Article History Received: 09-10-2022, Accepted: 03-01-2023, Published: 12-01-2023 ABSTRAK. Sekolah Menengah Kejuruan (SMK) Muhammadiyah 4 Surakarta memiliki jurusan Kimia Industri yang mempersiapkan tenaga terampil di industri kimia untuk mendukung dalam pengembangan teknologi. Salah satu Industri Kimia yang berkembang saat ini adalah Industri Baterai Ion Litium (LIB). LIB saat ini digunakan tidak hanya pada perangkat elektronik portabel, seperti komputer dan telepon seluler, tetapi juga untuk kendaraan listrik atau kendaraan hybrid. Oleh karena itu, SMK dengan Program Keahlian Kimia Industri hendaknya menyiapkan siswa yang lulus mempunyai kompetensi di bidang teknologi baterai. Dengan kompetensi dosen dalam teknologi produksi material aktif baterai pada skala mini plant dan fasilitas yang mewadai di Pusat Unggulan IPTEK (PUI) PT Teknologi Penyimpanan Energi Listrik, maka sangat memungkinkan dosen untuk mengenalkan teknologi dan fasilitas produksi baterai kepada siswa SMK Muhammadiyah 4 Surakarta. Metode yang dilakukan dalam pelatihan ini adalah metode ceramah, diskusi dan praktik. Adapun materi yang disampaikan mengenai baterai lithium ion (aplikasi, jenis, tipe, struktur, proses pembuatan material aktif, dan fabrikasi). Kegiatan praktik yang dilakukan yaitu praktik pembuatan material aktif katoda dengan metode ko-presipitasi dan fabrikasi sel baterai. Hasil kegiatan ini adalah kompetensi siswa dan guru meningkat yang ditunjukkan dengan nilai Pre-Test yang lebih baik dibandingkan dengan Post-Test. Kata kunci: baterai lithium ion, SMK, katoda ABSTRACT. Muhammadiyah 4 Surakarta Vocational High School has a Department of Industrial Chemistry which prepares skilled workers in the chemical industry to support the development of technology. One of the chemical industries that is currently developing is the Lithium Ion Battery (LIB) industry. LIBs are currently used not only in portable electronic devices, such as computers and cell phones but also for electric vehicles or hybrid vehicles. Therefore, Vocational High School with an industrial chemistry expertise program should prepare students who graduate to have competence in the field of battery technology. With the competence of lecturers in the production technology of battery active materials on a mini plant scale and adequate facilities at the Center of Excellence for Electrical Energy Storage Technology, it is very possible for lecturers to introduce technology and battery production facilities to students of Muhammadiyah 4 Surakarta Vocational High School. The methods used in this training are lecture, discussion and practice methods. The material presented was about LIB (application, type, type, structure, active material manufacturing process, and fabrication). EQUILIBRIUM JOURNAL OF CHEMICAL ENGINEERING EQUILIBRIUM JOURNAL OF CHEMICAL ENGINEERING Homepage:https://jurnal.uns.ac.id/equilibrium DOI: https://dx.doi.org/10.20961/equilibrium.v6i2.66334 The practical activities carried out are the practice of making cathode active materials with the co-precipitation method and battery cell fabrication. The result of this activity is that the competence of students and teachers increases as indicated by a better Pre-Test score compared to the Post-Test score. Keywords: lithium ion batteries, SMK, cathode Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 Peningkatan Kompetensi Siswa SMK Muhammadiyah 4 Surakarta mengenai Industri Baterai Lithium Ion y aProgram Studi Teknik Kimia, Fakultas Teknik, Universitas Sebelas Maret, Surakarta, Indonesia 57126 an IPTEK-Perguruan Tinggi Teknologi Penyimpanan Energi Listrik, Universitas Sebelas Maret, Surakarta, Indonesia, 57146 *Corresponding author: tikaparamitha@staff.uns.ac.id DOI: https://dx.doi.org/10.20961/equilibrium.v6i2.66334 DOI: https://dx.doi.org/10.20961/equilibrium.v6i2.66334 Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 1. PENDAHULUAN Sekolah Menengah Kejuruan (SMK) Muhammadiyah 4 Surakarta yang beralamat di Jl. Slamet Riyadi No.443 Kerten Surakarta, berdiri pada tanggal 19 April 2008, dengan visi “Mewujudkan SMK Muhammadiyah 4 Surakarta Sebagai Lembaga yang Kompeten Yang Membentuk Sumber Daya Insan Islami, Kreatif, Unggul, Berdaya Saing tinggi dan Peduli Lingkungan”. Sekolah ini memiliki 3 jurusan, yaitu Farmasi, Kimia Industri, dan Tata Kecantikan. Jurusan Kimia Industri merupakan salah satu jurusan di SMK Muhammadiyah 4 Surakarta yang mempersiapkan peserta didik dengan pengetahuan, keterampilan, dan sikap agar kompeten dalam mengoperasikan instrumen, melaksanakan proses, dan operasi teknik kimia serta proses pengolahan limbah sesuai Standar Operasional Procedure (SOP) sehingga mereka mampu membuat suatu produk dari bahan baku sampai produk jadi lengkap dengan uji mutu/kualitas dari produk yang telah dibuat. Selebihnya, jurusan Kimia Industri 163 Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 Paramitha / Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 menghasilkan tenaga terampil di industri kimia untuk mendukung dalam pengembangan dan penguasaan teknologi. Di Indonesia, Industri baterai merupakan salah satu industri kimia yang sedang berkembang. Baterai ion litium (LIB) sedang booming karena kepadatan energinya yang tinggi, perawatan yang rendah, self-discharge rendah, pengisian cepat, dan umur panjang [1]. LIB saat ini digunakan tidak hanya pada perangkat elektronik portabel, seperti komputer dan telepon seluler, tetapi juga untuk kendaraan listrik atau kendaraan hybrid [2]. Komponen dari LIB umumnya elektroda (katoda dan anoda), separator, dan elektrolit. Diantara komponen LIB yang sangat penting adalah katoda [3]. Material aktif katoda yang paling umum dipakai adalah golongan oksida logam transisi seperti litium kobalt (LCO), litium nikel kobalt aluminium oksida (NCA) dan litium nikel mangan kobalt (NMC). Namun, tingginya biaya kobalt dan terbatasnya kapasitas LCO telah mendorong substitusi kobalt (Co) dengan Mangan (Mn) dan Nikel (Ni) selama beberapa dekade terakhir, yang telah menyebabkan pengembangan NMC dan NCA dengan kepadatan energi yang lebih tinggi dan biaya yang lebih rendah. Selain itu, substitusi Co dipicu akibat permasalahan sosial ekonomi dan keberlanjutan karena 70% Co yang ditambang saat ini berasal dari Republik Demokratik Kongo (DRC). Dengan demikian, meningkatkan kandungan Ni dan menurunkan atau menghilangkan Co di material katoda menjadi lebih menonjol untuk aplikasi kendaraan listrik atau perangkat elektronik lainnya. Kebutuhan nikel dalam industri baterai didukung dengan ketersediaan nikel yang melimpah di Indonesia. Indonesia mengalami pertumbuhan produksi nikel tertinggi dalam beberapa tahun terakhir ini [4]. Beberapa pabrik peleburan logam telah beroperasi dan sedang dibangun untuk memasok bahan baku bagi industri baterai. 1. PENDAHULUAN Groundbreaking pembangunan pabrik baterai kendaraan listrik PT HKML Battery Indonesia telah dilaksanakan pada tanggal 15 September 2021 di Karawang, Jawa Barat. Sejalan dengan berkembangnya industri baterai, maka Sekolah Menengah Kejuruan (SMK) harus siap menghadapi perkembangan teknologi tersebut. Pengembangan teknologi baterai memiliki prospek yang baik di masa yang akan datang, sehingga SMK dengan Program Keahlian Kimia Industri hendaknya menyiapkan siswa yang lulus mempunyai kompetensi di bidang teknologi baterai dan aplikasinya. Keahliaan siswa SMK tersebut dalam bidang baterai, khususnya produksi material aktif katoda akan menjadi bekal dalam persaingan dunia kerja. Perkembangan industri baterai yang cukup baik saat ini tentunya akan membuka lapangan kerja yang besar. Siswa dengan keahlian tersebut memiliki peluang yang besar akan bekerja sesuai dengan bidangnya. Namun materi/pengetahuan mengenai teknologi di industri baterai khususnya produksi material aktif belum diberikan di SMK Muhammadiyah 4 Surakarta. Dengan pengenalan suatu proses di industri, siswa akan mempelajari spesifikasi bahan baku, tahapan proses untuk menghasilkan suatu produk, jenis-jenis alat proses, dan cara kerja alat proses. Dengan kompetensi dosen dalam teknologi produksi material aktif baterai pada skala mini plant dan fasilitas yang mewadai di Pusat Unggulan IPTEK (PUI) PT Teknologi Penyimpanan Energi Listrik, maka sangat memungkinkan dosen untuk mengenalkan teknologi dan fasilitas baterai kepada siswa SMK Muhammadiyah 4 Surakarta. Selain itu, melalui kegiatan ini juga dapat digunakan sebagai sarana untuk mengenalkan hasil riset di perguruan tinggi kepada masyarakat, khususnya siswa SMK Muhammadiyah 4 Surakarta. Oleh karena itu, penyampaian materi mengenai teknologi baterai sangat dibutuhkan oleh SMK Muhammadiyah 4 Surakarta agar dapat menyesuaikan dengan perkembangan teknologi dan mencetak siswa yang lulus SMK memiliki kompetensi sesuai dengan kebutuhan dunia kerja. 2. BAHAN DAN METODE Metode pelaksanaan pengabdian masyarakat yang diusulkan dibagi menjadi tiga tahapan [5] yang diuraikan sebagai berikut ini: 2.2. Tahapan Pelaksanaan Pertanyaan yang diberikan terdiri dari 6 soal pilihan ganda dan 2 soal uraian yang terlampir pada supplementary section. 2.1. Tahapan Persiapan Pada tahapan persiapan ini dilakukan dengan melakukan survei ke tempat mitra (SMK Muhammadiyah 4 Surakarta). Dari hasil survei, tim pengabdian masyarakat menganalisa kondisi di tempat mitra. Selanjutnya, tim pengabdian masyarakat menyampaikan solusi permasalahan kepada mitra. Solusi permasalahan yang diusulkan yaitu dengan diadakan pengenalan kepada siswa mengenai industri baterai. Siswa yang akan mengikuti kegiatan ini yaitu siswa kelas XI. Koordinasi lanjutan dengan mitra perlu dilakukan untuk menentukan jadwal pelaksanaan kegiatan pengabdian masyarakat, materi yang akan disampaikan, tempat yang akan digunakan, dan hal-hal lainnya agar kegiatan pengabdian masyarakat berjalan dengan lancar. Untuk menyusun materi yang akan disampaikan ke siswa, tim pengabdian masyarakat melakukan studi literatur agar diperoleh metode penyampaian materi yang baik dan materi yang berkualitas, sehingga kompetensi siswa akan meningkat. Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 164 Paramitha / Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 2.2. Tahapan Pelaksanaan Pada tahapan pelaksanaan, peserta terdiri dari 11 siswa dan 3 guru. Tim pengabdian masyarakat melakukan persiapan mengenai alat dan bahan yang akan digunakan oleh siswa selama pelaksanaan kegiatan masyarakat, seperti materi presentasi, alat dan bahan yang digunakan untuk membuat material aktif baterai, dan alat pelindung diri di laboratorium. Pada tahap pelaksanaan kegiatan masyarakat dilaksanakan selama satu hari yang terdiri dari : 1) pre-test (digunakan untuk mengetahui kemampuan awal siswa), 2) pengenalan industri baterai (keselamatan kerja di laboratorium, bahan baku material aktif katoda, tahapan proses pembuatan material aktif katoda, dan fabrikasi sel baterai), 3) praktek pembuatan material aktif baterai katoda dan mini plant tour fabrikasi sel baterai di PUI PT Teknologi Penyimpanan Energi Listrik, 4) post-test (digunakan untuk mengetahui peningkatan Pada tahapan pelaksanaan, peserta terdiri dari 11 siswa dan 3 guru. Tim pengabdian masyarakat melakukan persiapan mengenai alat dan bahan yang akan digunakan oleh siswa selama pelaksanaan kegiatan masyarakat, seperti materi presentasi, alat dan bahan yang digunakan untuk membuat material aktif baterai, dan alat pelindung diri di laboratorium. Pada tahap pelaksanaan kegiatan masyarakat dilaksanakan selama satu hari yang terdiri dari 1) t t (di k t k t h i k l i ) 2) l i d t i b t i (k l t persiapan mengenai alat dan bahan yang akan digunakan oleh siswa selama pelaksanaan kegiatan masyarakat, seperti materi presentasi, alat dan bahan yang digunakan untuk membuat material aktif baterai, dan alat pelindung diri di laboratorium. Pada tahap pelaksanaan kegiatan masyarakat dilaksanakan selama satu hari yang terdiri dari : 1) pre-test (digunakan untuk mengetahui kemampuan awal siswa), 2) pengenalan industri baterai (keselamatan kerja di laboratorium, bahan baku material aktif katoda, tahapan proses pembuatan material aktif katoda, dan fabrikasi sel baterai), 3) praktek pembuatan material aktif baterai katoda dan mini plant tour fabrikasi sel baterai di PUI PT Teknologi Penyimpanan Energi Listrik, 4) post-test (digunakan untuk mengetahui peningkatan kompetensi siswa mengenai industri baterai). Pertanyaan yang diberikan terdiri dari 6 soal pilihan ganda dan 2 soal uraian yang terlampir pada supplementary section. : 1) pre-test (digunakan untuk mengetahui kemampuan awal siswa), 2) pengenalan industri baterai (keselamatan kerja di laboratorium, bahan baku material aktif katoda, tahapan proses pembuatan material aktif katoda, dan fabrikasi sel baterai), 3) praktek pembuatan material aktif baterai katoda dan mini plant tour fabrikasi sel baterai di PUI PT Teknologi Penyimpanan Energi Listrik, 4) post-test (digunakan untuk mengetahui peningkatan kompetensi siswa mengenai industri baterai). 2.3. Tahapan Evaluasi Pada tahapan evaluasi dilakukan evaluasi pelaksanaan kegiatan pengabdian masyarakat yang diukur dari ketercapaian nilai kepuasan siswa. Mitra sekolah juga ikut mengevaluasi pelaksanaan kegiatan dalam bentuk saran yang dapat menjadi perbaikan untuk kegiatan selanjutnya. Evaluasi ini dilakukan dengan mengisi kuesioner yang telah disiapkan oleh panitia dengan skala penilaian STS=Sangat Tidak Setuju; TS=Tidak Setuju; S=Setuju; dan SS =Sangat Setuju, yang ditunjukkan pada Tabel 1 berikut: Tabel 1. Kuesioner Kepuasan dan Saran Perbaikan Kegiatan Tabel 1. Kuesioner Kepuasan dan Saran Perbaikan Kegiatan No Pernyataan 1 Materi yang disampaikan sesuai dengan kompetensi yang diharapkan 2 Penyampaian materi mudah dipahami dan sangat jelas 3 Metode ceramah, diskusi, dan praktik sesuai untuk penyampaian materi 4 Cara pemateri menyajikan materi menarik 5 Penyampaian materi sesuai dengan waktu yang disediakan 6 Media pembelajaran mendukung pemahaman (praktik sintesis material aktif dan fabrikasi sel silinder) 7 Penyelenggara kegiatan memberikan pelayanan sesuai dengan kebutuhan 8 Kegiatan dilaksanakan sesuai harapan 9 Kegiatan memberikan manfaat bagi peserta 10 Jika kegiatan ini diselenggarakan kembali, peserta bersedia untuk berpartisipasi 11 Saran dan perbaikan kegiatan selanjutnya ……………………………….. Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 3. HASIL DAN PEMBAHASAN 3.1 Penyampaian Materi mengenai Baterai Ion Litium 3.1 Penyampaian Materi mengenai Baterai Ion Litium Penyampaian materi disampaikan secara luring di PUI-PT Teknologi Penyimpanan Energi Listrik yang ditunjukkan pada Gambar 1. Pada kegiatan ini disampaikan materi meliputi keselamatan kerja di laboratorium, tipe LIB, bentuk-bentuk LIB, komponen penyusun LIB, bahan baku material aktif katoda, tahapan proses pembuatan material aktif katoda, dan fabrikasi sel baterai. Tipe baterai LIB yang digunakan di PUI-PT Teknologi Penyimpanan Energi Listrik adalah tipe 18650 (diameter dan tinggi sel baterai sebesar 18 mm dan 65 mm, berturut- turut). Bentuk-bentuk LIB yaitu cylindrical cell, prismatic cell, coin cell, dan pouch cell. Struktur sel baterai tipe silinder terdiri dari jelly roll (katoda, anoda, dan separator), current connector, dan safety devices. Salah satu material aktif katoda baterai yang banyak digunakan saat ini adalah Lithium Nickel Manganese Cobalt Oxide (LNMC). LNMC terdiri dari beberapa logam yaitu lithium, nikel, mangan, dan kobalt. Tahapan sintesis material aktif katoda dilakukan dengan metode ko-presipitasi. Sedangkan materi mengenai tahapan fabrikasi sel baterai juga disampaikan terkait dengan proses dan alat skala mini plant. 165 165 Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 Paramitha / Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 (a) (b) Gambar 1. Kegiatan Penyampaian Materi Industri Baterai di PUI-PT Teknologi Penyimpanan Energi Listrik (b) (b) (a) Gambar 1. Kegiatan Penyampaian Materi Industri Baterai di PUI-PT Teknologi Penyimpanan Energi Listrik Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 3.2 Praktek Pembuatan Material Aktif Katoda Pada kegiatan ini tim pengabdian mendemonstrasikan setiap tahapan pembuatan material aktif katoda. Selanjutnya siswa dan guru diberikan kesempatan bertanya jika ada yang kurang dipahami. Metode yang umum digunakan untuk mensintesis material katoda NMC ada berbagai macam diantaranya metode ko-presipitasi, sol- gel, molten salt, solid-state, hidrotermal dan lain-lain. Pada praktek pembuatan material aktif katoda ini dipilih metode ko-presipitasi. Keuntungan utama dari metode kopresipitasi ini dapat diringkas sebagai berikut: (1) biaya bahan baku (biasanya logam transisi sulfat) murah; (2) kelarutan tinggi dari bahan baku yang berbeda dengan kelarutan endapan yang sangat rendah; (3) rentang larutan padat yang luas dari endapan logam transisi; dan (4) kontrol morfologi dan distribusi ukuran partikel. Kegiatan proses pembuatan material katoda dengan metode ko presipitasi ditunjukkan pada Gambar 2. Gambar 2. Kegiatan Praktik Pembuatan Material Katoda di PUI-PT Teknologi Penyimpanan Energi Listrik mbar 2. Kegiatan Praktik Pembuatan Material Katoda di PUI-PT Teknologi Penyimpanan Energi Listrik 3.3 Praktek Fabrikasi Sel Baterai (a) (b) Gambar 3. Kegiatan Praktik Fabrikasi Sel Baterai di PUI-PT Teknologi Penyimpanan Energi Listrik (a) (b) di PUI PT Teknologi Pen impanan Energi Listrik (a) (b) ( ) ( ) Gambar 3. Kegiatan Praktik Fabrikasi Sel Baterai di PUI-PT Teknologi Penyimpanan Energi Listrik Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 166 Paramitha / Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 Pada kegiatan ini tim pengabdian mendemonstrasikan fabrikasi sel baterai. Selanjutnya siswa melakukan tahapan praktik fabrikasi sel baterai, sehingga dapat menambah tingkat pemahaman siswa mengenai materi fabrikasi sel baterai. Fabrikasi sel baterai meliputi proses pembuatan material aktif elektroda, pencampuran slurry elektroda (katoda dan anoda), pembuatan lembar elektroda, assembly baterai (penggulang elektroda dan separator, pemasukan dan pengelasan gulungan dengan casing, grooving dan penyambungan gulungan dengan tutup casing, pengisian elektrolit dalam glove box, penutupan casing), dan pengujian baterai. 3.4 Pengukuran Peningkatan Kompetensi Peserta Peningkatan kompetensi peserta dilihat dari hasil penilaian tes (pre-test dan post-test) serta kecakapan peserta pada saat praktik pembuatan material aktif katoda dan fabrikasi sel baterai. Jumlah jawaban benar dan salah pada post-test dan pre-test dari 13 peserta untuk soal pilihan ganda ditunjukkan pada Tabel 2. Tabel 2. Hasil Penilaian untuk Soal Pilihan Ganda Tabel 2. Hasil Penilaian untuk Soal Pilihan Ganda Pre-Test Post-Test Benar Salah Benar Salah Soal 1 8 6 14 0 Soal 2 3 11 14 0 Soal 3 14 0 14 0 Soal 4 2 12 14 0 Soal 5 2 12 9 5 Soal 6 2 12 10 4 Pada Tabel 2, jumlah jawaban yang benar meningkat pada Post-Test dibandingkan dengan Pre-Test. Pada Pre- Test, nilai terbaik yaitu 1 peserta dapat menjawab 4 soal dengan benar dari 6 soal yang diberikan. Sedangkan pada Post-Test, terdapat 4 peserta yang dapat menjawab semuanya dengan benar. Selanjutnya, pada Pre-Test untuk soal uraian mengenai bahan kimia yang digunakan untuk fabrikasi baterai dan proses fabrikasi sel silinder semua peserta tidak mampu menjawab dengan benar. Pada akhir kegiatan, peserta dapat menyebutkan beberapa bahan kimia yang digunakan seperti nikel sulfat, mangan sulfat, cobalt sulfat, litium karbonat, dan lain-lainnya. Berdasarkan analisa jawaban dari peserta di atas, peningkatan jumlah jawaban yang benar ini menunjukkan bahwa pemahaman peserta mengenai industri baterai lebih baik setelah diberikan materi dan dilakukan praktik. Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 PUSTAKA [1] Q. Wang, B. Mao, S. I. Stoliarov, and J. Sun, “A review of lithium ion battery failure mechanisms and fire prevention strategies,” Prog. Energy Combust. Sci., vol. 73, pp. 95–131, 2019, doi: 10.1016/j.pecs.2019.03.002. [1] Q. Wang, B. Mao, S. I. Stoliarov, and J. Sun, “A review of lithium ion battery failure mechanisms and fire prevention strategies,” Prog. Energy Combust. Sci., vol. 73, pp. 95–131, 2019, doi: 10.1016/j.pecs.2019.03.002. j p [2] Y. Chen et al., “A review of lithium-ion battery safety concerns: The issues, strategies, and testing standards,” J. Energy Chem., vol. 59, pp. 83–99, 2021, doi: 10.1016/j.jechem.2020.10.017. [2] Y. 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Zheng, “Controllable synthesis of spinel lithium nickel manganese oxide cathode material with enhanced electrochemical performances through a modified oxalate co-precipitation method,” J. Power Sources, vol. 274, pp. 1180–1187, 2015, doi: 10.1016/j.jpowsour.2014.10.154. [9] T. H. Cho, S. M. Park, M. Yoshio, T. 3.5 Evaluasi Kegiatan Hasil pengisian kuesioner mengenai kepuasan peserta terhadap kegiatan pengabdian masyarakat ini ditunjukkan pada Tabel 3. Berdasarkan data pada Tabel 3, peserta memberikan penilaian pada skala penilaian Sangat Setuju dan Setuju untuk semua penyataan yang diberikan. Hal ini menunjukkan bahwa peserta puas terhadap kegiatan pengabdian masyarakat dari berbagai aspek kegiatan seperti, materi yang disampaikan, metode penyampaian materi, waktu penyampaian materi, media pembelajaran, dan kebermanfaatan kegiatan. Tabel 3. Hasil Kuesioner Kepuasan Peserta Tabel 3. Hasil Kuesioner Kepuasan Peserta Tabel 3. Hasil Kuesioner Kepuasan Peserta No Pernyataan Jumlah Penilaian SS S TS STS 1 Materi yang disampaikan sesuai dengan kompetensi yang diharapkan 5 9 2 Penyampaian materi mudah dipahami dan sangat jelas 9 5 3 Metode ceramah, diskusi, dan praktik sesuai untuk penyampaian materi 6 8 4 Cara pemateri menyajikan materi menarik 7 7 5 Penyampaian materi sesuai dengan waktu yang disediakan 8 6 6 Media pembelajaran mendukung pemahaman (praktik sintesis material aktif dan fabrikasi sel silinder) 10 4 7 Penyelenggara kegiatan memberikan pelayanan sesuai dengan kebutuhan 4 10 8 Kegiatan dilaksanakan sesuai harapan 5 9 9 Kegiatan memberikan manfaat bagi peserta 11 3 10 Jika kegiatan ini diselenggarakan kembali, peserta bersedia untuk berpartisipasi 10 4 Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430 167 Paramitha / Equilibrium Journal of Chemical Engineering 6(2) (2022) 163–168 Selanjutnya mengenai saran dan perbaikan kegiatan yang disampaikan oleh peserta meliputi kegiatan bisa berkelanjutan dan kegiatan praktik diberikan waktu lebih panjang. 4. KESIMPULAN Peningkatan kompetensi siswa SMK Muhammadiyah 4 Surakarta mengenai industri baterai dilakukan melalui 3 rangkaian kegiatan yaitu penyampaian materi tentang baterai ion litium, praktik pembuatan material aktif katoda, dan praktik fabrikasi sel baterai. Kegiatan ini dapat meningkatkan pemahaman siswa dan guru tentang baterai ion litium dan keterampilan dalam membuat material aktif katoda dalam skala laboratorium. Peningkatan kompetensi siswa dan guru ditunjukkan dari peningkatan jumlah soal terjawab dengan benar dari hasil penilaian tes. Oleh karena itu, kegiatan penyampaian materi mengenai teknologi baterai diharapkan meluluskan siswa yang bisa berkompetisi dengan perkembangan teknologi yang semakin cepat. UCAPAN TERIMA KASIH Kegiatan pengabdian masyarakat berjudul “Peningkatan Kompetensi Siswa pada Bidang Industri Material Aktif Baterai di SMK Muhammadiyah 4 Surakarta” telah didanai oleh Universitas Sebelas Maret dengan NON APBN UNS Tahun 2022 dan surat kontrak Nomor: 255/UN27.22/PM.01.01/2022. Penulis juga mengucapkan terima kasih kepada SMK Muhammadiyah 4 Surakarta atas partisipasinya dan kepada PUI-PT Teknologi Penyimpanan Energi Listrik atas fasilitas yang disediakan dalam kegiatan ini. PUSTAKA Hirai, and Y. Hideshima, “Effect of synthesis condition on the structural and electrochemical properties of Li[Ni1/3Mn1/3Co1/3]O2 prepared by carbonate co-precipitation method,” J. Power Sources, vol. 142, no. 1–2, pp. 306–312, 2005, doi: 10.1016/j.jpowsour.2004.10.016. [10] K. M. Shaju, G. V. Subba Rao, and B. V. R. Chowdari, “Performance of layered Li(Ni1/3Co1/3Mn1/3)O2 as cathode for Li-ion batteries,” Electrochim. Acta, vol. 48, no. 2, pp. 145–151, 2002, doi: 10.1016/S0013- 4686(02)00593-5. [11] L. Xu et al., “Carbonate coprecipitation preparation of Li-rich layered oxides using the oxalate anion ligand as high-energy, high-power and durable cathode materials for lithium-ion batteries,” J. Mater. Chem. A, vol. 3, no. 42, pp. 21219–21226, 2015, doi: 10.1039/c5ta04157k. 168 Equilibrium Journal of Chemical Engineering, e-ISSN 2622-3430
https://openalex.org/W2556272690	https://repository.naturalis.nl/pub/614732/CTZ85-3_Njunji___337-359.pdf	English	null	The cave beetle genus Anthroherpon is polyphyletic; molecular phylogenetics and description of Graciliella n. gen. (Leiodidae, Leptodirini)	Contributions to zoology	2,016	cc-by	12,500	The cave beetle genus Anthroherpon is polyphyletic; molecular phylogenetics and description of Graciliella n. gen. (Leiodidae, Leptodirini) The cave beetle genus Anthroherpon is polyphyletic; molecular phylogenetics and description of Graciliella n. gen. (Leiodidae, Leptodirini) Iva Njunjić1, 2, 3, 7, Michel Perreau4, Kasper Hendriks3, 5, Menno Schilthuizen3, 6, Louis Deharveng2 1 Department of Biology and Ecology, University of Novi Sad, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia 2 Muséum National d’Histoire Naturelle, 45 Rue Buffon - CP50, 75005 Paris, France 3 Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands 4 IUT Paris Diderot, Université Paris Diderot, Sorbonne Paris Cité, 5, rue Thomas Mann, 75205, Paris cedex 13, France 5 Groningen Institute for Evolutionary Life Sciences, University of Groningen, Nijenborg 7, 9747 AG Groningen, The Netherlands 6 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Malaysia 7E mail: iva njunjic@dbe uns ac rs Iva Njunjić1, 2, 3, 7, Michel Perreau4, Kasper Hendriks3, 5, Menno Schilthuizen3, 6, Louis Deharveng2 1 Department of Biology and Ecology, University of Novi Sad, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia 2 Muséum National d’Histoire Naturelle, 45 Rue Buffon - CP50, 75005 Paris, France 3 Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands 4 IUT Paris Diderot, Université Paris Diderot, Sorbonne Paris Cité, 5, rue Thomas Mann, 75205, Paris cedex 13, France 5 Groningen Institute for Evolutionary Life Sciences, University of Groningen, Nijenborg 7, 9747 AG Groningen, The Netherlands 6 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Malaysia 7E mail: iva njunjic@dbe uns ac rs 7 E-mail: iva.njunjic@dbe.uns.ac.rs Key words: Coleoptera, convergent evolution, Dinaric Mountains, morphometrics, troglobites DNA extraction, PCR amplification, and sequencing ... 339 Phylogenetic analyses ............................................................ 340 Morphometric analysis .......................................................... 340 Results and Discussion ................................................................. 341 Acknowledgements ....................................................................... 344 References ....................................................................................... 345 Appendix ......................................................................................... 348 DNA extraction, PCR amplification, and sequencing ... 339 Phylogenetic analyses ............................................................ 340 Morphometric analysis .......................................................... 340 Results and Discussion ................................................................. 341 Acknowledgements ....................................................................... 344 References ....................................................................................... 345 Appendix ......................................................................................... 348 Abstract The subtribe Anthroherponina form an iconic group of obligate cave beetles, typical representatives of the Dinaric subterranean fauna, which is considered to be the richest in the world. Phylo- genetic studies within this subtribe are scarce and based only on morphological characters, which, due to troglomorphic conver- gence, are frequently unreliable. Moreover, morphological stasis and morphological polymorphism make classification of taxa difficult. To test if characters that have traditionally been ac- cepted as informative for Anthroherponina classification are indeed reliable, we evaluated the monophyly of the most species- rich genus of this subtribe - Anthroherpon Reitter, 1889. Our study, based on a molecular phylogenetic analysis of fragments of the 18S, 28S, and COI (both 5’ and 3’ end) loci revealed that the genus Anthroherpon as conventionally defined is polyphyl- etic. To resolve this polyphyly, we defined one new additional genus, Graciliella n. gen., for which we then examined the intra- generic diversity using molecular and morphometric approach- es. Molecular phylogenetic analysis of two COI mitochondrial gene fragments revealed the presence of four species inside Graciliella n. gen., including two new species, which we here describe as G. kosovaci n. sp. and G. ozimeci n. sp. To analyze interspecific morphological differences within Graciliella we performed a discriminant analysis based on 40 linear morpho- metric measurements. The results showed that differences be- tween species and subspecies inside Graciliella, however subtle they may seem, are measurable and reproducible. All species of the genus are briefly diagnosed, an identification key is proposed and a distribution map of all taxa of Graciliella is provided. Contributions to Zoology, 85 (3) 337-359 (2016) Contributions to Zoology, 85 (3) 337-359 (2016) Introduction Unrelated organisms that are placed under similar, severe selection pressures often converge on (or evolu- tionarily remain trapped in) a limited set of body shapes, and may therefore be hard to classify morpho- logically. Extreme environments (e.g. subterranean habitats, deep sea, polar regions, deserts) exert strong directional selection upon their inhabitants, leading to convergent and generally predictable outcomes (Cloudsley-Thompson, 1988; Trontelj et al., 2012). As a consequence, extremophiles found in Arctic tundra (Grundt et al., 2006), subterranean habitats (Lefébure et al., 2006) and deep-sea environments (Vrijenhoek et al., 1994) are well-known for their morphological uniformity and cryptic diversity. Subterranean environments are considered to be ex- treme because of highly stable abiotic conditions and low nutrient availability. Species that have colonized these habitats evolve a somewhat predictable suite of morphological, physiological and behavioural charac- teristics (Gross, 2012; Trontelj et al., 2012), including eye degeneration, depigmentation, development of sen- sory organs, longer life cycles, lower metabolic rate, Contents Introduction .................................................................................... 337 Material and methods ................................................................... 338 Acronyms ................................................................................... 338 Taxon sampling and morphological study ........................ 339 Introduction .................................................................................... 337 Material and methods ................................................................... 338 Acronyms ................................................................................... 338 Taxon sampling and morphological study ........................ 339 Njunjić et al. – The genus Anthroherpon is polyphyletic 338 and body shape modifications (Racovitza, 1907; Van- del, 1964; Culver et al., 1990; Faille et al., 2009). This set of characteristics is known as ‘troglobiomorphy’ or ‘troglomorphy’ and organisms characterized by these features are often restricted to subterranean environ- ment (troglobites). prises taxa of exclusively leptodiroid morphological type, showing the most pronounced troglomorphic characters among Leptodirini (Njunjić et al., 2015). To date, taxonomic studies of Anthroherponina were based exclusively on morphological traits, but possible phenotypic convergence leaves these conclusions doubt- ful. g The troglomorphic phenotype shared by diverse cave-adapted animals is often regarded as a classical example of convergent evolution (Stemmer, 2015). Many studies have shown troglomorphic convergence in different taxa (Christiansen, 1961; Hedin and Thom- as, 2010; Protas et al., 2006), which may complicate phylogenetic reconstruction by morphological ap- proaches. Molecular techniques have become a crucial tool to overcome this problem and to efficiently reveal phylogenetic relationships among different taxonomic categories. They have been recently involved in eluci- dating the phylogeny of different groups with subter- ranean representatives such as Bivalvia (Stepien et al., 2001), Dytiscidae (Leys et al., 2003), Decapoda (Zak šek et al., 2007), Parabathynellidae (Abrams et al., 2012), Trechini (Faille et al., 2009; 2013) and Leptodi- rini (Caccone and Sbordoni, 2001; Ribera et al., 2010; Fresneda et al., 2011; Cieslak et al., 2014a; 2014b). Molecular techniques have helped in distinguishing morphological characters inherited from a common ancestor from those resulting from recent, independent adaptation to a specific biotope. To test if characters that have traditionally been ac- cepted as indicative of Anthroherponina classification are indeed reliable, we evaluated the monophyly of the most species-rich genus of this subtribe - Anthroher- pon Reitter, 1889. Taxonomy of this genus has always been difficult. Most taxa were described in the late 19th century and in the second half of the 20th century, on a small number of specimens and without an explicit diagnosis, so many were subsequently synonymized. Moreover, the geographical distribution of some taxa is mysteriously disjunct (Njunjić et al., 2015). Contents i The present work provides a first phylogenetic anal- ysis of three closely related genera of the subtribe Anthroherponina: Anthroherpon, Leptomeson Jeannel, 1924, and Hadesia Müller, 1911, combining morpho- logical and molecular approaches. We clarify their ge- neric status and we reveal the existence of the fourth, new genus, Graciliella, for which we then examine the intrageneric diversity. Additionally, two new species belonging to this new genus are described. Among Insecta, Coleoptera are the dominant group that have colonized subterranean habitats (Sket, 2005). Two groups of Coleoptera are particularly diverse in caves: the subfamily Cholevinae in the suborder Poly- phaga, and the subfamily Trechinae in the suborder Adephaga (Casale et al., 1998; Faille et al., 2009). With more than 230 genera and 900 (mostly polytypic) species, the tribe Leptodirini (Cholevinae) forms one of the most species-rich tribes of cave-adapted Co- leoptera, surpassed only by Trechini (Carabidae). Most are subterranean, living in caves and fissures, except a few species inhabiting underground termite nests or superficial litter layers. Within the Leptodirini, four morphological types are recognized: bathyscioid, pho- leuonoid, scaphoid, and leptodiroid (Jeannel, 1924). The leptodiroid morphological type, named after Leptodi- rus hochenwartii Schmidt, 1832 from Slovenia, Croa- tia, and Italy, is the most derived morphologically, compared to the presumed ancestral body plan (Sket, 2005). Species belonging to this morphological type have extremely long appendages, extremely elongated head and pronotum, hemispherical elytra, and are ful- ly anophthalmic. The subtribe Anthroherponina com- Acronyms The following abbreviations for collections and insti- tutions are used: CNHM: Croatian Natural History Museum, Zagreb, Croatia; MNHN: Muséum National d’Histoire Naturelle, Paris, France; Naturalis: Naturalis Biodiversity Center, Leiden, The Netherlands; NMP: Narodni Museum, Prague, Czech Republic; NHM: Naturhistorisches Museum Wien; CINJ: Iva Njunjić Collection, Belgrade, Serbia; CMPR: Michel Perreau Collection, Paris, France; CDPV: Dragan Pavićević Collection, Belgrade, Serbia. The following abbreviations for morphological de- tails are used: HL: head length (measured from the posterior margin of the clypeus); HW: maximum width of the head; PL: pronotum length (measured along the median line); Pwmax: maximum width of the prono- tum (largest transverse width); Pwmin: minimum width of the pronotum (shortest transverse width), PL/ Pwmax: length of the pronotum divided by maximum Contributions to Zoology, 85 (3) – 2016 339 need to be resolved among them. As outgroups we chose several subterranean species or subspecies from other genera of the tribe Leptodirini: Parapropus seri- ceus muelleri Jeannel, 1924, Charonites Apfelbeck, 1907 sp., and Apholeuonus nudus sturanyi Apfelbeck, 1906 of which we sequenced a single individual per population. To root the tree, we used Speonesiotes Jean- nel, 1910 sp. belonging to the subtribe Bathysciotina. In total we sampled 36 specimens of 18 species of Anthro- herpon, 4 specimens of 4 species of Hadesia, and 4 specimens of 4 species of Leptomeson (Table 1; S1). width of the pronotum; MPl: mesothoracic pedunculus length, MPwmin: minimal width of the mesothoracic pedunculus; PL/MPl: length of the pronotum divided by the length of mesothoracic pedunculus; EL: elytra length (as linear distance measured along the median line from the base to the apex); EW: maximum width of the elytra; EL/EW: length of the elytra divided by maximum width of the elytra; MP2/MP3: length of the second maxillary palp divided by the length of the third maxillary palp. Reference to type material is ab- breviated as HT: holotype. After extraction, male genitalia were cleared in clove oil, mounted in Euparal on glass slides and pinned beneath the specimens. The external morphol- ogy of specimens was examined using Leica MZ 75 and Leica M10 stereomicroscopes. Microphotographs were taken on a Leica DIAPLAN or a Zeiss AXIO- LAB microscope with a camera diagnostic instrument Spot INSIGHT IN1820. Macrophotographs were taken using a Canon Eos 70D camera with Canon EF MP-E 65mm f/2.8 macro lens and Canon macro twin lite mt-24ex flash. Acronyms Digital pictures were processed using the Helicon Focus software 5.3 (http://www.helicon- soft.com/heliconsoft-products/helicon-focus/). Taxon sampling and morphological study Specimens were collected in caves of the Dinaric range, in Montenegro, Bosnia and Herzegovina, and Croatia as listed in S1. For amplification and sequenc- ing we used two specimens per population of the genus Graciliella n. gen. and Anthroherpon, and one speci- men per population of the genus Hadesia Müller, 1911. Since specimens from the same population had almost identical 28S, 18S and COI gene fragments, we used only one individual per population for the phylogenetic tree. A total of 44 specimens representing 27 taxa from three genera (Anthroherpon, Hadesia, and Leptome- son) were included in the molecular analysis. We chose these genera because they are the most species-rich Anthroherponina and because major taxonomic issues DNA extraction, PCR amplification, and sequencing The specimens used in the study were collected alive in the field and preserved in 96% ethanol. DNA was extracted from whole specimens or from one leg with a standard phenol-chloroform extraction (Blin and Staf ford, 1976) or the DNeasy Tissue Kit (Qiagen GmbH, Hilden, Germany). Voucher specimens are stored in the MNHN (Paris, France), CNHM (Zagreb, Croatia) and DNA aliquots are kept in the tissue collections of Naturalis (Leiden, Netherlands).i Table 1. Genera with total number of species and number of spe- cies included in the study. Taxonomy follows Perreau (2004), updated. Genus N. spp. Sampled spp. Anthroherpon 30 18 Hadesia 4 4 Leptomeson 9 4 Table 1. Genera with total number of species and number of spe- cies included in the study. Taxonomy follows Perreau (2004), updated. We amplified fragments of two nuclear genes: 5’ end of the small ribosomal unit, 18S rRNA (SSU) and Table 2. Primers used in the study. Fragment Name Sense Sequence 5’-3’ Reference COIa LCOI-1490 F GGTCAACAAATCATAAAGATATTG Folmer et al. (1994) COIa HCOI-2198 R TAAACTTCAGGGTGACCAAAAAATCA Folmer et al. (1994) COIb Jerry F CAACATTTATTTTGATTTTTTGG Simon et al. (1994) COIb Pat R TCCAATGCACTAATCTGCCATATTA Simon et al. (1994) 18S 5’ F GACAACCTGGTTGATCCTGCCAGT Shull et al. (2001) 18S b5.0 R TAACCGCAACAACTTTAAT Shull et al. (2001) 28S Ka F ACACGGACCAAGGAGTCTAGCATG Ribera et al. (2010) 28S Kb R CGTCCTGCTGTCTTAAGTTAC Ribera et al. (2010) Table 2. Primers used in the study. 340 Njunjić et al. – The genus Anthroherpon is polyphyletic an internal fragment of the large ribosomal unit, 28S rRNA (LSU), and of two non-overlapping sections of mitochondrial gene fragments–the 5’ and 3’ halves of cytochrome c oxidase subunit 1 (which we here term COIa and COIb, respectively). Primers used are given in Table 2, and PCR protocols are given in S2. Se- quences were assembled and edited using Geneious version 8.0.5 (http://www.geneious.com/, Kearse et al., 2012). DNA sequences obtained for each genetic marker were aligned separately using MAFFT version 7 (Katoh and Standley, 2013). Sequences have been deposited in GenBank with Acc. Nos (S1). did two separate phylogenetic analyses - one at the ge- nus level, using all three loci, and one at the species level within Graciliella using only COI. Phylogenetic analysis was performed using MrBayes 3.2.2 (Ron- quist and Huelsenbeck, 2003) on CIPRES (Miller et al., 2010), with settings for multiple markers unlinked, running two replicates of 15 × 106 generations each. DNA extraction, PCR amplification, and sequencing Sample frequency was set to 5000. Convergence diag- nostics were run using Tracer version 1.5 (Rambaut et al., 2014), where ESS values for all parameters were >>200. After discarding a 25% burn-in, the resulting majority-rule consensus tree was visualized using FigTree version 1.4 (Rambaut, 2012). Morphometric analysis For each sequence alignment, the optimal model of nucleotide substitution was determined using jModel- Test2 (Darriba et al., 2012; Guindon and Gascuel, 2003), run on the CIPRES webportal (Miller et al., 2010). Selection was based on the Akaike Information Criterion (AIC), and resulted in selection of TIM2ef+I, GTR+G, TPM1uf+I+G and TPM3uf+I+G substitution models for 18S, 28S, COIa and COIb, respectively. We We subjected a total of 41 individuals, all males, be- longing to G. apfelbecki apfelbecki (14 specimens), G. apfelbecki scutulatum (1 specimen), G. metohiensis (14 specimens), G. absoloni (1 specimen), G. lahneri (2 specimens), G. ozimeci (9 specimens), and G. koso- vaci (1 specimen) to linear morphometric measure- ment with a Micro-Vu Vertex 251HC (https://www. Fig 1. Bayesian inference of combined 18S, 28S, COIa, and COIb data sets for Hadesia, Anthroherpon, Leptomeson, and Graciliella. Numbers above nodes are posterior probabilities. Fig 1. Bayesian inference of combined 18S, 28S, COIa, and COIb data sets for Hadesia, Anthroherpon, Leptomeson, and Graciliella. Numbers above nodes are posterior probabilities. 341 Contributions to Zoology, 85 (3) – 2016 groups: H. vasiceki + H. lakotai and H. asamo + H. weiratheri as sister groups at the same level of boot- strap value (Perreau and Pavićević, 2008). Our phylo- genetic tree based on molecular data has a different topology – H. asamo and H. vasiceki are early-branch- ing and H. lakotai and H. weiratheri are forming a clade. These results are surprising regarding the distri- bution of the species. Namely, H. asamo and H. weiratheri are both located in the Orjen massif in Montenegro while H. weiratheri and H. lakotai were found in remote massifs, in caves which are more than 50 km apart. Moreover, H. asamo and H. weiratheri are morphologically extremely similar (Perreau and Pavićević, 2008) which can easily mislead phyloge- netic reconstruction by morphological approaches. microvu.com/), three-dimensional set-up, using Inspec Metrology Software (https://www.inspec-inc.com/). We used 40 landmarks on antennae, maxillary palps, head, thorax, abdomen, and legs. Each individual was measured three times. In a small number of cases, ob- vious measurement errors were detected a posteriori (values differing by a factor of >2 from the other two replicates of the same individual). These were then re- placed by the average value of the other two replicates. The full list of measured material and their respective morphometrics is given in S3 and S5; the derivations of the 40 linear measurements are given in S4. Morphometric analysis We first checked, for those species for which we had DNA ex- tracts, whether the species that had been recognized by traditional, qualitative morphological study, could also be recovered as monophyletic groups in the molecular phylogeny (see below). As this appeared to be the case, we then subjected this set of independently obtained quantitative measurements to a Discriminant Analysis in PAST 3.10 (Hammer et al., 2001), to investigate whether a more objective morphometric analysis would allow us to recognize the same groups. y p g pp The genus Leptomeson was originally established as a subgenus of Anthroherpon (Jeannel, 1924) and then subsequently raised to genus rank by Guéorguiev (1990). This separation was based on the following characters of Leptomeson: more developed mesotho- racic pedunculus, with constriction in the middle part and wider in the base, endophallus with differentiated basal ‘armature’ (sclerified stylet-shaped phanera, Giachino et al., 2011) and parameres bearing 4 setae. Further investigations and discoveries of several new species belonging to this genus have shown that num- ber of setae on parameres varies in fact from 3 to 4 (Giachino et al., 2011). The molecular phylogenetic reconstruction is in accordance with the morphology regarding the separation of the genus Leptomeson from Anthroherpon. The clade Graciliella + Leptome- son, in addition to being clearly supported by the mo- lecular analysis, is also morphologically supported by the following characters that are likely synapomor- phies: very elongated pronotum and mesothoracic pe- dunculus, and mesoventrum without processus be- tween mesocoxae. Mesoventral processus is normally entire (it completely separates the two mesocoxal cavities) in Leptodirini, except in some highly evolved groups such as Anthoherponina in which it is reduced or absent (Jeannel, 1911). We observed that the degree of reduction of this processus is a distinguishing char- acter among Anthroherponina: it is present in the genus Anthroherpon, and absent in Leptomeson and Graci liella. However, this character is still insufficiently ex- plored in other Anthroherponina to assess its relevance in non-Anthroherponine Leptodirini. For instance, An- throherpon stenocephalum (Apfelbeck, 1910) is the only species of the genus Anthroherpon in which we noticed intrapopulation variability of this character: some specimens have a mesoventral processus between Results and Discussion Our study, based on morphological and molecular approaches, reveals that the genus Anthroherpon as conventionally defined (Jeannel, 1924; Guéorguiev 1990; Giachino and Gueorguiev, 1993; Giachino and Vailati, 2005; Perreau and Pavićević, 2008) is poly- phyletic (Fig. 1). The genus Leptomeson forms the highly supported sister clade of one polytypic Anthro- herpon species, namely A. apfelbecki sensu lato and the genus Hadesia forms the sister clade of the re- maining Anthroherpon species. To resolve this poly- phyly, we defined one new additional genus, Graci liella, comprising A. apfelbecki sensu lato and one species, A. absoloni (Guéorguiev, 1990), for which we did not have genetic data, but which morphologically closely resembles A. apfelbecki (see below under the genus description). Morphological phylogenetic analy- sis of the species of Hadesia was obtained by Perreau and Pavićević (2008). Their parsimony analysis gave a well-supported monophyletic group for H. vasiceki Müller, 1911 and H. lakotai Perreau and Pavićević, 2008 (bootstrap value 100 %), but the possible sister group Hadesia asamo Perreau and Pavićević, 2008 + H. weiratheri Zariquiey, 1927 was weakly supported (bootstrap value 50 %). However, analysis based on distances as optimality criterion supported both 342 Njunjić et al. – The genus Anthroherpon is polyphyletic Fig. 2. Genital structures of Graciliella and Leptomeson, complete aedeagus + detail of paramere apex: A-B. G. apfelbecki apfelbecki; C-D. G. metohijensis; E-F. G. absoloni; G-H. G. kosovaci sp. n.; I-J. G. ozimeci sp. n.; L-M. G. lahneri; N-O. L. leonhardi; ss. sclerified structure in the endophallus; K. spermatheca of G. ozimeci sp. n. Scale bar is 0.1 mm Fig. 2. Genital structures of Graciliella and Leptomeson, complete aedeagus + detail of paramere apex: A-B. G. apfelbecki apfelbecki; C-D. G. metohijensis; E-F. G. absoloni; G-H. G. kosovaci sp. n.; I-J. G. ozimeci sp. n.; L-M. G. lahneri; N-O. L. leonhardi; ss. sclerified structure in the endophallus; K. spermatheca of G. ozimeci sp. n. Scale bar is 0.1 mm A dorsal comb of very short bristles on the terminal maxillary palpomere is present in three genera that we analyzed in this study (Anthroherpon, Leptomeson, and Graciliella). However, it is absent in Hadesia. We could find no previous literature regarding this charac- ter so that we believe it was not previously observed. Given the scarcity of nutrients in the subterranean the mesocoxae while it is absent in others, but no inter- mediate state was observed. Results and Discussion The monophyly of the ge- nus Leptomeson is morphologically supported by the presence of the sclerotized structures in the endophallus (Fig. 2O), which are absent in Graciliella, and the shape of mesothoracic pedunculus with the constriction in the mid-section or posterior of the mid-section (Fig. 3C). the mesocoxae while it is absent in others, but no inter- mediate state was observed. The monophyly of the ge- nus Leptomeson is morphologically supported by the presence of the sclerotized structures in the endophallus (Fig. 2O), which are absent in Graciliella, and the shape of mesothoracic pedunculus with the constriction in the mid-section or posterior of the mid-section (Fig. 3C). Contributions to Zoology, 85 (3) – 2016 343 Fig. 3. Pronotum and mesothoracic pedunculus: A. Graciliella metohijensis; B. Anthroherpon cylindricolle cylindricolle; C. Leptome- son bujasi; mp. mesothoracic pedunculus. Scale bar is 1 mm. othoracic pedunculus: A. Graciliella metohijensis; B. Anthroherpon cylindricolle cylindricolle; C. Leptome- acic pedunculus. Scale bar is 1 mm. Fig. 3. Pronotum and mesothoracic pedunculus: A. Graciliella metohijensis; B. Anthroherpon cylindricolle cylindricolle; C. Leptome- son bujasi; mp. mesothoracic pedunculus. Scale bar is 1 mm. (Fig. 5B). Based on this independent, initial morpho- metric separation, we then detected additional qualita- tive diagnostic characters in setation, shape of prono- tum, aedeagus, and microsculpture. The diagnoses and identification keys are largely based on these qualita- tive characters, as they are more easily accessible. environment, this comb is possibly an extra sensory organ that has a yet to be determined role in detecting food. Our molecular analysis shows that the four Graci liella species are genetically well-separated, with COI- differences between 3.9 and 12.4%, values that are normally found at or above the species level in Cholevinae (Schilthuizen et al., 2011) (Fig. 4). To con- firm that our mostly genetically circumscribed species indeed correspond with morphologically recognizable units, we carried out a linear discriminant analysis (LDA) based on 40 linear morphometric measure- ments. An LDA is especially suited for maximizing inter-group separation and minimizing intra-group separation for pre-defined units (McLachlan, 2004). This LDA confirmed that the interspecific morpho- logical differences, however subtle and minute as they may seem, are measurable and reproducible. Along the first two axes (with major loadings for tibia lengths, lengths of antennomeres 4 and 11, and elytra length), all species can be distinguished, except the two new species, G. kosovaci and G. ozimeci (Fig. 5A). Fig. 4. Bayesian inference of combined COIa and COIb data sets for Graciliella. Numbers above nodes are posterior probabilities. Results and Discussion These latter two species, however, are well-separated along the third discriminant axis, in which there are addi- tional loadings for the first meso- and metatarsomere Perreau and Pavićević (2008) reconstructed the phy- logeny of the subtribe Anthroherponina based on 11 morphological characters. Their most parsimonious tree shows Leptomeson and Anthroherpon as sister genera, and Hadesia distantly related, as sister genus of Kircheria Giachino and Vailati (2006). The character states that Perreau and Pavićević defined for Anthro- herpon also apply to all species of Graciliella. If we rather accept our molecular phylogeny, in which Gra- ciliella is a sister clade of Leptomeson, and Anthroher- pon s.str. sister clade of Hadesia, this would imply that a large number of morphological characters are strong- ly homoplasious. These characters are: female ventrite VIII with an anterior apophysis versus without, aedea- gus with internal sclerified structure versus without, abdominal ventrites with glabrous plates versus with- out glabrous plates, female first ventrite with lateral hollows versus without, third maxillary palpomere short versus long, claws narrow versus wide. Although 344 Njunjić et al. – The genus Anthroherpon is polyphyletic Fig. 4. Bayesian inference of comb COIa and COIb data sets for Gracili Numbers above nodes are post probabilities. Fig. 4. Bayesian inf COIa and COIb data Numbers above n probabilities. many of these characters seem reliable and have tradi- tionally been accepted as indicative of Anthroherponi- na classification, our results suggest that perhaps this could be attributed to convergence due to shared selec- tive pressures rather than to shared ancestry. Fig. 5. Linear discriminant analysis (LDA) based on 40 linear morphometric measurements. A. Axes 1 and 2; B. Axes 1 and 3. References Giachino PM, Bregović P, Jalžić B. 2011. Five new species of the genus Leptomeson Jeannel, 1924 from Croatia and Bos- nia and Herzegovina (Coleoptera, Cholevidae, Leptodiri- nae). Natura Croatica 20: 355-374. Abrams MK, Guzik TM, Cooper BJS, Humphreys FW, King AR, Cho LJ, Austin DA. 2012. What lies beneath: Molecular phylogenetics and ancestral state reconstruction of the an- cient subterranean Australian Parabathynellidae (Syncarida, Crustacea). Molecular Phylogenetics and Evolution 64: 130-144. doi: 10.1016/j.ympev.2012.03.010 Giachino PM, Guéorguiev VB. 1993. Note sul genere Antroher- pon Reitter (Coleoptera Cholevidae Leptodirinae). Annali del Museo civico di Storia naturale di Brescia 28: 287-329. Apfelbeck V. 1911. Contributiones ad Coleopterum faunam peninsulae balcanicae. Glasnik Zemaljskog Muzeja u Bosni i Hercegovini 23: 209-223. Giachino PM, Vailati D. 2005. 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Christiansen KA. 1961. Convergence and parallelism in cave Entomobryinae. Evolution 15: 288-301. Guéorguiev VB. 1990. Recherches sur les Bathysciinae (Co- leoptera: Catopidae) de Yougoslavie. I. Antroherponini. Acta Entomologica Musei nationalis Pragae 42: 37-273. Cieslak A, Fresneda J, Ribera I. 2014a. Life-history specializa- tion was not an evolutionary dead-end in Pyrenean cave bee- tles. Proceedings of the Royal Society B 281: 20132978. http://dx.doi.org/10.1098/rspb.2013.2978 Guindon S, Gascuel O. 2003. A simple, fast, and accurate algo- rithm to estimate large phylogenies by maximum likeli- hood. Systematic Biology 52: 696-704. doi: 10.1080/1063515 0390235520 Cieslak A, Fresneda J, Ribera I. 2014b. Developmental con- straints in cave beetles. Biology letters 10: 20140712. http:// dx.doi.org/10.1098/rsbl.2014.0712 Hajna ZN. 2012. Dinaric karst: Geography and geology. Pp. 195-203 in: Culver DC, White WB, eds, Encyclopedia of Caves. 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FigTree v1.4. http://tree.bio.ed.ac.uk/software/ figtree/ Published online: 30 August 2016 Editor: J.C. Biesmeijer Editor: J.C. Biesmeijer 347 Contributions to Zoology, 85 (3) – 2016 Type species: Graciliella apfelbecki apfelbecki (Mül- ler, 1910) Type species: Graciliella apfelbecki apfelbecki (Mül- ler, 1910) Description. With a body length from 7.09 to 8.63 mm (HT 8.33 mm), this new genus is one of the largest- bodied Leptodirini known from the Dinaric Moun- tains. General morphology leptodiroid; pronotum and mesothoracic pedunculus very elongated, elytra phy- sogastric; eyeless, wingless, and depigmented. Mesothoracic pedunculus. Elongated, PL/MPl: 4-5.3 mm (HT 5.06); slightly wider (minimal width) than long or as long as wide; minimal width in the anterior part; MPl: 0.31-0.48 mm (HT 0.43), MPwmin: 0.42-0.55 mm (HT 0.51). Ecusson (scutum+scutellum; Jeannel, 1911) rough, ‘U’-shaped and with microsculp- tural mesh resembling honeycomb pattern. Head. Elongated and hypognathous, wider in the anterior part and wider than the pronotum, HL 1.33- 1.58 mm (HT 1.55 mm), HW 0.75-0.93 mm (HT 0.83 mm). Dorsal surface shiny or matte, with superficial microreticulation visible at 40 x magnification under a stereomicroscope and with sparse and short hairs. Few random punctures present on the dorsal surface of the head and most numerous on the frons. Dorsal side of the occiput bearing two patches of punctures with very short hairs on both sides of the head. Labrum trape- zoid with setae longer than on the frons. Maxillae long, both galea and lacinia elongated and with brushes of setae. Maxillary palps with 4 articles; second maxil- lary palpomere arched and about the same length as the third one; terminal maxillary palpomere is the shortest and conical in shape; mean MP2/MP3: 1.33. The base of the terminal maxillary palpomere with a dorsal comb of very short bristles (Fig. 6). Labium with trapezoid submentum bearing two long setae. Mandibles angulate in dorsal view, with sharp apex and with 3 or 4 small teeth between the apex and the last big tooth. Antennal insertions are located in the posterior quarter of the length of the head. Antennae Elytra. Elliptic, physogastric, and wide; maximum width in the anterior part of the mid-section, at the mid-section, or slightly posterior of the mid-section, EL: 3.48-4.54 mm (HT 4.16), EW: 2.19-3.42 (HT 2.78). Surface smooth, shiny or matte, generally with no mi- croreticulation except in some specimens of Gracili- ella ozimeci sp. n. Punctuation superficial, spaced, and irregular; if present, hairs are sparse, short or long. Abdomen. Transversal carina on the posterior margin of mesoventrum non-interrupted, without mesoventral processus; mesocoxal cavities widely confluent (Fig. 8B). Appendix longer than the body, second antennomere is the short- est and last one is longer than the penultimate in both sexes. All antennomeres are covered with hairs of me- dium length, but only antennomeres 7-11 bear up to 8 long, erected setae arranged circularly and symmetri- cally in apical part (Fig. 7A). Three last antennomeres (9, 10, 11) bear very short and thin hairs inserted be- tween the hairs of medium length and long apical se- tae. These short, thin hairs are the most numerous on the last antennomere where they form dense covering (Fig. 7B). Systematic part Above, where we discuss the results of molecular phy- logenetics, we show that the genus Anthroherpon is polyphyletic. For this reason we erect a new genus, Graciliella. In this systematic section, we describe and diagnose this genus and its constituent species. For full treatment of molecular phylogenetics we refer to the Results section. http://zoobank.org/urn:lsid:zoobank.org:pub: 0A49048 5-E40F-4422-8D34-D78A92716FB0 Pronotum. Distinctly longer than wide, narrow, and with a strong constriction in the posterior quarter. Maximal width is in the second third of its length; Pw- max: 0.6-0.8 mm (HT 0.72 mm); Pwmin: 0.3-0.45 mm (HT 0.36); PL: 1.77-2.27 mm (HT 2.18). Sides diver- gent in the anterior part of the pronotum, before its maximal width and convergent in the posterior quarter of the length of the pronotum. Surface shiny or matte, with evident microreticulation visible at 40 x magnifi- cation under a stereomicroscope. Without or with few very short hairs, mostly in the posterior part. Online supplementary information Sequenced specimens, with depository, locality, collectors, and sequence accession numbers. S1. Sequenced specimens, with depository, locality, collectors, and sequence accession numbers. S2. PCR cycling conditions. S1. Sequenced specimens, with depository, locality, collectors, and sequence accession numbers. S3. The list of material included in the morphometric analysis (Sheet 1) and list of measured traits (Sheet 2). S4. The derivations of the 40 linear measurements. S5. Landmarks recorded on the body of Graciliella spp. S5. Landmarks recorded on the body of Graciliella spp. Njunjić et al. – The genus Anthroherpon is polyphyletic Njunjić et al. – The genus Anthroherpon is polyphyletic 348 Type species: Graciliella apfelbecki apfelbecki (Mül- ler, 1910) Abdominal ventrites densely covered with short hairs except in the anterior part where they are smooth. Legs. Long, slender, and pubescent. Femora en- larged in the basal parts, tibiae straight or slightly curved inwards (protibiae). Male protarsi with five protarsomeres, female protarsi with four protarsomer- es, mesotarsi and metatarsi with five tarsomeres in both sexes. Tarsi not dilated, tarsal empodium with two long setae. 349 Contributions to Zoology, 85 (3) – 2016 Fig. 6. Terminal maxillary palpomere with a dorsal comb of bristles (c). Fig. 6. Terminal maxillary palpomere with a dorsal comb of bristles (c). Fig. 6. Terminal maxillary palpomere with a dorsal comb of bristles (c). Aedeagus (Fig. 2A-J, L, M). Slender, median lobe with rounded, lanceolate or acute apex, endophallus without ‘sclerified stylet-shaped phanera in the median part’ (Giachino et al., 2011) i.e., endophallus without sclerotized structures. Parameres as long as or shorter than the median lobe (setae excluded), bearing three apical setae. Urite IX reduced to a ring, as in most Leptodirini. Graciliella apfelbecki schwienbacheri (Giachino and Vailati, 2005) n. comb. Graciliella apfelbecki sculptifrons (Winkler, 1925) n. comb. Graciliella apfelbecki schwienbacheri (Giachino a Vailati, 2005) n. comb. Graciliella apfelbecki sculptifrons (Winkler, 1925) n. comb. Graciliella lahneri (Matcha, 1916) n. comb., n. stat Graciliella metohijensis (Zariquiey, 1927) n. comb n. stat Graciliella kosovaci sp n Graciliella lahneri (Matcha, 1916) n. comb., n. stat Graciliella metohijensis (Zariquiey, 1927) n. comb., n. stat Graciliella lahneri (Matcha, 1916) n. comb., n. stat Graciliella metohijensis (Zariquiey, 1927) n. comb., n. stat Graciliella apfelbecki scutulatum (Giachino and Guéorguiev, 1993) n. comb. Njunjić et al. – The genus Anthroherpon is polyphyletic Njunjić et al. – The genus Anthroherpon is polyphyletic n. stat Graciliella kosovaci sp. n. Female genitalia. Abdominal ventrite VIII with a long and narrow anterior expansion. Appendicular parts (gonocoxites and gonosubcoxites, cf. Deuve, 2001 for terminology) of the urite IX absent. Sper- matheca: weakly sclerified, ‘J’-shaped, short and slightly widened in the apical region (Fig. 2K). Graciliella ozimeci sp. n. After removing these taxa, the ‘hoermanni’ species group comprises: Anthroherpon hoermanni hoerman- ni (Apfelbeck, 1889), A. hoermanni hoffmanni Giachi no and Guéorguiev, 1993, A. hoermanni hypsophilum Apfelbeck, 1907, A. hoermanni orlovacensis, Guéorg uiev, 1990, A. hoermanni sericeum Jeannel, 1930, A. scutariensis Giachino and Guéorguiev, 1993. The genus comprises the following taxa that were pre- viously placed in ‘hoermanni’ species group of the genus Anthroherpon: Cross diagnosis. The new genus can be easily distin- guished from the genus Anthroherpon, even with the naked eye, by the very elongated mesothoracic pedun- culus, narrowest in the anterior part, shape of prono- tum (Fig. 3) and more spherical-shaped elytra. Addi- tional difference is in the mesoventral processus which Graciliella absoloni (Guéorguiev, 1990) n. comb. Graciliella apfelbecki apfelbecki (Müller, 1910) n. comb. Graciliella apfelbecki scutulatum (Giachino and Guéorguiev, 1993) n. comb. Graciliella apfelbecki scutulatum (Giachino and Guéorguiev, 1993) n. comb. 350 Identification key to separate genera is absent in Graciliella and present in Anthroherpon (Fig. 8). It differs from the genus Leptomeson by the following combination of characters: larger body size (from 7.09 to 8.63 mm in Graciliella and from 4.63 to 7.22 mm in Leptomeson), shape of mesothoracic pe- dunculus and elongated male genitalia without scle- rotized structures in the endophallus (Fig. 2A-J, L, M). In Graciliella, the narrowest part of the mesothoracic pedunculus is in the anterior part while in Leptomeson it is in the mid-section or posterior of the mid-section (Fig. 3). The following identification key of the genera of the subtribe Anthroherponina modifies and updates the respective section of the key given by Perreau and Pavićević (2008): 8. Pronotum and mesothoracic pedunculus less elon- gated. Mesothoracic pedunculus short or absent; PL/MPl > 5.3 mm (Fig. 3B). Transversal carina on the posterior margin of mesoventrum interrupted, mesoventrum with long processus between meso- coxae (there is an exception; see: Discussion) (Fig. 8A) ...................................................... Anthroherpon Reitter Distribution. The distribution areas are shown on the map of Fig. 9. Etymology. The new generic name derives from the Latin word ‘gracilis’ which means slender and refers to the habitus of the genus. - Pronotum and mesothoracic pedunculus very elongated. Mesothoracic pedunculus long, always 10 and 11 Fig. 7. Antennomeres: A. antennomeres 7 and 8; B. antennomeres 10 and 11. Fig. 7. Antennomeres: A. antennomeres 7 and 8; B. antennomeres 10 and 11. Fig. 8. Mesoventrum of Anthroherpon and Graciliella: A. Anthroherpon cylindricolle cylindricolle, B. Graciliella metohijensis; mp. mesoventral processus. Fig. 7. Antennomeres: A. antennomeres 7 and 8; B. antennomeres 10 and 11. Fig. 8. Mesoventrum of Anthroherpon and Graciliella: A. Anthroherpon cylindricolle cylindricolle, B. Graciliella metohijensis; mp. mesoventral processus. Fig. 8. Mesoventrum of Anthroherpon and Graciliella: A. Anthroherpon cylindricolle cylindricolle, B. Graciliella metohijensis; mp mesoventral processus Fig. 8. Mesoventrum of Anthroherpon and Graciliella: A. Anthroherpon cylindricolle cylindricolle, B. Graciliella metohijensis; mp. mesoventral processus. 351 Contributions to Zoology, 85 (3) – 2016 (CNHM). Paratype is labeled with white, printed local- ity labels and with red printed labels ‘PARATYPUS Graciliella kosovaci sp. n. I. Njunjić, M. Perreau, K. Hendriks, M. Schilthuizen and L. Deharveng det. 2015’. (CNHM). Paratype is labeled with white, printed local- ity labels and with red printed labels ‘PARATYPUS Graciliella kosovaci sp. n. I. Njunjić, M. Perreau, K. Hendriks, M. Schilthuizen and L. Deharveng det. 2015’. present; PL/MPl < 5.3 mm. Identification key to separate genera Transversal carina on the posterior margin of mesoventrum non-inter- rupted, mesoventrum without processus between mesocoxae ................................................................................. 9 Type locality. Croatia, Popovići, Gruda, Konavle, Kaverna 781, 50 m a.s.l. 9. Mesothoracic pedunculus narrowest in the anterior part (Fig. 3A). Aedeagus slender, without scle- rotized structures in the endophallus, parameres bearing 3 setae, (Fig. 2A-J, L, M), body size from 7.09 to 8.63 mm ................................. Graciliella n. gen. 9. Mesothoracic pedunculus narrowest in the anterior part (Fig. 3A). Aedeagus slender, without scle- rotized structures in the endophallus, parameres bearing 3 setae, (Fig. 2A-J, L, M), body size from 7.09 to 8.63 mm ................................. Graciliella n. gen. Diagnosis. Habitus illustrated in Fig. 10A. Distinct from the other species of the genus by the following characters: small size of the body (7.09-7.36 mm); lat- eral sides of pronotum slightly concave in the anterior part before the maximal width; lateral sides of the mesothoracic pedunculus almost parallel on the ante- rior third of its length; long hairs on the dorsal surface of the elytra; aedeagus small, median lobe with acute apex, parameres strongly arched inwards, of uniform width, bearing very short setae (Fig. 2G, H). - Mesothoracic pedunculus narrowest in the mid- section or posterior of the mid-section (Fig. 3C). Aedeagus robust, with sclerotized structures in the endophallus, parameres bearing 3 or 4 setae, (Fig. 2N,O), body size from 4.63 to 7.22 mm ......................... - Mesothoracic pedunculus narrowest in the mid- section or posterior of the mid-section (Fig. 3C). Aedeagus robust, with sclerotized structures in the endophallus, parameres bearing 3 or 4 setae, (Fig. 2N,O), body size from 4.63 to 7.22 mm ......................... Leptomeson Jeannel Graciliella kosovaci sp. n. urn:lsid:zoobank.org:act:CCB91AA9-E5D4-4DCE- 9070-C5A3A89C2BFB Description. Body length from 7.09 to 7.36 mm (HT 7.09). General morphology leptodiroid; head wid- er than pronotum; eyeless; elytra physogastric; wing- less, and depigmented. Type series. Holotype male labeled as follows: ‘CROA- TIA, Popovići, Gruda, Konavle, Kaverna 781, 50 m, 09.11.2014, leg. R. Ozimec’ (white label, printed)/ ‘HOLOTYPUS Graciliella kosovaci sp. n. I. Njunjić, M. Perreau, K. Hendriks, M. Schilthuizen and L. De- harveng det. 2015’ (red label, printed), (CNHM). Para- type: 1♀, 09.11.2014, same cave, leg. R. Ozimec, Head. hypognathous, wider in the anterior part and wider than the pronotum, HL 1.34-1.39 mm (HT 1.39 mm), HW 0.75-0.78 mm (HT 0.78 mm). Surface smooth and shiny with superficial microreticulation and with sparse and short hairs. Few random punc- tures present on clypeus and along both sides of fron- to-genal sulcus. Fig. 9. Distribution map of the species of Graciliella in relation to the main geo- morphological units of the Dinaric Mountains. Dotted lines separate the three main belts, from south to north: Maritime Belt or Southwestern Belt, the Central Belt or High Dinaric Mountains, and the Northeastern Belt. Fig. 9. Distribution map of the species of Graciliella in relation to the main geo- morphological units of the Dinaric Mountains. Dotted lines separate the three main belts, from south to north: Maritime Belt or Southwestern Belt, the Central Belt or High Dinaric Mountains, and the Northeastern Belt. Njunjić et al. – The genus Anthroherpon is polyphyletic 352 Table 3. Antennal formula of the holotype of Graciliella kosovaci sp. n. Article I II III IV V VI VII VIII IX X XI Length (mm) 0.47 0.30 1.02 0.86 1.21 1.08 1.00 0.79 0.88 0.75 1.03 Table 4. Antennal formula of the holotype of Graciliella ozimeci sp. n. Article I II III IV V VI VII VIII IX X XI Length (mm) 0.51 0.37 1.00 0.92 1.28 1.11 1.07 0.85 0.93 0.76 1.06 Table 3. Antennal formula of the holotype of Graciliella kosovaci sp. n. Table 4. Antennal formula of the holotype of Graciliella ozimeci sp. n. Article I II III IV V VI VII VIII IX X XI Length (mm) 0.51 0.37 1.00 0.92 1.28 1.11 1.07 0.85 0.93 0.76 1.06 Table 4. Antennal formula of the holotype of Graciliella ozimeci sp. n. Pronotum. Narrow and elongated, approximately three times longer than wide, mean PL/Pwmax 3.06 (HT 3.11). Graciliella kosovaci sp. n. urn:lsid:zoobank.org:act:CCB91AA9-E5D4-4DCE- 9070-C5A3A89C2BFB Sides divergent and slightly concave in the anterior part before the maximal width, and conver- gent between the maximal and minimal width of the pronotum. After the minimal width of pronotum, sides are divergent and with rounded posterior margin. Dor- sal surface shiny, with superficial microreticulation, and numerous, random and shallow punctures; hairs are not present. the remains of the freshwater cave-dwelling tube worm Marifugia clavatica observed on the rock indicates its recent hydrological activity (Cvitanović, 2015). Etymology. We are glad to dedicate this interesting species to a dear friend, speleologist Petar Kosovac from Belgrade, in recognition for his enthusiastic help in the field and in collecting material. Graciliella ozimeci sp. n. urn:lsid:zoobank.org:act:37DB2F08-D301-4637-9023 -E948491ABEAA Mesothoracic pedunculus. 1.16 times (HT 1.09) wider than long; lateral sides parallel in the anterior third of the length (measured from the posterior mar- gin of pronotum to the posterior margin of écusson). Ecusson rough, ‘U’-shaped and with microsculptural mesh resembling honeycomb pattern. Type series. Holotype male labeled as follows: ‘CRO- ATIA, Dubrovačko primorje, Točionik, Vranja peć, 230 m, 08.11.2014., leg. R. Ozimec’ (white label, print- ed)/ ‘HOLOTYPUS Graciliella ozimeci sp. n. I. Njun jić, M. Perreau, K. Hendriks, M. Schilthuizen and L. Deharveng det. 2015’ (red label, printed), (CNHM). Paratypes: 27♂ 30♀, 08.11.2014, same cave, leg. R. Ozimec, (CNHM, MNHN, CINJ, CMPR, Naturalis). Paratypes are labeled with white, printed locality la- bels and with red printed labels ‘PARATYPUS Gra- ciliella ozimeci sp. n. I. Njunjić, M. Perreau, K. Hen- driks, M. Schilthuizen and L. Deharveng det. 2015’. Type locality. Croatia, Dubrovačko primorje, Točionik, Vranja peć, 230 m a.s.l. Elytra. Physogastric and thick in lateral view, maxi- mal width slightly posterior of the mid-section; mean EL/EW: 1.45 mm (HT 1.52). Surface smooth and shiny, microreticulation not visible; punctuation sparse and superficial. Hairs, sparse and present mostly on the dorsal surface, very rare laterally; as long as the medi- um-sized hairs on the antennae. Legs. Like in other species of this genus (see genus diagnosis), protibia straight. Aedeagus. Small, median lobe longer than para- meres (setae included) and with acute apex in dorsal view (Fig. 2G, H). Parameres strongly arched inwards, of uniform width and bearing three short apical setae. Diagnosis. Habitus illustrated in Fig. 10B. Distinct from the other species of the genus by the following characters: body size 7.13-7.5 mm; elytra more elon- gated, EL/EW: 1.47-1.64 mm; shorter mesothoracic pedunculus (1.38 times wider than long); surface of the body matte. Aedeagus with rounded apex in dorsal view and almost straight parameres. Distribution. Known only from the type locality (Fig. 9). Kaverna 781 is 142 m long and located in south Croatia, in the tunnel ‘Konavosko polje-Sea’ and is probably a part of the upper floor system of under- ground channels which naturally drain the water of Ko- navle polje to the sea. Lack of stalactite deposits indi- cates that the cave is a relatively young foundation, and Description. Body length from 7.13 to 7.50 mm (HT 7.45). General morphology leptodiroid; head Contributions to Zoology, 85 (3) – 2016 353 Fig. 10. Habitus of Graciliella. A. G. kosovaci sp. Graciliella ozimeci sp. n. urn:lsid:zoobank.org:act:37DB2F08-D301-4637-9023 -E948491ABEAA n.; B. G. ozmeci sp. n. Scale bar is 1 mm (photos by: Theodoor Hijerman). us of Graciliella. A. G. kosovaci sp. n.; B. G. ozmeci sp. n. Scale bar is 1 mm (photos by: Theodoor Hijerman Fig. 10. Habitus of Graciliella. A. G. kosovaci sp. n.; B. G. ozmeci sp. n. Scale bar is 1 mm (photos by: Theodoor Hijerman). Njunjić et al. – The genus Anthroherpon is polyphyletic 354 wider than pronotum; eyeless; elytra physogastric; wingless, and depigmented. Antroherpon Apfelbecki J. Müller: Jeannel, 1911: 560. A t h ( t ) A f lb ki J Müll 1910 J Antroherpon Apfelbecki J. Müller: Jeannel, 1911: 560. Antroherpon (s. str.) Apfelbecki J. Müller, 1910: Jean- nel, 1924: 420. Head. Hypognathous, wider in the anterior part and wider than pronotum, HL 1.35-1.46 mm (HT 1.46 mm), HW 0.75-0.81 mm (HT 0.81 mm). Surface matte, with superficial microreticulation and with sparse and short hairs on clypeus, genae, and frons, particularly along the dorsal side of fronto-genal sulcus. Last an- tennomere is shorter in females. Antroherpon Apfelbecki Müll: Winkler, 1925: 144. Antroherpon (s. str.) Apfelbecki subsp. Apfelbecki Müller: Jeannel, 1930: 147. Antroherpon apfelbecki J. Müller: V.B. Guéorguiev, 1990: 264. Antroherpon apfelbecki apfelbecki J. Müller, 1910: Giachino and Guéorguiev, 1993: 316. Pronotum. Narrow and elongated, mean PL/Pw- max 2.85 (HT 3.01). Sides almost straight in the ante- rior part before the maximal width; posterior margin almost straight. Dorsal surface matte and hairless, with superficial microreticulation, and numerous, ran- dom and shallow punctures. Ventral side of the prono- tum completely smooth behind the first pair of legs. Antroherpon apfelbecki J. Müller: Perreau and Pavi ćević, 2008: 211. Antroherpon Kauti Apfelbeck, 1911: 216. (synonymy in Jeannel, 1914). Type locality: Herzegowina: in der Vjeternica höhle bei Zavala. Antroherpon Kauti Apfelbeck, 1916: 346. Mesothoracic pedunculus. 1.38 times (HT 1.53) wider than long; lateral sides parallel in the anterior quarter of the length (measured from the posterior margin of pronotum to the posterior margin of écus- son). Ecusson rough, ‘U’-shaped and with microsculp- tural mesh resembling honeycomb pattern. Material examined. 1♂: LECTOTYPE (here desig- nated), Hercegovina, ‘Vjeternica H.’, 11.07.1914., leg. K. Absolon, RMNH.INS.627472 (Naturalis); 1♀: PA- RALECTOTYPE (here designated), Herzegovina Vje ternica, 1931, leg. K. Absolon (MNHN); 1♂: COTYPE, Höhle bei Savala, Herzegovina (MNHN); 1♂, 1♀: Her- zegovina Vjeternica, leg. K. Absolon (MNHN); 1♀: Za- vala, Hr., leg. Novak, 5.9.1925 (MNHN); 1♀: Zavala, Herzeg. Graciliella ozimeci sp. n. urn:lsid:zoobank.org:act:37DB2F08-D301-4637-9023 -E948491ABEAA (MNHN); 1♂: Hercegovina, Neretva, Ravno, Zavala, Vjeternica pećina, Popovo polje, 42.88-17.98 03/ VI/2011., leg. M. Perreau, RMHN.INS.550268 (Natura- lis); 1♀: same data, RMNH.INS.550269 (Naturalis); 1♀: Vjeternica pećina, Collectie G. Paganetti-Hummler acq. 1937, ZMA.INS.1236005 (Naturalis); 1♀: same data, ZMA.INS.1236007 (Naturalis); 1♂: Hercegovina, Zava- la, Dr. Grabowski, Coll. Dr. S. Breuning, ZMA. INS.1235988 (Naturalis); 2♀: BiH, Zavala, Vjeternica., leg. R. Ozimec (CNHM); 1♀: same locality, 21.08.2008., leg. Bakšić Darko (CNHM); 2♂: same locality, 20.08.2007., leg. M. Perreau (CMPR); 1♀: Zavala, Popo- vo polje, Vjetrenica, Hercegovina, 12.09.1975., leg. Pret- ner (CMPR); 1♀: Dr. Absolon, Vjeternica (CMPR); 1♂: Vjeternica, Absolon, 1912, Hercegovina (CMPR); 1♀: Kali pecina apud Grebci, Herzegovina, Absolon (CMPR); 3♀: Grabovica bei Grepci, Herzegovina, Abso- lon (CMPR); 3♂ 3♀: Gjurkovina pećina apud Grepci, Herzegovina, Absolon (CMPR); 2♂ 4♀: Močiljska pec, Rjeka-Dubrov., Svircev, 5.1.931. (MNHN); 1♂ 1♀: Močiljska pec. Dubrovnik, Svircev, 1.9.923. (MNHN); 2♂: Croatia, Dubrovačko primorje, Dubrovnik, Osojnik, Močiljska špilja, 12.11.2014., leg. Hanžek Nikola (CNHM); 2♀: same locality, leg., R. Ozimec (CNHM); 1♀: Močiljska pećina, Močilje, Ombla, Dalmatia, leg. E. Elytra. Physogastric but narrow; maximal width slightly posterior of the mid-section; mean EL/EW: 1.47-1.64 mm (HT 1.49). Surface irregular and matte, microreticulation and punctures visible in some speci- mens. Hairs very sparse and present mostly on the dor- sal surface, very rare or absent laterally. Legs. Like in other species of the genus (see genus description), protibia straight. Aedeagus. Median lobe longer than parameres (se- tae excluded) and with rounded apex in dorsal view (Fig. 2I, J). Parameres almost straight, of uniform width and bearing three apical setae. Distribution. Known only from the type locality (Fig. 9). Vranja peć is situated in south Croatia, in the municipality Dubrovačko primorje, close to the village Točionik. It is a cave with a vertical entrance, 188 m long and 50 m deep. The bottom of the channel is cov- ered with rock debris and large blocks of rocks broken off from the ceiling. The cave is hydrologically inac- tive except the small drippings at the bottom (Cvitanović, 2015). Graciliella apfelbecki apfelbecki (Müller, 1910) n. comb. Graciliella apfelbecki apfelbecki (Müller, 1910) n. comb. Antroherpon apfelbecki Müller, 1910: 186. Graciliella apfelbecki apfelbecki (Müller, 1910) n. comb. Antroherpon apfelbecki Müller, 1910: 186. Graciliella apfelbecki sculptifrons (Winkler, 1925) n. comb. Graciliella apfelbecki sculptifrons (Winkler, 1925) n. comb. Antroherpon Apfelbecki sculptifrons Winkler, 1925: 144. Type locality: In einem Hallenschacht bei der Žaba planina südlich von Metković [=Žaba Mt, Golubinka] Antroherpon (s. str.) Apfelbecki subsp. sculptifrons Winkler: Jeannel, 1930: 147. Antroherpon apfelbecki sculptifrons Winkler: Pret- ner, 1974: 11. Material examined: 1♂: TYPE, Žaba plan., Golu binka (NHM). Diagnosis. Habitus illustrated in Fig. 11A. Body length from 7.3 to 8.63 mm. Median lobe with lanceo- late apex in dorsal view and considerably longer than parameres (setae excluded). Parameres of uniform width, slightly curved inwards in the apical part and bearing three long apical setae (Fig. 2A, B). Graciliella metohijensis (Zariquiey, 1927) n. stat., n. comb. Graciliella metohijensis (Zariquiey, 1927) n. stat., n. comb. Antroherpon Apfelbecki subsp. Metohijensis Zari quiey, 1927: 163. Type locality: Höhle 216, Urvol P., Nordostherzeg., Karstlande [= Đatlo pećina, Korita, Bosnia and Herze- govina; N 43˚03.710’ E 18˚29.678’, alt. 1013 m a.s.l.] Antroherpon (s str) Apfelbecki subsp metohiensis Type locality: Höhle 216, Urvol P., Nordostherzeg., Karstlande [= Đatlo pećina, Korita, Bosnia and Herze- Remarks. Two specimens, one in Naturalis and the other one in MHNH were labelled as types, so we des- ignated the male specimen from Naturalis as lectotype and female specimen from MNHN as paralectotype. govina; N 43˚03.710’ E 18˚29.678’, alt. 1013 m a.s.l.] Antroherpon (s. str.) Apfelbecki subsp. metohiensis Zariquiey: Jeannel, 1930: 147. Antroherpon apfelbecki metohijense Zariquiey: We do not have molecular data for the three following subspecies of G. apfelbecki, so we follow the classifi- cation of Giachino and Vailati (2005) on this point. Pretner, 1974: 11. Antroherpon apfelbecki metohijensis Zariquiey: Guéorguiev, 1990: 265. Guéorguiev, 1990: 265. Antroherpon apfelbecki metohijensis Zariquiey, 1928: Giachino and Guéorguiev, 1993, Natura brescia- na: 317. Graciliella apfelbecki scutulatum (Giachino and Guéorguiev, 1993) n. comb. Antroherpon scutulatum Giachino and V.B. Guéor- guiev, 1993: 317. Graciliella apfelbecki scutulatum (Giachino and Guéorguiev, 1993) n. comb. Antroherpon scutulatum Giachino and V.B. Guéor- guiev, 1993: 317. Material examined. 1♂: TYPE, H 216 Urvol p. Weirather, NordostHerzeg. 1922., Karstlande (MNHN); 1♂: BiH, Korita, Kobilja Glava, Đatlo, alt. 1013m, 13.09.2006., leg. M. Perreau (CMPR); 1♀: same local- ity, 05.08.2013., leg. M. Perreau (CMPR); 1♀: same locality, 15.03.2008, leg. S. Ognjenović (CMPR); 1♀: same locality, 13.07.2007., leg. M. Đokić (CMPR); 3♂: same locality, 23.06.2003., leg. M. Perreau (CMPR); 3♂ 3♀: BiH, Korita, Brestica, Jametina, 14.07.2007., leg. M. Perreau (CMPR); 10♂ 8♀: BiH, Balabani, Drvendžina pećina, 26.08.2013., leg. I. Njunjić (CMPR, CINJ); 3♂ 3♀: Montenegro, Golija Mt, Donje Čarađe, Prljača, 07.08.2010., leg. S. Ognjenović (CDPV); 2♂, same locality, 28.08.2010., leg. I. Njunjić (CINJ); 2♀, same locality, 13.08.2010, M. Plećaš (CDPV); 1♂, same locality, same date, leg. M. Perreau (CMPR); 3♀: Montenegro, Kazanci, Bijela pećina, 10.08.2010, leg., M. Perreau; 1♀: Hercegovina, grot Provalija, 22.07.1962., RMNH.INS.627543 (Naturalis); 1♀: same Type locality: Erzegovina, Bravenik [= Hercegovina, Zupci, Bravenik; N 42˚37.143’ E 18˚25.969’, alt. 740 m a.s.l.] Antroherpon apfelbecki scutulatum Giachino and Guéorguiev: Perreau and Pavićević, 2008: 212. Antroherpon apfelbecki scutulatum Giachino and Guéorguiev: Perreau and Pavićević, 2008: 212. Material examined: 1♂ 3♀: BiH, Trebinje, Zupci, Bravenik (= Jama u Braveniku), alt. 740 m a.s.l., 19.06.2003., leg. M. Perreau (CMPR). Antroherpon apfelbecki Müller, 1910: 186. Antroherpon apfelbecki Müller, 1910: 186. Type locality. Höhle zwischen Jasenica und Zavala (Herzegowina) [= pećina Vjetrenica; N 42° 50’ 28.34’’ E 17° 59’ 11.87’’, alt. 268 m] 355 Contributions to Zoology, 85 (3) – 2016 Pretner (CMPR); 1♂: Croatia, Dubrovačko primorje, Dubrovnik, Gromača, Špilja za Gromačkom vlakom, 28.08.2014., leg. Slapnik Rajko (CNHM); 1♀: same lo- cality, 25.09.2014., leg. Slapnik Rajko (CNHM); 1♀: Bosnia and Herzegovina, Bobani, Grabovica, 08.05.2011., leg. R. Ozimec (CNHM); 1♀: Grotte Grabo- vica, Herzegovina Weirather, 1-14 (MNHN); 1♂: Kali pećina, Grebci, Herceg., Svircev, 2.1.931. (MNHN); 1♂: Gr. Močilje, pr. Ragusa, 30.11.921., G. Müller (MNHN); 2♀: Orlica pecina, Popovo polje, Herzegovina, Absolon (CMPR); 1♀: Ravno Hercegov. Vl. Zoufal (CMPR); 1♂: Bihovo, Trebinje, Herz, Collectie G. Paganetti-Hummler acq. 1937, ZMA.INS.1236013 (Naturalis). Pretner (CMPR); 1♂: Croatia, Dubrovačko primorje, Dubrovnik, Gromača, Špilja za Gromačkom vlakom, 28.08.2014., leg. Slapnik Rajko (CNHM); 1♀: same lo- cality, 25.09.2014., leg. Slapnik Rajko (CNHM); 1♀: Bosnia and Herzegovina, Bobani, Grabovica, 08.05.2011., leg. R. Ozimec (CNHM); 1♀: Grotte Grabo- vica, Herzegovina Weirather, 1-14 (MNHN); 1♂: Kali pećina, Grebci, Herceg., Svircev, 2.1.931. (MNHN); 1♂: Gr. Močilje, pr. Ragusa, 30.11.921., G. Müller (MNHN); 2♀: Orlica pecina, Popovo polje, Herzegovina, Absolon (CMPR); 1♀: Ravno Hercegov. Vl. Zoufal (CMPR); 1♂: Bihovo, Trebinje, Herz, Collectie G. Paganetti-Hummler acq. 1937, ZMA.INS.1236013 (Naturalis). Graciliella metohijensis (Zariquiey, 1927) n. stat., n. comb. Material examined: 1♂ 3♀: BiH, Trebinje, Zupci, Bravenik (= Jama u Braveniku), alt. 740 m a.s.l., 19.06.2003., leg. M. Perreau (CMPR). Graciliella apfelbecki schwienbacheri (Giachino and Vailati, 2005) n. comb. Anthroherpon apfelbecki schwienbacheri Giachino and Vailati, 2005: 159. Type locality: Albania, Kelmend, Tamara umg., Shpel- la Siper Rruges, 185m a.s.l. Material examined: We did not examine this sub- species. Type locality: Albania, Kelmend, Tamara umg., Shpel- la Siper Rruges, 185m a.s.l. Material examined: We did not examine this sub- species. 356 Njunjić et al. – The genus Anthroherpon is polyphyletic data, Hercegovina, grot Provalija, 22.07.1962., RMNH. INS.627700 (Naturalis); 1♂ 5♀: Montenegro, Stozi, Stoška pećina, 10.08.2010., leg. I. Njunjić (CINJ); 2♀: same data, leg. S. Ognjenović (CDPV). Antroherpon apfelbecki lahneri Matcha: Guéor- guiev, 1990: 265. Antroherpon apfelbecki lahneri Matcha, 1916: Gia chino and Guéorguiev, 1993: 317. Diagnosis. Habitus illustrated in Fig. 11B. Large spe- cies, body length from 7.76 to 8.53 mm. Distinct from the other species of the genus by the following charac- ters: elytra strongly physogastric and very wide, mean EL/EW: 1.33, with the largest width slightly before the mid-section and dense but shallow punctuation. Sparse hairs are present mostly on the dorsal surface of the elytra. Pronotum with rounded anterior margin. Para- meres shorter than the median lobe (setae excluded), with strong constriction in the preapical region (Fig. 2C, D) in some populations, or without the constric- tion. Median lobe of the aedeagus with lanceolate apex in dorsal view. This combination of characters sup- ports the change of the status from subspecies to spe- cies rank. New status is consistent with the molecular phylogeny, morphometrics and geographic location of this species mostly in the Central Belt of the Dinaric Mountains (Golija Mt., Vojnik Mt., Crvanj Mt., Somina Mt., Korita), rather than in the Southwestern (Mari- time) Belt as most of the other species and subspecies of the genus Graciliella. Material examined. 1♀: TYPE, Lovćen, Geb. Mal. 1916, Montenegro, Rittm. Matcha, Duboki do (MNHN); 1♀: Montenegro, Lovćen Mt, Bjeloši, Jankova jama, 27.06.2013., leg. I. Njunjić (CINJ); 2♂: Montenegro, Virpazar, Trnovo, Grbočica, 30.05.2004., leg. R. Mle- jnek (CMPR); 1♂: Montenegro, Virpazar, Trnovo, Grbočica pećina, cave traps, 21.03-16.07.2008., leg. R. Lohaj, ZMA.INS.1236016 (Naturalis); 1♀: same data, ZMA.INS.1235992 (Naturalis). Diagnosis. Elytra slightly convex on the disc. Median lobe of the aedeagus elongated and narrow, with acute apex. Antroherpon (s. str.) Lahneri Matcha: Jeannel, 1930: 147. Antroherpon (s. str.) Lahneri Matcha, 1916: 420. A h L h i M t h F i 1927 23 Type locality: Montenegro: Lottspeich Grotte bei Njeguši Antroherpon (s. str.) Lahneri Matcha, 1916: 420. Antroherpon Lahneri Matcha: Fagniez, 1927: 23. Antroherpon (s. str.) Lahneri Matcha: Jeannel, 1930: 147. Graciliella metohijensis (Zariquiey, 1927) n. stat., n. comb. Parameres as long as the median lobe, with weak preapical constriction and significantly wider in the apical part. Apex of the parameres acute and bearing three setae (Fig. 2L, M). The distinctive shape of the aedeagus allows the change of the status from subspecies to species rank. New status is con- sistent with the morphometrics and the geographic location of this species in Mountains of Maritime Montenegro. Remarks. There is inter- and intrapopulation vari- ability in the shape of parameres: In some popula- tions (e.g., Drvendžina pećina in Balabani) the para- meres have a strong preapical constriction while in others (e.g., Prljača on Golija Mt., Stoška pećina on Somina Mt.), the parameres are of equal width in full-length. In the population from Drvendžina pećina there is a gradient in this character: from evi- dent preapical constriction to parameres without con- striction. Some specimens from this population have asymmetric aedeagi: one paramere with constriction and other without. Since we have sequences from only two specimens from the population of Drvendžina pećina, we are unable to assess the taxo- nomic significance of this variability. Graciliella absoloni (Guéorguiev, 1990) n. comb. Antroherpon absoloni Guéorguiev, 1990: 265. Type locality: Montenegro, Lipska pećina près de Ce tinje [N 42° 22.433’ E 018° 57.207’, alt. 456 m a.s.l.] Material examined. 1♂: TYPE, Montenegro, Lipska pećina près de Cetinje, 10.VIII 1912., leg. J. Matcha (NMP); 1♀: PARATYPE, same data (NMP). Diagnosis. Body length 7.53 mm (HT). Elongated mesothoracic pedunculus, narrowest in the anterior part, PL/MPl: 4.13 (HT). Pronotum distinctly longer than wide, narrow, and with a strong constriction in the posterior quarter. Maximal width is in the second third of its length; PL: 1.98 mm (HT). Elytra elliptic and physogastric; EL: 3.5 mm (HT). Aedeagus slender, en- dophallus without sclerotized structures, elongated and with lanceolate apex in dorsal view. Parameres shorter than the median lobe, with weak preapical con- striction and slightly wider in the apical part, bearing three setae (Fig. 2E, F). These characters fit in the de- scription of Graciliella which allows us to place this species in this new genus. Graciliella lahneri (Matcha, 1916) n. stat. n. comb Antroherpon Lahneri Matcha, 1916: 63. Type locality: Montenegro: Lottspeich Grotte bei Njeguši Type locality: Montenegro: Lottspeich Grotte bei Njeguši Antroherpon (s. str.) Lahneri Matcha, 1916: 420. Antroherpon Lahneri Matcha: Fagniez, 1927: 23. Contributions to Zoology, 85 (3) – 2016 357 Fig. 11. Habitus of Graciliella. A. Biogeography and ecology 1. Elytra strongly physogastric and very wide, EL/ EW: 1.18-1.43 mm, with the largest width slightly before the middle, depressed and attenuated to- wards the apex ....................... metohijensis (Zariquiey) - Elytra less physogastric and less wide, EL/EW: 1.3- 1.64 mm, with the largest width in the mid-section or slightly in the posterior part of the mid-section, not depressed and not attenuated towards the apex . .......................................................................................................... 2 2. Anterior part of the pronotum ovoid. Parameres as long as the median lobe, significantly wider in the apical part ................................................ lahneri (Matcha) - Anterior part of the pronotum subcylindrical. Para- meres shorter than the median lobe, same width in the full length or slightly wider in the apical part .... .......................................................................................................... 3 3. Protibiae weakly curved inwards. Elytra with rounded apex and with the largest width in the mid- dle. Parameres with weak preapical constriction and slightly wider in the apical part. (6.7-7.1 mm) ... ............................................................ absoloni (Guéorguiev) - Protibiae straight. Elytra with acute apex and with the largest width slightly posterior of the mid-sec- tion. Parameres of uniform width, without constric- tion in the apical part ........................................................... 4 4. Lateral sides of pronotum convex in the anterior part. Median lobe with lanceolate apex in dorsal view and considerably longer than parameres (setae excluded). Parameres of uniform width, slightly curved inwards in the apical part. Body length 7.3- 8.63 mm ............................................... apfelbecki (Müller) - Lateral sides of pronotum concave or almost straight in the anterior part. Median lobe with acute apex or rounded in the apical part, longer than para- meres (setae excluded). Parameres strongly arched inwards or almost straight. Body length 7.09-7.5 mm ................................................................................................. 5 5. Lateral sides of the pronotum concave in the ante- rior part before the maximal width. Dorsal surface of the body smooth and shiny. Long hairs on the 1. Elytra strongly physogastric and very wide, EL/ EW: 1.18-1.43 mm, with the largest width slightly before the middle, depressed and attenuated to- wards the apex ....................... metohijensis (Zariquiey) The genus Graciliella is distributed in subterranean habitats of the Dinaric Mountains, from Crvanj moun- tain (Bosnia and Herzegovina) in the north, to Trnovo (Montenegro) in the south, and from Žaba mountain (Croatia) in the west to Prokletije mountain (Montene- gro) in the east. It is distributed from an altitude of 50 m (G. kosovaci in Croatia, Konavle, Kaverna 781) to 1270 m (G. Identification key dorsal surface of the elytra. Aedeagus with acute apex in dorsal view, parameres strongly arched in- wards, bearing very short setae .......... kosovaci sp.n. Here we present the identification key to the genus Graciliella, excluding the subspecies. It is dedicated to practical identification of species and does not corre- spond to the molecular phylogeny. Although we have used a few characters previously mentioned in the An- throherpon key of Giachino and Vailati (2005), the present key is based chiefly on the diagnoses provided above. - Lateral sides of pronotum almost straight in the an- terior part before the maximal width. Dorsal sur- face of the body matte. Short hairs on the dorsal surface of the elytra. Aedeagus with rounded apex in dorsal view, parameres almost straight, bearing long setae .......................................................... ozimeci sp.n. - Lateral sides of pronotum almost straight in the an- terior part before the maximal width. Dorsal sur- face of the body matte. Short hairs on the dorsal surface of the elytra. Aedeagus with rounded apex in dorsal view, parameres almost straight, bearing long setae .......................................................... ozimeci sp.n. Graciliella metohijensis (Zariquiey, 1927) n. stat., n. comb. G. apfelbecki apfelbecki; B. G. metohijensis. Scale bar is 1 mm (photos by: Theodoor Hijerman) aciliella. A. G. apfelbecki apfelbecki; B. G. metohijensis. Scale bar is 1 mm (photos by: Theodoor Hijerman) Fig. 11. Habitus of Graciliella. A. G. apfelbecki apfelbecki; B. G. metohijensis. Scale bar is 1 mm (photos by Fig. 11. Habitus of Graciliella. A. G. apfelbecki apfelbecki; B. G. metohijensis. Scale bar is 1 mm (photos by: Theodoor Hijerman). 358 Njunjić et al. – The genus Anthroherpon is polyphyletic 5. Lateral sides of the pronotum concave in the ante- rior part before the maximal width. Dorsal surface of the body smooth and shiny. Long hairs on the Biogeography and ecology metohijensis in Montenegro, Somina, Stoška pećina) above sea level. p Figure 9 shows the geographic distribution of the species of the genus Graciliella, in relation to the three main geomorphological units of the Dinaric Moun- tains: the Maritime Belt or Southwestern Belt, the Central Belt or High Dinaric Mountains, and the Northeastern Belt (Hajna, 2012). The genus Graciliel- la is present only in the Maritime and Central Belt. Even though the cave fauna of the Northeastern Belt is fairly well known, Graciliella have never been found in this region. The distribution of G. apfelbecki schwienbacheri far from other apfelbecki subspecies and in the Central Belt rather than in the Maritime Belt of the Dinaric Mountains where other apfelbecki sub- species are distributed indicates that the systematic position of this subspecies should be analyzed using molecular approaches. In the region of Velež Mt. in Bosnia and Herzego- vina all three genera were found – Leptomeson loreki Zoufal, 1904, Anthroherpon ganglbaueri ganglbau- eri (Apfelbeck, 1894) and Graciliella metohijensis (Zariquiey, 1927), but never in syntopy. However, in a few caves in Bosnia and Herzegovina (e.g. Vjeternica in Zavala, Bravenik in Zupci, Veliko Đatlo in Korita) and Croatia (Špilja za Gromačkom Vlakom in Du brovačko primorje), Graciliella and Hadesia coexist in the same cave. The biotope of Hadesia differs in many aspects from most other troglobitic species of Leptodirini – these beetles inhabit a semi-aquatic cave habitat called ‘hygropetric’ – they are always found in the vicinity of strong flows of running water 359 Contributions to Zoology, 85 (3) – 2016 tites and stalagmites with almost no visible siltation. Their diet is understudied but it is assumed that they are saprophagous like many other Cholevinae (Beutel and Leschen, 2016) while Hadesia is considered to filter water or brush the surface of stalagmitic walls with its specialized mouthparts (Perreau and Pavićević, 2008). The different ecological niches in Hadesia and Graciliella may allow coexistence with- out competition. on stalagmitic walls and often inside the film of run- ning water (Jeannel, 1924, Remy, 1940, Sket, 2004, Perreau and Pavićević, 2008). Graciliella, Anthroher- pon and Leptomeson, however, prefer cave walls without water flow and they have never been found inside the water film itself. Also, they have never been observed on deposits of guano and almost never under the rocks on the cave floor. The representatives of these three genera inhabit humid cave walls, stalac-
W2042298143.txt	https://www.jstage.jst.go.jp/article/jea/23/2/23_JE20120229/_pdf	en		New Challenges in Cancer Control in Japan	Journal of epidemiology	2,013	cc-by	514	J Epidemiol 2013;23(2):153-154 doi:10.2188/jea.JE20120229 Health Policy News New Challenges in Cancer Control in Japan Hiroyuki Noda and Manabu Sumi Cancer Control and Health Promotion Division, Health Service Bureau, Ministry of Health, Labour and Welfare, Tokyo, Japan Received December 22, 2012; accepted December 22, 2012; released online February 2, 2013 have radiotherapy equipment, an outpatient chemotherapy center, and a center for cancer-related consultation and support. In addition, the number of prefectures with a regional cancer registry increased from 35 to 47 (ie, to all prefectures). Moreover, 30 000 medical doctors engaged in cancer treatment received training in palliative therapy. In an attempt to add to these improvements, the second term of the basic plan will further improve the quality of cancer control, in both the medical and social contexts, by setting overall goals, namely, decreasing the age-standardized cancer On June 8, 2012 the Japanese Cabinet decided to launch the second term of the “Basic Plan for Promotion of Cancer Measures”.1 The ﬁrst term was established in 2007 and was scheduled to be revised every 5 years under the terms of the Cancer Control Basic Act. During the ﬁrst term of the basic plan (2007–2012), the essential infrastructure for cancer control was successfully improved. The number of designated cancer hospitals increased from 286 to 397, and now 68% of medical districts have such a hospital. All designated cancer hospitals Basic Plan to Promote Cancer Control Programs (approved in June 2012) Figure. Address for correspondence. Hiroyuki Noda, M.D., Ph.D., Cancer Control and Health Promotion Division, Health Service Bureau, Ministry of Health, Labour and Welfare, 1-2-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-8916, Japan (e-mail: noda-hiroyukiaa@mhlw.go.jp). Copyright © 2013 by the Japan Epidemiological Association 153 154 New Challenges in Cancer Control in Japan mortality rate, reducing pain and improving the lives of all cancer patients and their relatives, and creating a society in which people with cancer can live comfortably. To reach these goals, the new basic plan addresses the rapid decrease in the number of surgeons, so as to improve surgical treatment of cancer, and calls for continued improvements in radiotherapy and chemotherapy. The plan also promotes alleviation of pain immediately after a cancer diagnosis and cancer control among infants and people of working age. Cancer survivors often have difﬁculty ﬁnding work and supporting themselves ﬁnancially after successful cancer treatment: 34% of Japanese workers living with cancer had lost their jobs or had retired. Furthermore, the new basic plan calls for further development of an accurate cancer registry and numerical targets for active and passive smoking and the percentage of the population receiving cancer screening. After the launch of the second term, discussions are already underway regarding enactment of new legislation and the development of more-ambitious cancer control. J Epidemiol 2013;23(2):153-154 By focusing on the social and medical context of cancer, the new basic plan launched by the Japanese government will address challenges in global cancer control amongst cancer patients and cancer survivors. ONLINE ONLY MATERIALS Abstract in Japanese. ACKNOWLEDGMENTS Conﬂicts of interest: None declared. REFERENCES 1. Japanese Government. Basic Plan for Promotion of Cancer Measures. 2012. Available: http://www.mhlw.go.jp/bunya/kenkou/ gan_keikaku.html.
https://openalex.org/W4366202484	https://bmcbiol.biomedcentral.com/counter/pdf/10.1186/s12915-023-01552-y	English	null	Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation	BMC biology	2,023	cc-by	13,318	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation Chen Wang1†, Dong‑Dong Wu2,3,4†, Yao‑Hua Yuan5, Meng‑Cheng Yao2,3,4, Jian‑Lin Han6,7, Ya‑Jiang Wu1, Fen Shan1, Wan‑Ping Li1, Jun‑Qiong Zhai1, Mian Huang1, Shi‑Ming Peng1, Qin‑Hui Cai1, Jian‑Yi Yu5, Qun‑Xiu Liu5, Zhao‑Yang Liu8, Lin‑Xiang Li9, Ming‑Sheng Teng10, Wei Huang11, Jun‑Ying Zhou12, Chi Zhang13, Wu Chen1* and Xiao‑Long Tu2,3,4* Abstract Background Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. Results In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious muta‑ tions, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. Conclusions The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a BMC Biology BMC Biology Wang et al. BMC Biology (2023) 21:64 https://doi.org/10.1186/s12915-023-01552-y Open Access Backgroundh g The tiger (Panthera tigris) is one of the largest felids and a widely recognized flagship species of wildlife conser- vation in the world. There are six commonly accepted living tiger subspecies, including the South China tiger (P. t. amoyensis), Amur tiger (P. t. altaica), Indochinese tiger (P. t. corbetti), Malayan tiger (P. t. jacksoni), Bengal tiger (P. t. tigris), and Sumatran tiger (P. t. sumatrae) [1– 3]. Among them, the Sumatran tiger is the only island population to be distinctive from all continental tigers and the Amur tiger splits last within mainland Asia [4], while the South China tiger is the rarest tiger subspe- cies [5]. In the 1950s, around 4000 South China tigers were found in 13 provinces in China [6]. Unfortunately, up to 3000 tigers were hunted as a pest and killed mer- cilessly during that period. Habitat loss/fragmentation further accelerated the decline of these tigers [7]. Only in 1979, the Chinese government banned the hunting of the tigers, whereas the number of the South China tiger was estimated to be only 30–80 in 1996. No South China tiger was directly sighted in the wild since the 1990s [7], it was therefore believed to be “functionally extinct” by many scientists, followed by an official dec- laration of its extinction in the wild in 2012.f y y Because of the speculated inbreeding depression and extinction of the South China tiger in the wild, some experts suggested to introduce genetic supplementation from other tiger subspecies into the captive South China tiger population to enlarge its gene pool [8]. Hybridi- zation between the South China and Amur tigers was believed to have occurred in some Chinese zoos [13], which was supported by genetic characterization using mitochondrial and microsatellite DNA markers [14, 15]. It is evident that one allele at individual genetic loci throughout the genomes of a small and managed popu- lation can be fixed rapidly, leading to an increase in genomic homozygosity and subsequently the inbreed- ing load [16, 17]. Considering the positive correlation between genetic heterozygosity and fitness [18, 19], it is urgently needed to examine the genomic landscape of existing genetic variations that have driven the past res- cue of the South China tiger, in reference to large-scale population genomic studies on other tiger subspecies [1, 4, 20]. genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos. Keywords South China tiger, Chromosome-level genome, Whole genome sequencing, Genomic inbreeding, Deleterious mutations Keywords South China tiger, Chromosome-level genome, Whole genome sequencing, Genomic inbreeding, Deleterious mutations The Chinese Association of Zoological Gardens com- menced a coordinated South China tiger captive breeding and management program in 1994. From 1995, the South China Tiger Committee (renamed as Tiger Taxonomy Advisory Group, the Tiger TAG, in 2015) has been organ- izing an annual review of the captive population and designing breeding and exchange plans [12]. The Tiger TAG set a goal to maintain 90% of the genetic variations present in the captive population in 1995 and targeted to sustain 70% of the variations over the next 100 years. Since then, the breeding tigers began to be exchanged between the zoos based on their pedigree records and the number of the South China tigers increased rapidly. There were 205 South China tigers managed in captivity by 16 Chinese zoos and 18 individuals in the Laohu Val- ley Reserve in South Africa by 2020. †Chen Wang and Dong-Dong Wu contributed equally to this work. Correspondence: Wu Chen guangzhouchenwu@sina.com Xiao‑Long Tu tuxiaolong@mail.kiz.ac.cn Full list of author information is available at the end of the article Correspondence: Wu Chen guangzhouchenwu@sina.com Xiao‑Long Tu tuxiaolong@mail.kiz.ac.cn Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 16 Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology Backgroundh After being polished with the PacBio long reads and corrected using the Illu- mina short reads [26], the PacBio-based initial assembly resulted in a contig N50 at 6.20 Mb. We scaffolded the PacBio contigs using the Bionano optical mapping data. Backgroundh Considerable efforts have been made to rescue the South China tiger through a captive breeding pro- gram in China [8]. Since 1955, a total of 27 male and 20 female South China tigers were kept in captivity according to the studbook [9]. In 1963, the first suc- cess of captive breeding of this tiger subspecies was achieved in the Guiyang Qianling Zoo. Detailed pedi- gree records indicated that all captive South China tigers were the descendants of two male and four female wild-caught tigers, which were managed in the Shanghai Zoo (one male and one female from Guizhou as well as one female from Fujian provinces) and Gui- yang Qianling Zoo (one male and two females from Guizhou province) [9]. These descendants formed two founder lineages managed independently by the Shang- hai and Guiyang Qianling Zoos over the 1970s. During 1972-1984, the captive population expanded quickly from 13 to 49 tigers. However, fewer cubs were born during 1985–1995 and the average survival rate of new- born cubs was low [10, 11], following the rapid loss of genetic variations and possible inbreeding depression within this small population. In this study, we assembled a de novo chromosome- level genome and re-sequenced 29 whole genomes of the captive South China tigers collected from four major zoos. Combined with other whole-genome data of six tiger subspecies, we characterized the genetic diver- sity, population genetic structure, demographic history, genomic inbreeding, and deleterious mutation load of the captive South China tiger population. These findings do not only explain the successful breeding history of the South China tigers in captivity, but also pave the way for Page 3 of 16 Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology (2023) 21:64 a genomics-informed management by applying genome- wide markers to routinely monitor and sustain their criti- cal genetic variations in the future. [22], optical mapping [23], and Hi-C [24] technologies. We generated 122.31 Gb (50.96×) of PacBio long reads, 1,011.73 Gb (421.55×) of Illumina paired-end short reads, 440.32 Gb (183.47×) of Bionano optical molecules, and 532.46 Gb (221.86×) of Hi-C data (Additional file 1: Tables S1 and S2). The K-mer [25] analysis revealed its genome size to be 2.47 Gb (Additional file 1: Table S3 and Additional file 2: Figure S1). De novo genome assembly and whole‑genome re‑sequencing of the South China tigers To investigate the genetic variations of the South China tiger (Fig. 1a), we first constructed a high-quality de novo assembly of the South China tiger genome using a combination of high-fidelity short-read sequencing [21], long-read single-molecule real-time sequencing Fig. 1 Genome of South China tiger. a A male South China tiger. b Circos plot of South China tiger genomic features. Outer to inner: pseudo-chromosome, gene density (500-kb window) (with higher gene density indicated by higher blue column), GC content (500-kb window), and SNP density (500-kb window). c Distribution of the captive South China tigers in China. Yellow circles show 15 city locations of the captive South China tigers, including Luoyang, Zhengzhou, Linyi, Suzhou, Shanghai, Hangzhou, Nanchang, Chongqing, Chengdu, Guiyang, Changsha, Fuzhou, Longyan, Shaoguan, and Guangzhou. Circle size is proportional to the number of South China tigers in each city. All data are from the South China tiger studbook (2020). Red cross represents the sampling site of the South China tigers in our study Fig. 1 Genome of South China tiger. a A male South China tiger. b Circos plot of South China tiger genomic features. Outer to inner: pseudo-chromosome, gene density (500-kb window) (with higher gene density indicated by higher blue column), GC content (500-kb window), and SNP density (500-kb window). c Distribution of the captive South China tigers in China. Yellow circles show 15 city locations of the captive South China tigers, including Luoyang, Zhengzhou, Linyi, Suzhou, Shanghai, Hangzhou, Nanchang, Chongqing, Chengdu, Guiyang, Changsha, Fuzhou, Longyan, Shaoguan, and Guangzhou. Circle size is proportional to the number of South China tigers in each city. All data are from the South China tiger studbook (2020). Red cross represents the sampling site of the South China tigers in our study Page 4 of 16 Wang et al. BMC Biology (2023) 21:64 The resulting scaffolds were further clustered into chro- mosome-scale scaffolds using the Hi-C data (Additional file 2: Figure S2). Finally, the de novo assembly yielded 2.44 Gb of genomic sequences with a contig N50 at 6.13 Mb and a scaffold N50 at 150.19 Mb (Fig. 1b and Addi- tional file 1: Table S4). De novo genome assembly and whole‑genome re‑sequencing of the South China tigers The de novo assembly contained 19 pseudo-chromosomes anchored with 2.40 Gb of con- tigs (99.35%) and 2.42 Gb of scaffolds (99.36%), show- ing a high collinearity with the domestic cat (Felis catus) reference genome (FelCat9.0, Ensembl release 98, last access in September 2019), except for E3 chromosome (Additional file 1: Table S5 and Additional file 2: Figure S3). In the analysis of complete Benchmarking Univer- sal Single-Copy Orthologs (BUSCO), our assembled genome covered 95.5% of the BUSCO genes [27] (Addi- tional file 1: Table S6). By integrating the homology- and de novo-based predictions, 20,908 protein-coding genes were annotated (Additional file 1: Tables S7-S9). There were 844.92 Mb (34.98% of the genome size) of repeti- tive elements as well as 568 microRNAs (miRNA), 6,309 transfer RNAs (tRNA), 993 ribosomal RNAs (rRNA), and 1410 small nuclear RNAs (snRNA) (Additional file 1: Table S10). Altogether, we assembled and annotated the South China tiger genome (Amotig1.0) [28] as one of the top-quality chromosome-level genomes of all big cats (Additional file 1: Table S4) [29–36]. from other tiger subspecies (FST = 0.324–0.459) (Addi- tional file 1: Table S17 and Additional file 2: Figure S9), confirming the observations of previous studies [1, 4]. However, the genetic diversity of the South China tiger was not as low (π = 0.657 × 10−3) (Additional file 1: Table S16 and Additional file 2: Figure S7) as what was inferred from its pedigree records [9], and the genomic heterozygosity of the South China tiger was moderate among six tiger subspecies (Additional file 1: Table S15 and Additional file 2: Figure S8). Nonetheless, the South China tiger also showed a significant genetic differen- tiation from other tiger subspecies (FST = 0.278–0.459) (Additional file 1: Table S17 and Additional file 2: Figure S9). To clarify the phylogenetic relationships among six tiger subspecies, we performed neighbor-joining (NJ) phylogenetic reconstructions, principal component analysis (PCA), and a model-based ancestry estimation using ADMIXTURE software to infer their population genetic structure. We reconstructed the NJ trees based on pairwise genetic distances with the domestic cat as an outgroup, which supported the taxonomic status of six distinct tiger subspecies [1, 3, 40, 41] (Fig. 2a). The PCA (Additional file 2: Figure S10), maximum-likeli- hood tree (Additional file 2: Figure S11), and identity- by-state analysis (Additional file 2: Figures S12 and S13) all verified their phylogenetic relationships at the subspecies level. De novo genome assembly and whole‑genome re‑sequencing of the South China tigers However, the South China tigers were further differentiated into two lineages (e.g., lin- eages 1 and 2), except one particular tiger labeled as the ptam_1 to be a potential hybrid, while the Amur, Sumatran, and Malayan tigers formed three addi- tional genetic clusters at K = 5 (Fig. 2b). All six tiger subspecies were differentiated from each other at K = 9, despite potential gene flow among some tiger sub- species (Fig. 2b). Additionally, the population genetic structure of only captive South China tigers verified the two genomic lineages at K = 2 (Additional file 2: Figure S14), which mirrored the Shanghai and Gui- yang founder lineages [10, 11]. Meanwhile, the pair- wise sequential Markovian coalescent (PSMC) plots showed that all tigers experienced continuous bottle- necks since the onset of the Last Glacial Period (Fig. 2c and Additional file 2: Figure S15), a pattern that was observed in a previous study [1]. i To explore genetic variations in the captive South China tiger population, we re-sequenced the whole genomes of 29 South China tigers collected from four major zoos in China (Fig. 1c). A domestic cat was also re-sequenced and used as an outgroup. We generated around 1200 Gb of whole-genome sequencing data with an average coverage depth of 15.63× per genome [37]. We combined our data with the 40 published genomes of six tiger subspecies (10.38-29.59× coverage depths) [38, 39] (Additional file 1: Tables S11-S13 and Additional file 2: Figures S4 and S5). Altogether, we identified 10.21 million high-quality single nucleotide polymorphisms (SNPs) in these 69 tiger genomes after stringent quality control and alignment of all sequencing data against the Amotig1.0 genome (Additional file 1: Tables S14 and S15 and Additional file 2: Figure S6). Genetic variations among and demographic history of tiger subspecies All tiger subspecies have experienced severe population bottlenecks due to human hunting and habitat loss/frag- mentation, we thus compared the levels of their genetic variations. It was evident that the Sumatran tiger carried the least genetic diversity in terms of genome-wide het- erozygosity and nucleotide diversity (π = 0.553 × 10−3) (Fig. 3a, Additional file 1: Table S16, and Additional file 2: Figures S7 and S8), but the highest genetic differentiation Limited gene flow from other tiger subspecies into the captive South China tiger population To detect a signal of potential genetic admixture/intro- gression from other tiger subspecies into the South China tiger population (e.g., the lineages 1 and 2 as well as the ptam_1 tiger), we applied several popula- tion genetic analyses, including the ABBA-BABA Page 5 of 16 Wang et al. BMC Biology (2023) 21:64 Fig. 2 Genetic components of extant South China tigers. a Phylogenetic relationship of the South China tiger with other tiger subspecies, with a domestic cat as an outgroup, all nodes are 100% of reliability. b Population genetic structuring of different tiger subspecies. c PSMC plot of the inferred demographic histories of each tiger subspecies. d Average ratio of introgression between the South China tiger and other tiger subspecies Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology Page 5 of 16 Fig. 2 Genetic components of extant South China tigers. a Phylogenetic relationship of the South China tiger with other tiger subspecies, with a domestic cat as an outgroup, all nodes are 100% of reliability. b Population genetic structuring of different tiger subspecies. c PSMC plot of the inferred demographic histories of each tiger subspecies. d Average ratio of introgression between the South China tiger and other tiger subspecies Fig. 2 Genetic components of extant South China tigers. a Phylogenetic relationship of the South China tiger with other tiger subspecies, with a domestic cat as an outgroup, all nodes are 100% of reliability. b Population genetic structuring of different tiger subspecies. c PSMC plot of the inferred demographic histories of each tiger subspecies. d Average ratio of introgression between the South China tiger and other tiger subspecies Genomic inbreeding among tiger subspecies [42, 43], Dsuite [44], and TreeMix methods [45]. All results clearly indicated the highest admixture in the ptam_1 tiger [1] as a hybrid from the Indochinese tiger (Fig. 2b, d, and Additional file 2: Figures S16 and S20). Although genetic admixture occurred among tiger subspecies, there was limited gene flow from other tiger subspecies into the lineages 1 and 2 of the South China tiger (Additional file 1: Tables S16 and S17 and Additional file 2: Figures S17-S19). Altogether, we observed very limited introgression (0.13–1.50%) among the South China tiger genomes (Fig. 2d), indi- cating their genetic uniqueness to be warranted for a full protection. To characterize the speculated inbreeding depression among the South China tigers, we determined pairwise relatedness between individuals within each tiger subspe- cies using allelic identity-by-descent (IBD) [46] (Fig. 3b and Additional file 1: Table S20). Further, we calculated individual inbreeding based on genome-wide SNPs using the inbreeding coefficients FH (a measure of the increase in individual SNP homozygosity compared with mean Hardy-Weinberg expected homozygosity) and FROH [47] (based on the runs of homozygosity (ROH) ≥ 100 kb). Among all tiger subspecies, the highest levels of the relatedness, FH, and FROH in ROH < 1 Mb were observed Wang et al. BMC Biology (2023) 21:64 Page 6 of 16 Wang et al. BMC Biology Fig. 3 Comparison of genetic variations among different tiger subspecies. a Genome-wide heterozygosity per individual. b Pairwise relatednes based on allelic identity-by-descent (IBD), that is, genetic identity because of a recent common ancestor. Z0, Z1, and Z2 are the probabilities tha two individuals share neither, one or two alleles IBD. c Genomic inbreeding coefficients (FROH) based on different lengths of runs of homozygosi (ROH), with a minimum length of 100 kb Fig. 3 Comparison of genetic variations among different tiger subspecies. a Genome-wide heterozygosity per individual. b Pairwise relatedness b d ll li id i b d (IBD) h i i id i b f Z0 Z d Z2 h b bili i h Fig. 3 Comparison of genetic variations among different tiger subspecies. a Genome-wide heterozygosity per individual. b Pairwise relatedness based on allelic identity-by-descent (IBD), that is, genetic identity because of a recent common ancestor. Z0, Z1, and Z2 are the probabilities that two individuals share neither, one or two alleles IBD. Genomic inbreeding among tiger subspecies in the Sumatran tiger, however, its FROH values were reduced down to the lowest in ROH > 2 Mb, indicating the effects of strong founder and/or bottleneck events occurred in the recent past (Additional file 1: Table S21 and Additional file 2: Figure S21), while the Amur tiger seemed to have experienced constant inbreeding pres- sure as evidenced by their second highest relatedness, FH, and FROH values in ROH > 100 kb, which were increased to the highest in ROH > 2 Mb. On the other hand, the South China tigers shared a similar pattern of their relat- edness as what was observed among other tiger subspe- cies, but they had relatively high FROH values in ROH > 100 kb and 1 Mb, compared with the Bengal, Indochi- nese, and Malayan tigers, which were likely the impact of recent inbreeding/founder events coupled with contrac- tion of their effective population size (Fig. 3c). Among the four major zoos, the South China tigers in the Shang- hai Zoo had the most number of ROH (average number = 3293, ROH = 100 kb–1 Mb), but only all the South China tigers in the Shanghai Zoo and the hybrid ptam_1 did not have any long ROH (> 2 Mb) (Additional file 1: Table S21), suggesting the captive tigers in the Shanghai Zoo to be least inbred. Furthermore, the South China tiger had the lowest FROH (average value = 0.33, ROH ≥ 100 kb) (Additional file 1: Table S21). Although the pedigree-based inbreeding coefficients (Fp) among the South China tigers were as high as 0.1796–0.5048 [9], their relatedness was more strongly correlated with both FH and FROH than Fp when high-density SNPs were avail- able [48] (Additional file 2: Figure S22). The genome-wide SNP-based individual inbreeding estimates are thus rec- ommended for assisting the decision-making of captive breeding of the South China tiger. Accumulation and purging of deleterious mutations among tiger subspecies Because of the importance of managing deleterious muta- tions in conserved genomic elements for species conser- vation [49], we computed the proportion of deleterious mutations retained in potential coding regions of six tiger subspecies and obtained a total of 70,273 SNPs in three categories of high-, moderate-, and low-impact (nearly neutral) mutations, of which only 0.51% were highly deleterious as most of them were stop-gain mutations across six tiger subspecies (Additional file 1: Table S22). All tiger subspecies shared a similar distribution pattern in the proportions of these three categories of mutations (Fig. 4a and Additional file 1: Table S22). Nevertheless, the average number of homozygous sites with high- and moderate-impact mutations of the South China tiger (9.27 and 632.51, respectively) was significantly lower than other tiger subspecies (19.17–29.66 and 1255.67– 2198.42, respectively) (t test, P < 0.01), while the average Genomic inbreeding among tiger subspecies c Genomic inbreeding coefficients (FROH) based on different lengths of runs of homozygosity (ROH), with a minimum length of 100 kb Wang et al. BMC Biology (2023) 21:64 Page 7 of 16 number of homozygous sites with low-impact mutations of the South China tiger (13131.88) was close to other tiger subspecies (11127.83–12914.29) (Additional file 1: Table S23), an indication of a stronger genetic purging of the high- and moderate-impact mutations from the South China tiger. Additionally, the average proportions of high- and moderate-impact mutations in a homozy- gous state were the lowest (20.24% and 20.47%, respec- tively) among the South China tiger compared with other tiger subspecies (23.59–42.94% and 25.29–48.34% for high- and moderate-impact mutations, respectively) (Fig. 4b and Additional file 1: Table S23), indicating a relatively effective genetic purging of homozygous geno- types of such mutations. Moreover, this was particularly true that the average proportion of homozygous geno- types of both high- and moderate-impact mutations to all deleterious mutations was much lower than that of the low-impact mutations in the South China tiger, a pattern largely shared by other tiger subspecies (Additional file 1: Table. S23 and Additional file 2: Figure S23). The distribu- tion of derived alleles of such high- and moderate-impact mutations displayed a downward shift compared to the low-impact mutations in the South China tiger (Fig. 4c). Besides, we calculated Rxy [50] to estimate whether there was an excess or deficit of deleterious mutations in the South China tiger compared with the other tiger subspe- cies. The relative mutation loads of Rxy were reduced below 1 in both high- and moderate-impact mutations in the South China tiger compared with both Amur and Sumatran tigers (Fig. 4d). Altogether, these results indi- cated a relatively effective genetic purging of both high- and moderate-impact mutations in a homozygous state from the South China tiger following its population con- traction with a controlled increase in inbreeding based on pedigree records, a phenomenon that was observed in other species [51–54]. Discussion To facilitate the best practice of conservation genom- ics based on high-density markers across the entire genomes [55] to mitigate potential inbreeding loads associated with deleterious mutations of all endan- gered tigers, we first assembled the best South China tiger genome and applied it as a reference to the analy- ses of all tiger genomic data [13, 20–23]. We analyzed the largest population of the South China tiger com- bined with other tiger subspecies and found that all six tiger subspecies phylogenetically differentiated from each other, despite gene flow between some tiger sub- species [1–3]. The admixture from the Amur, Indochi- nese, and Bengal tigers was found in the captive South China tiger population by previous genomic studies [1, 15], so the South China tiger was thought to be the Wang et al. BMC Biology (2023) 21:64 Page 8 of 16 Wang et al. BMC Biology Fig. 4 Comparison of deleterious mutations among different tiger subspecies. a Percentage of three categories of nearly neutral (low), mildly (moderate), and highly deleterious (high impact) mutations in different tiger subspecies. b Individual homozygote percentage per mutation category of different tiger subspecies. c Site frequency spectra for SNPs per mutation category in the South China tiger. Derived allele counts ≥ 10 are displayed as mean counts per interval. d Rxy analysis compares the South China tiger with the Sumatran and Amur tigers per mutation category. Rxy < 1 indicates a relative frequency deficit of the corresponding category Fig. 4 Comparison of deleterious mutations among different tiger subspecies. a Percentage of three categories of nearly neutral (low), mildly (moderate), and highly deleterious (high impact) mutations in different tiger subspecies. b Individual homozygote percentage per mutation category of different tiger subspecies. c Site frequency spectra for SNPs per mutation category in the South China tiger. Derived allele counts ≥ 10 are displayed as mean counts per interval. d Rxy analysis compares the South China tiger with the Sumatran and Amur tigers per mutation category. Rxy < 1 indicates a relative frequency deficit of the corresponding category Fig. 4 Comparison of deleterious mutations among different tiger subspecies. a Percentage of three categories of nearly neutral (low), mildly (moderate), and highly deleterious (high impact) mutations in different tiger subspecies. b Individual homozygote percentage per mutation category of different tiger subspecies. c Site frequency spectra for SNPs per mutation category in the South China tiger. Discussion We also recognized that the tigers of the two lineages were not evenly distributed among the zoos, for example, the seven tigers sampled from the Guangzhou Zoo for this study all belonged to the Shanghai lineage. This calls for a genomics-assisted exchange plan of breed- ing tigers across all major zoos. In fact, the Shanghai and Guiyang lineages showed different reproductive perfor- mance and fitness among the newborn tigers [62]. Our genomic analysis detected the lowest genomic inbreed- ing of the South China tigers in the Shanghai Zoo. We therefore recommend a genomics-informed exchange of breeding tigers between these two founder/genomic lineages, so that to maximize the benefit of maintaining existing unique and critical genetic variations that are expected to outweigh the cost of increased genetic load within the captive South China tiger population [20]. unique genetic variations and putatively deleterious mutations. Evaluation of genomic variations in all tiger subspecies showed that the Sumatran tiger had the lowest genetic diversity, while the South China tiger harbored moder- ate genetic variations. We also found genomic inbreed- ing in the Sumatran, Amur, and South China tigers. Moreover, the accumulation of large ROH (> 1 Mb) since the beginning of captive breeding has been resulting in relatively high genomic inbreeding and genetic burden in the South China tiger [56]. It was reported that the cap- tive South China tiger population suffered from inbreed- ing depression in terms of high juvenile mortality and impaired adult fertility [12]. To control the inbreeding based on pedigree records, the captive South China tigers were permitted to mate only if their mate suitability indexes (MSI, ranging from 1 to 6 across all tigers) cal- culated by PMx software [57], were below 4. The MSI is a composite score that integrates four genetic components into a single index, including Delta GD, differences in Mk values, inbreeding coefficient, and unknown ancestry [58]. Therefore, the breeding plan based on the MSI may have assisted most deleterious mutations to be inherited as recessive alleles masked in heterozygous states and thus free of purifying selection [59, 60]. Our genomic analysis, however, revealed an efficient genetic purging of both high- and moderate-impact deleterious mutations from the South China tiger genomes. It is known that the purging reduces the frequency of deleterious mutations, depending on the degree of dominance and the magni- tude of the deleterious effects. Discussion Derived allele counts ≥ 10 are displayed as mean counts per interval. d Rxy analysis compares the South China tiger with the Sumatran and Amur tigers per mutation category. Rxy < 1 indicates a relative frequency deficit of the corresponding category validating very little or negligible genetic contami- nation in the captive South China tiger population. However, we only re-sequenced the whole genomes of 29 South China tigers collected from the four major zoos in China. As such, we shall continue and focus our efforts on collecting and re-sequencing addi- tional whole genomes of most, if not all, reproductive South China tigers, to further fine-map and effectively manage their viable genomic landscapes along with most taxonomically controversial among all the tiger subspecies. From 2004, the Tiger TAG collected blood samples from all newborn tigers for genetic analy- sis to remove apparently admixed tigers from breed- ing, including the ptam_1 from the Chongqing Zoo [1] that was obviously a hybrid from the Indochinese tiger (Additional file 2: Figures S16 and S20). In this study, we found that the South China tiger had very limited genetic admixture from other tiger subspecies, Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology (2023) 21:64 Page 9 of 16 Page 9 of 16 Shanghai and Guiyang founder lineages were formed during the 1970s, but the two lineages began to merge for breeding to minimize potential inbreeding depres- sion since the mid-1980s [11]. The two genetic lineages based on current genome-wide SNPs and early mtDNA and microsatellite analyses [15] clearly mirrored the two founder lineages, due probably to historical allelic seg- regation and/or genetic drift among limited founders of separate geographical origins and relatively independent reproduction of the South China tigers in isolated zoos, as what was observed in the killer whale ecotypes [46] and highly inbred pigs [61]. The Tiger TAG has been in charge of the breeding and transfer plan for South China tiger, following a principle of priority to allow the tigers to breed in their original facilities first, which may have lim- ited the exchange of breeding tigers among the zoos [11]. According to the studbook, the descendants of the Gui- yang lineage were much fewer than those of the Shanghai lineage [9]. Discussion Because most high-impact mutations were stop-gain variants leading to loss of gene functions, the high-impact mutations were exposed to the highest strength of purging [51]. It was true that the average number of homozygous sites with high- impact mutations per individual (9.27) was much lower than those of the moderate- (632.51) and low-impact (13,131.88) mutations in the South China tiger genomes. Surprisingly, the lowest proportion of homozygous geno- types and relatively low Rxy estimates (< 1) were associ- ated with both high- and moderate-impact mutations in the South China tiger genomes. This verified that both high- and moderate-impact mutations were more likely to be inherited as recessive alleles than low-impact muta- tions in the captive South China tiger population [12]. Based on recent founder effect and ongoing inbreeding, these deleterious mutations are expected to be continu- ously accumulated in the South China tiger genomes, therefore their potential impacts on fitness should be evaluated across generations of the captive South China tiger population [10, 11]. It is certain that all captive South China tigers are the descendants of two males and four female tigers [9] The Construction of optical genome mapsfi g DNA of sufficient quality was extracted and labeled from the Tuantuan blood cells according to standard BioNano protocols (BioNano Genomics), after which nicking, labeling, repairing, and staining processes were implemented. We digested DNA using a specific single- stranded nicking endonuclease (Nt.BspQI). BioNano Solve (v3.0.1) [66] was used to produce optical maps with single molecules above 100 kb in size and six labels per molecule. The scaffold-level assembly had a N50 length at 31.62 Mb. De novo genome sequencing Blood samples of two male (Tuantuan and Kangkang) and ione female (Huanhuan) South China tiger were acquired from the Guangzhou Zoo for genome sequenc- ing. All the DNA was extracted from blood using phenol- chloroform method [63]. All libraries were constructed at BGI (Shenzhen, China). For PacBio sequencing, genomic DNA from the Tuantuan was used to construct three libraries of 20-kb insert size using the SMRTbell Tem- plate Prep Kit 1.0 (Pacific Biosciences, USA), and the fragment size was selected using Blue Pippin (Labgene Scientific SA, Switzerland). Then, the library sequencing was performed on a PacBio RS II sequencer. For short reads sequencing, three short insert libraries (270, 500, and 800 bp) were constructed for Tuantuan and four long mate-paired insert libraries (2, 5, 10, and 20 kb) were constructed for Huanhuan. The libraries were con- structed as per the manufacturer’s instructions (MGIEasy universal DNA Library Preparation Kit, BGI, China). All libraries were sequenced on the Illumina HiSeq X Ten Platform using 150-bp paired-end reads according to the Illumina protocols, except for the 800-bp library, which was sequenced on the HiSeq 2500 System using 125-bp paired-end reads. Estimation of genome size We applied K-mer (17-mer) distribution analysis to esti- mate the South China tiger genome size using clean reads from the short-insert libraries. Genome size was calcu- lated using the formula: Genome size = K-mer_number / K_depth of peak. Hi‑C analysish The Hi-C library was constructed from the blood cells of Kangkang according to the standard procedures of BGI and sequenced using the MGISEQ-2000 Platform. The Bionano-based scaffolds were anchored into a chro- mosome-scale assembly using a Hi-C proximity-based assembly approach. We aligned the Hi-C reads to scaf- folds using bowtie2 (v2.2.5) [67] and interaction maps were generated following HiC-Pro (v2.5.0) [68] pipelines. The uniquely mapped read pairs were used as input for Juicer [69] and 3d-DNA [70] Hi-C analysis and scaffold- ing pipelines. The resulting Hi-C contact maps were visualized using Juicebox [71], and mis-assemblies and mis-joins were manually corrected based on neighboring interactions. The preliminary chromosome assembly was then generated and named as Amotig1.0. The Amotig1.0 genome was assessed by calculating the number of Hi-C read pairs in any two bins of 500 kb. The synteny analy- sis between the Amotig1.0 and the domestic cat genomes (Ensembl release 98, accessed in September 2019) was performed using the Mummer-4.0.0 software (https:// github.com/gmarcais/mummer). The chromosome IDs in the Amotig1.0 genome were determined according to the synteny relationship with the cat genome. To evaluate the quality of the Amotig1.0 genome, we performed the BUSCO pipeline (version 5.0) [27] with orthologs data- base of mammalia_odb10. Conclusions In this study, we de novo assembled the high-quality chromosome-level reference genome of a South China tiger and re-sequenced the whole genomes of 29 South China tigers collected from four major breeding zoos in China. The results indicated that the captive South China tigers included in our study (expect the pam_1) had lim- ited genetic admixture from other tiger subspecies. The genetic diversity was slightly higher in the South China tiger than in the Sumatran tiger, while the South China tiger had high FROH values under longer ROH (> 1 Mb), indicating its recent inbreeding/founder events and/or population bottleneck/isolation. Although most high- and moderate-impact deleterious mutations in the South China tiger genomes may be masked as recessive alleles for their inheritance in heterozygous states, such del- eterious SNPs have been effectively purged, when they were in homozygous states, from the South China tiger It is certain that all captive South China tigers are the descendants of two males and four female tigers [9]. The Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology (2023) 21:64 Page 10 of 16 population. We recommend that individuals of the two genomic lineages could be merged to breed for mini- mizing a further loss of the unique and critical genetic variations. All the new findings from our present study demonstrated the power and effectiveness of concerted efforts to conserve the captive South China tiger in the past and shed light into a potentially bright future of these critically endangered cats. The captive South China tigers are the last hope of the tiger subspecies, we wish that they would have a success of reintroduction program in the future and to be another case as what is achieved in the protection of the Giant Panda, which was downlisted to ’vulnerable’ on the IUCN Red List. Gb in length) with N50 size at 6.24 Mb. We also aligned the Illumina X Ten data to the assembly using BWA (v0.7.15) [64] for error correction by Pilon (v1.22) [65] with the parameter “--mindepth 6”, which is an integrated tool for comprehensive variant detection and genome assembly improvement. The final assembly generated a total length of 2.42 Gb and N50 length at 6.20 Mb. Genome annotationi The PacBio data were de novo assembled using WTDBG- 1.2.8 (https://github.com/ruanjue/wtdbg-1.2.8) and genomic contigs were polished with the Arrow program (https://www.pacb.com/support/software-downloads/) by aligning SMRT reads, which yielded an assembly (2.43 We identified repetitive sequences in the Amotig1.0 genome using a combination of homology- and de novo- based methods. First, RepeatMasker [72] and Repeat- ProteinMask [72] were used to search repeat sequences Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology (2023) 21:64 Page 11 of 16 Page 11 of 16 against with Repbase database [73, 74]. Second, LTRhar- vest (GenomeTools v1.5.9) [75] and RepeatModeler (http://www.repeatmasker.org/RepeatModeler/) were used to build a de novo repeat library, and then repeats were annotated using RepeatMasker [72] with default parameters. Last, tandem repeats were detected using Tandem Repeats Finder (TRF) [76]. against with Repbase database [73, 74]. Second, LTRhar- vest (GenomeTools v1.5.9) [75] and RepeatModeler (http://www.repeatmasker.org/RepeatModeler/) were used to build a de novo repeat library, and then repeats were annotated using RepeatMasker [72] with default parameters. Last, tandem repeats were detected using Tandem Repeats Finder (TRF) [76]. Quality control To ensure reads were reliable and without artificial bias (e.g., low-quality paired reads that result from base-call- ing duplicates and adapter contamination), we conducted a series of quality control (QC) procedures, as follows: (1) Removed reads with ≥ 1% unidentified nucleotides (N). For gene structure prediction, we used homology-based prediction based on the protein sequences from five species (Felis catus, Homo sapiens, Mus musculus, Pan- thera pardus, and Panthera tigris altaica) downloaded from the Ensembl database (release 93). These protein sequences were mapped to the Amotig1.0 genome using TBLASTN (E-value cutoff: 1e−5) [77]. High-scoring seg- ment pairs (HSPs) were concatenated using Solar (v0.9.6) [25]. GeneWise (v2.4.1) [78] was used to define accurate gene models. We then merged and filtered redundancy from different homology results based on the GeneWise score (≥ 0.4). To obtain the final gene set, transposons and single-exon genes without functional annotations were filtered out. (2) Removed reads with > 40% bases having phred quality < 20. (3) Removed reads with > 10 nucleotides overlapping the adapter (allowing ≤ 10% mismatches). Sampling information and whole genome re‑sequencing Sampling information and whole genome re‑sequencing We collected a total of 30 specimens, including 29 South China tigers (P. t. amoyensis) from four major zoos in China and a domestic cat (Felis catus) from the Guang- zhou Zoo, China (Supplementary Table S11). The 29 South China tigers were born in 1999 to 2018, and the blood samples of the individuals were collected in four major city zoos in 2018 and 2019. Genomic DNA was extracted from whole blood using the DNeasy Blood & Tissue Kit (QIAGEN, Valencia, California, USA) follow- ing the manufacturer’s protocols. For each sample, one 350 bp size of DNA library was constructed according to the manufacturer’s protocols (Illumina). The constructed libraries were sequenced using the Illumina Hiseq X Ten platform for 150 bp paired-end reads. Read alignment and variant calling We used BWA [86] to align the clean reads of each sample against our newly assembled South China tiger genome (settings: mem -t 4 -k 32 -M -R). Alignment files were converted to BAM files using SAMtools (settings: -bS -t) (v-0.1.19) [87]. In addition, potential PCR dupli- cations were removed using Picard (http://broadinstitute. github.io/picard/). We called SNPs using the Haplotype- Caller approach implemented in the Genome Analysis Toolkit (GATK) package [88]. Filtering criteria were as follows: i Gene functional annotations were assigned using BLASTP (BLAST+ v2.2.26) [77] against public data- bases, including the Swiss-Prot (release-2017_09) [79], TrEMBL (release-2017_09) [79], KEGG (v84.0) [80], COG [81], and NCBI nucleotide collection nr/nt (https:// www.ncbi.nlm.nih.gov/nucleotide/, v20170924). The motifs and domains in the protein sequences were anno- tated using InterProScan (v5.16-55.0) [82].h (1) SNPs with QD < 2.0; FS > 60.0; MQ < 40.0; QUAL < 30; DP < 4.0; MQRankSum < -12.5; and ReadPos- RankSum < -8.0 were filtered. The tRNA genes were predicted using tRNAscan-SE (v1.3.1) [83] with eukaryote parameters. The rRNA frag- ments were identified by aligning the rRNA template sequences from the Human Rfam database [84] using BLASTN (BLAST+ v2.2.26) [77] (E-value 1e−5). The snR- NAs and miRNAs were searched via a two-step method: i.e., aligned with BLAST and then searched with INFER- NAL (infernal-1.1.1) [85] against the Rfam database. i (2) Multi-nucleotide polymorphisms were ignored.i (3) SNPs within 5 bp of a gap were filtered. i (4) Overall depth (for all individuals) was > 1/3× and < 3×. (5) Unobserved variant allele constituted < 10%. A total of 54,067,600 high-quality SNPs were retained for subsequent analyses after filtering. Gene-based SNP annotation was performed using ANNOVAR [89]. Inbreeding coefficient We measured individual inbreeding using the genomic inbreeding coefficients FH [48], which is the fraction of IBD of the two alleles in a diploid individual from a com- mon ancestor. FH was calculated using PLINK (v1.9) [90]. Alternatively, individual genomic inbreeding coefficients was also measured using FROH [99], which is an estimate of ROH proportion in an individual genome. Abbreviations Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12915-023-01552-y. We used the PSMC [98] model to reconstruct demo- graphic history. To estimate effective ancestral popu- lation size changes for individual tiger subspecies, we selected individuals with high sequencing depth to ensure the quality of the consensus sequence. SNPs were detected using SAMtools [87], sites were filtered based on a minimum depth (DP = 4) and the highest depth (DP = 50) and mapping quality (Q = 20). We only retained autosomal SNPs. Parameters were set to: -N30, -t15, -r5, -p‘4+25*2+4+6’. A mutation rate of 3.5 × 10−9 per base per generation and generation time of 5 years was used. Calculation of genetic diversity PLINK (v1.9) [90] was used to detect ROH via a slid- ing window approach, with the following parameters: ‘--homozyg-window-snp 100 --homozyg-window-het 2 --homozyg-window-missing 5 --homozyg-snp 100 --homozyg-kb 100 --homozyg-density 10 --homozyg-gap 100’. Pairwise nucleotide diversity θπ and Watterson’s esti- mator θw [93] within a tiger subspecies were calculated using a sliding-window approach (20-kb windows slid- ing in 10-kb steps). Genetic differentiation between tiger subspecies was calculated using the pairwise fixation index FST [94]. Additional file 1: Table S1. Sample characteristics and genome sequenc‑ ing information before and after filtering. Table S2. Sequencing data statistics. Table S3. 17-mer statistic information. Table S4. Genome assembly information. Table S5. Sizes of individual chromosomes. Table S6. Assessment of completeness of the South China tiger genome assembly. Table S7. Summary of repeat contents. Table S8. Statistics of gene structure prediction. Table S9. Result of gene functional annota‑ tion. Table S10. Summary of non-coding RNA in genome. Table S11. Sampling information included in the analyses. Table S12. Re-sequencing data statistics in this study. Table S13. Genome mapping and coverage information for all accessions analyzed in this study. Table S14. Number and distribution of SNPs in each genome. Table S15. Distribution of whole-genome SNPs in different subspecies/species. Table S16. Average genome-wide nucleotide diversity in six tiger subspecies. Table S17. Pairwise FST values between six tiger subspecies. Table S18. ABBA-BABA estimates. Only results with \|Z score\| > 3 were remained. Table S19. f4- ratio calculated between individual South China tigers and other tiger subspecies. Only results with FDR < 0.05 were retained. Table S20. Identity‑by‑state (IBS) and identity‑by‑descent (IBD) analyses To evaluate the similarity between two tigers within a tiger subspecies, genome-wide IBS pairwise identities were calculated using the toolset SNPRelate [95] in the R package. Based on the matrix of genome-wide IBS pair- wise distances, we performed multidimensional scaling and cluster analyses and determined the groups using a permutation score. We also calculated the pairwise IBD using PLINK (v1.9) (settings: --file --genome --min 0.05) [90]. Phylogenetic and population genetic analysis y y We selected genome-wide 54,067,600 SNPs for phyloge- netic construction with a cat as outgroup and 10,205,707 SNPs for PCA and population structure analyses compo- nents. PCA was performed using PLINK (v1.9) (settings: --bfile --pca –noweb) [90]. Genetic structure was inferred using ADMIXTURE (v1.3) [91], with implementation of a block-relaxation algorithm (settings: --cv -m=block). To explore convergence of individuals, we predefined the number of genetic clusters K from 2 to 9. We calculated the p-distance matrix using VCF2Dis (https://github. com/BGI-shenzhen/VCF2Dis) and a NJ tree was gener- ated using the R package APE and 100 bootstraps were run for a reliable tree [92]. We selected genome-wide 54,067,600 SNPs for phyloge- netic construction with a cat as outgroup and 10,205,707 SNPs for PCA and population structure analyses compo- nents. PCA was performed using PLINK (v1.9) (settings: --bfile --pca –noweb) [90]. Genetic structure was inferred using ADMIXTURE (v1.3) [91], with implementation of a block-relaxation algorithm (settings: --cv -m=block). To explore convergence of individuals, we predefined the number of genetic clusters K from 2 to 9. We calculated the p-distance matrix using VCF2Dis (https://github. com/BGI-shenzhen/VCF2Dis) and a NJ tree was gener- ated using the R package APE and 100 bootstraps were run for a reliable tree [92]. Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology (2023) 21:64 Page 12 of 16 Identification of deleterious mutationsh The variants leading to functional changes were regarded as candidates of deleterious mutations. Thus, we only analyzed the SNPs in all potential coding regions, while the domestic cat allele was regarded as an ancestral allele. SnpEff (v4.3t) [100] was used for genetic variant annota- tion and functional effect prediction. Introgression and demographic history analyses We separated the South China tiger population into three classes (i.e., lineage 1, lineage 2, and ptam1) based on phylogeny. TreeMix (v1.13) [96] was used to detect gene flow between the tiger subspecies based on genome- wide allele frequency data at individual SNPs. We first constructed a maximum-likelihood tree for six tiger subspecies using blocks of 10,000 SNPs. The number of migration events was set from 1 to 6. We calculated introgression among six tiger subspecies using Patter- son’s D-statistic (ABBA-BABA test) [97], with the cat as the outgroup, and tested the proportions of admixture events (f4-ratio) within each South China tiger using Dsuite (v0.4) [44], we calculated the mean introgression ratio between each South China tiger and other tiger subspecies. Abbreviations SNPs Single nucleotide polymorphisms PCA Principal component analysis NJ Neighbor-joining IBD Identity-by-descent ROH Runs of homozygosity Fp Pedigree-based inbreeding coefficients TRF Tandem Repeats Finder HSPs High-scoring segment pairs QC Quality control GATK Genome Analysis Toolkit IBS Identity-by-state Abbreviations SNPs Single nucleotide polymorphisms PCA Principal component analysis NJ Neighbor-joining IBD Identity-by-descent ROH Runs of homozygosity Fp Pedigree-based inbreeding coefficients TRF Tandem Repeats Finder HSPs High-scoring segment pairs QC Quality control GATK Genome Analysis Toolkit IBS Identity-by-state Analysis of runs of homozygosity Regions of homozygosity were extracted for all chro- mosomes of all individuals based on SNP information. Page 13 of 16 Wang et al. BMC Biology ( Wang et al. BMC Biology (2023) 21:64 Wang et al. BMC Biology Pairwise relatedness estimates based on allelic identity-by-descent (IBD). Table S21. Inbreeding coefficients and ROH information. Table S22. Deleterious mutations segregating across and within six tiger subspecies. Table S23. Individual homozygote and heterozygote SNP counts per impact category in each tiger subspecies. S20. Plot of inferred introgression between the South China tiger ptam_1 individual and other tiger subspecies populations detected by the TreeMix method. The scale bar shows 10 times the average standard error of the entries in the sample covariance matrix. Figure S21. Genomic inbreed‑ ing coefficients FH in each tiger subspecies. Figure S22. The plot of three inbreeding coefficients (FP, FH, and FROH) of the South China tiger. Figure S23. Comparison of homozygote and heterozygote percentage of per mutation category among six tiger subspecies. The significant value was calculated by t-test. S20. Plot of inferred introgression between the South China tiger ptam_1 individual and other tiger subspecies populations detected by the TreeMix method. The scale bar shows 10 times the average standard error of the entries in the sample covariance matrix. Figure S21. Genomic inbreed‑ ing coefficients FH in each tiger subspecies. Figure S22. The plot of three inbreeding coefficients (FP, FH, and FROH) of the South China tiger. Figure S23. Comparison of homozygote and heterozygote percentage of per mutation category among six tiger subspecies. The significant value was calculated by t-test. Additional file 2: Figure S1. Distribution of 17-mer depth for estimat‑ ing the genome size. The x-axis represents k-mer depths while y-axis represents the proportions. The blue line represents the proportion of 17-mer in each depth. The peak depth is at 25-fold and total number of 17-mer is 61,791,522,108. The South China tiger (P. t. amoyensis) genome size was estimated to be 2471.66 Mb from the formula: Genome size = K-mer_number/K_depth of peak. Figure S2. Hi-C chromosomal contact heat map. A 500 kb resolution was used to calculate the number of Hi-C read pairs in any two bins. Figure S3. The collinearity between the South China tiger and domestic cat (Felis catus) genomes. Each dot represents an aligned region while the minimum length is 10 kb. The red dot represents forward comparison and the blue dot reverse comparison. Analysis of runs of homozygosity Figure S4. Q30 and GC content for each sample. The average Q30 and GC content are 93.24% and 42.04%, respectively. Their very low variation reflected our re-sequencing data to be high quality. Figure S5. The distribution of mapping rates and average mapping depths for each individual. The depths ranged from 12.91× to 18.96 × while the rates varied from 96.03% to 98.92%. Figure S6. The distribution of the SNP number and frequency. Number of genome-wide SNPs (top) and frequency of SNPs per 1 kb of each tiger sample (bottom). Figure S7. Nucleotide diversity π estimates of six tiger subspecies. Figure S8. Heterozygosity statistics of genome-wide SNPs. (a) Observed heterozygosity across all individual genomes of each tiger subspecies. (b) Genomic heterozygosity in each tiger subspecies at population level. Figure S9. Pairwise FST values between six tiger subspe‑ cies. The weighted FST values are shown above the diagonal while their standard deviations below the diagonal. Figure S10. Principal component analysis using genome-wide SNPs of six tiger subspecies. Figure S11. A maximum likelihood tree was built using TreeMix software with whole- genome sequencing data of six tiger subspecies and cat. Figure S12. Cluster analysis based on the matrix of genome-wide identity-by-state (IBS) pairwise distances between six tiger subspecies determined by a per‑ mutation score. Figure S13. Analysis of genome-wide average identity- by-state (IBS) pairwise identities between six tiger subspecies. Figure S14. Population genetic structure of the South China tigers estimated by the ADMIXTURE. Figure S15. Demographic histories of tiger subspecies. PSMC analysis shows the change in effective population size over time. The dash lines represent 100 bootstraps. The representative individuals sequenced at a high read coverage were selected for each graph, South China tiger (ptam_4, mean 25.38×), Amur tiger (ptal_1, mean 14.76×), Indochinese tiger (ptco_2, mean 13.98×), Malayan tiger (ptja_7, mean 13.13×), Bengal tiger (ptti_3, mean 13.79×), and Sumatran tiger (ptsu_1, mean 13.41×). Figure S16. The results of Dsuite. Heatmap showing statistical support for introgression between pairs of tiger subspecies. Cells in the heatmap indicate the pairwise Z score values between the branch b identified on the expanded tree on the Y axis (relative to its sister branch) and the taxa P3 identified on the X-axis. The grey color indicates the none. (a) Result of branch for South China tiger (lineage 1) with the other tiger subspecies populations. Availability of data and materials Genome assemblies and DNA sequencing data were deposited in the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn) under Project no. PRJCA006384. The de novo genome of the South China tiger was depos‑ ited under Accession ID GWHBEIN00000000 [28]. The re-sequencing genomic data of 29 South China tigers and a domestic cat were deposited under Acces‑ sion ID CRA004909 [37]. Consent for publication Not applicable. Consent for publication Not applicable. 13.13×), Bengal tiger (ptti_3, mean 13.79×), and Sumatran tiger (ptsu_1, mean 13.41×). Figure S16. The results of Dsuite. Heatmap showing statistical support for introgression between pairs of tiger subspecies. Cells in the heatmap indicate the pairwise Z score values between the branch b identified on the expanded tree on the Y axis (relative to its sister branch) and the taxa P3 identified on the X-axis. The grey color indicates the none. (a) Result of branch for South China tiger (lineage 1) with the other tiger subspecies populations. (b) Result of branch for South China tiger (lineage 2) with the other tiger subspecies populations. (c) Result of branch for South China tiger (ptam1 individual) with the other tiger subspecies populations. Figure S17. The graph of ABBA-BABA test. The values of D (pop1, pop2; pop3, pop4) >0 indicates that there are gene flows between pop1 and pop3. Here, only the introgression occurred in South China tiger will be shown. (a) D-statistic for South China tiger (lineage 1) with other tiger subspecies. (b) D-statistic for South China tiger (lineage 2) with other tiger subspecies. (c) D-statistic for South China tiger (ptam1 individual) with other tiger subspecies. Figure S18. Plot of inferred introgression between the South China tiger lineage 1 and other tiger subspecies populations detected by the TreeMix method. The scale bar shows 10 times the average standard error of the entries in the sample covariance matrix. Figure S19. Plot of inferred introgression between the South China tiger lineage 2 and other tiger subspecies populations detected by the TreeMix method. The scale bar shows 10 times the aver‑ age standard error of the entries in the sample covariance matrix. Figure Funding This work was supported by the Science and Technology Program of Guang‑ zhou, China (202102020815) and Guangdong Provincial Key R&D Program (2022B1111040001). Competing interests The authors declare that they have no competing interests. Analysis of runs of homozygosity (b) Result of branch for South China tiger (lineage 2) with the other tiger subspecies populations. (c) Result of branch for South China tiger (ptam1 individual) with the other tiger subspecies populations. Figure S17. The graph of ABBA-BABA test. The values of D (pop1, pop2; pop3, pop4) >0 indicates that there are gene flows between pop1 and pop3. Here, only the introgression occurred in South China tiger will be shown. (a) D-statistic for South China tiger (lineage 1) with other tiger subspecies. (b) D-statistic for South China tiger (lineage 2) with other tiger subspecies. (c) D-statistic for South China tiger (ptam1 individual) with other tiger subspecies. Figure S18. Plot of inferred introgression between the South China tiger lineage 1 and other tiger subspecies populations detected by the TreeMix method. The scale bar shows 10 times the average standard error of the entries in the sample covariance matrix. Figure S19. Plot of inferred introgression between the South China tiger lineage 2 and other tiger subspecies populations detected by the TreeMix method. The scale bar shows 10 times the aver‑ age standard error of the entries in the sample covariance matrix. Figure Authors’ contributions WC conceived and initiated the project. YHY, YJW, FS, WPL, JQZ, MH, SMP, QHC, JYY, QXL, ZYL, LXL, MST, WH, and JYZ organized and collected the samples. CZ performed DNA extraction, library construction, and sequencing. XLT and MCY performed data analysis. CW and DDW drafted the manuscript. JLH extensively revised the manuscript. All authors have read and approved the final version of the manuscript. Acknowledgements We thank Zujin Chen, Aiping Jiang, Qirui Wu, and Xuanjiao Chen from the Guangzhou Zoo for providing support during this study. We thank Zujin Chen, Aiping Jiang, Qirui Wu, and Xuanjiao Chen from the Guangzhou Zoo for providing support during this study. Ethics approval and consent to participate All necessary research permits and ethics approvals for this study were granted by Guangzhou Zoo, China (Project number GZ_20180216). Received: 29 March 2022 Accepted: 21 February 2023 References The struggle to save the last South China tigers. 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Author details 1 Guangzhou Zoo & Guangzhou Wildlife Research Center, Guangzhou 510070, China. 2 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650201, China. 3 Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. 4 Kunming College of Life Science, University of the Chinese Academy of Sciences, Kun‑ ming 650204, China. 5 Shanghai Zoo, Shanghai 200336, China. 6 CAAS‑ILRI Joint Laboratory on Livestock and Forage Genetic Resources, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, China. 7 International Livestock Research Institute (ILRI), Nairobi 00100, Kenya. 8 Wangcheng Park, Luoyang 471000, China. 9 Suzhou Shangfangshan Forest Zoo, Suzhou 215009, China. 10 Chongqing Zoo, Chongqing 401326, China. 11 Nanchang Zoo, Nanchang 330025, China. 12 Chinese Association of Zoologi‑ cal Gardens, Beijing 100037, China. 13 Qinghai Province Key Laboratory of Crop Molecular Breeding, Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai, China. 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https://openalex.org/W4379053947	https://www.mdpi.com/2227-9067/10/6/988/pdf?version=1685601915	English	null	Nationwide Survey Reveals High Prevalence of Non-Swimmers among Children with Congenital Heart Defects	Children	2,023	cc-by	4,407	Christian Apitz 1,, Dominik Tobias 1, Paul Helm 2, Ulrike M. Bauer 2, Claudia Niessner 3 and Jannos Siaplaouras 1,4 Christian Apitz 1,, Dominik Tobias 1, Paul Helm 2, Ulrike M. Bauer 2, Claudia Niessner 3 and Jannos Siaplaouras 1,4 Christian Apitz 1,, Dominik Tobias 1, Paul Helm 2, Ulrike M. Bauer 2, Claudia Niessner 3 and Jannos Siaplaouras 1,4 1 Division of Pediatric Cardiology, Children’s Hospital, University of Ulm, 89075 Ulm, Germany 2 National Register for Congenital Heart Defects, 13353 Berlin, Germany 3 Institute for Sport and Sport Science, Karlsruhe Institute for Technology, 76131 Karlsruhe, Germany 4 Praxis am Herz-Jesu-Krankenhaus, 36037 Fulda, Germany Correspondence: christian.apitz@uniklinik-ulm.de; Tel.: +49-73150057127; Fax: +49-73150057105 1 Division of Pediatric Cardiology, Children’s Hospital, University of Ulm, 89075 Ulm, Germany 2 National Register for Congenital Heart Defects, 13353 Berlin, Germany 3 Institute for Sport and Sport Science, Karlsruhe Institute for Technology, 76131 Karlsruhe, Germany 4 Praxis am Herz-Jesu-Krankenhaus, 36037 Fulda, Germany * Correspondence: christian.apitz@uniklinik-ulm.de; Tel.: +49-73150057127; Fax: +49-73150057105 Abstract: Background: Physical activity is important for children with congenital heart defects (CHD), not only for somatic health, but also for neurologic, emotional, and psychosocial development. Swimming is a popular endurance sport which is in general suitable for most children with CHD. Since we have previously shown that children with CHD are less frequently physically active than their healthy peers, we hypothesized that the prevalence of non-swimmers is higher in CHD patients than in healthy children. Methods: To obtain representative data, we performed a nationwide survey in collaboration with the German National Register of Congenital Heart Defects (NRCHD) and the Institute for Sport Sciences of the Karlsruhe Institute for Technology (KIT). The questionnaire included questions capturing the prevalence of swimming skills and the timing of swim learning and was part of the “Motorik-Modul” (MoMo) from the German Health Interview and Examination Survey for Children and Adolescents (KiGGS). A representative age-matched subset of 4569 participants of the MoMo wave two study served as a healthy control group. Results: From 894 CHD-patients (mean age of 12.5 ± 3.1 years), the proportion of non-swimmers in children with CHD was signiﬁcantly higher (16% versus 4.3%; p < 0.001) compared to healthy children and was dependent on CHD severity: Children with complex CHD had an almost ﬁve-fold increased risk (20.4%) of being unable to swim, whereas in children with simple CHD, the ability to swim did not differ signiﬁcantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not signiﬁcant). Citation: Apitz, C.; Tobias, D.; Helm, P.; Bauer, U.M.; Niessner, C.; Siaplaouras, J. Nationwide Survey Reveals High Prevalence of Non-Swimmers among Children with Congenital Heart Defects. Children 2023, 10, 988. https:// doi.org/10.3390/children10060988 Keywords: congenital heart defects; pediatric cardiology; sports; swimming; prevention Academic Editors: P. Syamasundar Rao and Bibhuti B. Das Received: 27 March 2023 Revised: 23 April 2023 Accepted: 29 May 2023 Published: 31 May 2023 Christian Apitz 1,*, Dominik Tobias 1, Paul Helm 2, Ulrike M. Bauer 2, Claudia Niessner 3 and Jannos Siaplaouras 1,4 Conclusions: According to our results, one in ﬁve patients with complex CHD are non-swimmers, a situation that is concerning in regard of motoric development, inclusion and integration, as well as prevention of drowning accidents. Implementation of swim learning interventions for children with CHD would be a reasonable approach. children children children children 1. Introduction Physical exercise and sports are of paramount importance for children with congenital heart disease (CHD), not just for the acquisition of motoric skills, but also for their cognitive, emotional, and psychosocial development. Current recommendations for physical activity, recreational sports, and exercise training in pediatric patients with CHD advise “to comply with public health recommendations of daily participation in 60 min or more of moderate-to- vigorous physical activity that is developmentally appropriate and enjoyable and involves a variety of activities” [1]. Swimming is a popular endurance sport, which is usually suitable for most children with CHD because of its mainly dynamic characteristics. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). y y However, it has previously shown that children with CHD only rarely follow the recommended levels of physical activity. Various causes can be attributed to this: Cautious- ness or even overprotection of parents, caregivers, and sports teachers, and furthermore, https://www.mdpi.com/journal/children Children 2023, 10, 988. https://doi.org/10.3390/children10060988 Children 2023, 10, 988 2 of 7 misperception of physicians and health care professionals regarding the risks and beneﬁts of physical activity [2–9]. These uncertainties might result in the avoidance of contact with various sports, not least swimming. However, being unable to swim is not only concerning in regard to development and integration but can also have dramatic consequences. Drowning remains a leading cause of evitable death in children [10–12]. According to statistical data from the German Life Saving Association (Deutsche Lebens-Rettungs-Gesellschaft, DLRG), 355 people died by drowning in Germany in 2022, and 46 of those were children or adolescents [13]. In addition, there were more than 100 near-drowning accidents making inpatient treatment necessary. Among children who survive near-drowning, health sequelae occur in about 7.5% of cases. The main reason for drowning or near-drowning in children over four years is minimal swim training or even a complete lack of training [11,12]. Steps to prevent drowning, therefore, include teaching children to swim, complemented by education on water safety [11]. In a previous national survey study, we were able to show that children with CHD in Germany were less frequently physically active than their healthy peers [14]. Therefore, we hypothesized that the prevalence of non-swimmers might also be higher in CHD patients than in healthy children. 2. Methods To obtain representative data, we performed a nationwide survey in collaboration with the German National Register of Congenital Heart Defects (NRCHD) and the Institute for Sport Sciences of the Karlsruhe Institute for Technology (KIT). The cross-sectional online survey was conducted from November to December 2021. For patient recruitment, the database of the NRCHD was scanned for patients with an age range from 6 to 17 years on the date of launching the survey. Patients and their respective families were contacted by email and invited to participate in the study. The protocol was approved by the institutional ethical committee. The questionnaire included questions capturing the prevalence of swimming skills and the timing of swim learning and was part of the “Motorik-Modul” (MoMo) from the German Health Interview and Examination Survey for Children and Adolescents (KiGGS). Design and results of the MoMo Baseline and Longitudinal Study have been published previously [15]. A healthy control group served a representative age-matched subset of 4569 participants of the MoMo wave 2 study. Statistical analysis was performed by IBM SPSS statistics version 25.0 (IBM Inc., Ar- monk, NY, USA). Values of continuous variables are reported as mean ± standard deviation. The Pearson’s chi-square test was used for group comparisons. A p value less than 0.05 was considered statistically signiﬁcant. 3. Results A total of 1647 patients agreed to participate in the online survey, and 894 patients (mean age of 12.5 ± 3.1 years; 47.2% female) completed the questionnaire. The study participants were allocated according to the anatomic complexity into simple (23.8%), moderate (37.8%), and complex CHD (38.4%) (Table 1) [16]. Genetic syndromes and chromosomal disorders were present in 72 patients (8%), most frequently Down syndrome in 45 patients (5%) and Di-George-Syndrome in 12 patients (1.3%) (Table 2). Children 2023, 10, 988 3 of 7 Table 1. Classiﬁcation of congenital heart defects (CHD) of Task Force 1 of the 32nd Bethesda Conference categorizing according to the anatomic complexity into mild, moderate, and complex CHD (adapted with permission from Ref. [16], from Copyright Clearance Centers Rightslink service (Date 30 May 2023; No 5558971108981), Licensee: Christian Apitz). Table 1. Classiﬁcation of congenital heart defects (CHD) of Task Force 1 of the 32nd Bethesda Conference categorizing according to the anatomic complexity into mild, moderate, and complex CHD (adapted with permission from Ref. [16], from Copyright Clearance Centers Rightslink service (Date 30 May 2023; No 5558971108981), Licensee: Christian Apitz). 3. Results Simple CHD Moderate CHD Complex CHD Isolated congenital aortic valve disease Aorto-left ventricular ﬁstulas Conduits, valved or nonvalved Isolated congenital mitral valve disease (e.g., except parachute valve, cleft leaﬂet) Anomalous pulmonary venous drainage, partial or total Cyanotic congenital heart (all forms) Small atrial septal defect Atrioventricular septal defects (partial or complete) Double-outlet ventricle Isolated small ventricular septal defect (no associated lesions) Coarctation of the aorta Eisenmenger syndrome Mild pulmonary stenosis Ebstein’s anomaly Fontan procedure Small patent ductus arteriosus Infundibular right ventricular outﬂow obstruction of signiﬁcance Mitral atresia Previously ligated or occluded ductus arteriosus Ostium primum atrial septal defect Single ventricle (also called double inlet or outlet, common, or primitive) Repaired secundum or sinus venosus atrial septal defect without residua Patent ductus arteriosus (not closed) Pulmonary atresia (all forms) Repaired ventricular septal defect without residua Pulmonary valve regurgitation (moderate to severe) Pulmonary vascular obstructive disease Pulmonary valve stenosis (moderate to severe) Transposition of the great arteries Sinus of Valsalva ﬁstula/aneurysm Tricuspid atresia Sinus venosus atrial septal defect Truncus arteriosus/hemitruncus Subvalvular AS or SupraAS (except HOCM) Other abnormalities of atrioventricular or ventriculoarterial connection not included above (ie, crisscross heart, isomerism, heterotaxy syndromes, ventricular inversion) Tetralogy of Fallot Ventricular septal defect with: Absent valve or valves, Aortic regurgitation, Coarctation of the aorta, Mitral disease, Right ventricular outﬂow tract obstruction, Straddling tricuspid/mitral valve, Subaortic stenosis Children 2023, 10, 988 4 of 7 Table 2. Prevalence of Syndromes among the included CHD-patients. Syndrome Numbers of Patients Percentage of Total (n = 894) Down-Syndrome 45 5% Di George-Syndrome 12 1.3% Noonan-Syndrome 3 0.3% Williams-Beuren-Syndrome 2 0.2% Vacterl-Syndrome 2 0.2% Edwards-Syndrome 2 0.2% Charge-Syndrome 1 0.1% Kartagener-Syndrome 1 0.1% Holt-Oram-Syndrome 1 0.1% XXY-Klinefelter-Syndrome 1 0.1% Goldenhar-Syndrome 1 0.1% Leopard-Syndrome 1 0.1% Total 72 8% Syndrome Numbers of Patients Percentage of Total (n = 894) Down-Syndrome 45 5% Di George-Syndrome 12 1.3% Noonan-Syndrome 3 0.3% Williams-Beuren-Syndrome 2 0.2% Vacterl-Syndrome 2 0.2% Edwards-Syndrome 2 0.2% Charge-Syndrome 1 0.1% Kartagener-Syndrome 1 0.1% Holt-Oram-Syndrome 1 0.1% XXY-Klinefelter-Syndrome 1 0.1% Goldenhar-Syndrome 1 0.1% Leopard-Syndrome 1 0.1% Total 72 8% Compared to healthy children, the prevalence of non-swimmers in children with CHD i ifi l hi h (16% 4 3% 0 001) d d d CHD Table 2. Prevalence of Syndromes among the included CHD-patients. 3. Results Syndrome Numbers of Patients Perc Down-Syndrome 45 Compared to healthy children, the prevalence of non-swimmers in children with CHD was signiﬁcantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD-severity: Children with complex CHD had an almost ﬁve-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to swim, while in children with simple CHD, the ability to swim did not differ signiﬁcantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not signiﬁcant) (Figure 1). CHD was significantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD- severity: Children with complex CHD had an almost five-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable o swim, while in children with simple CHD, the ability to swim did not differ significantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not significant) (Figure 1). Figure 1. Prevalence of non-swimmers in children with congenital heart defects (CHD) and in the healthy reference group (in percent). Compared to healthy children, the proportion of non- wimmers in children with CHD was significantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD severity: Children with complex CHD had an almost five-fold increased risk 20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to wim, while in children with simple CHD, the ability to swim did not differ significantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not significant). Figure 1. Prevalence of non-swimmers in children with congenital heart defects (CHD) and in the healthy reference group (in percent). Compared to healthy children, the proportion of non-swimmers in children with CHD was signiﬁcantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD severity: Children with complex CHD had an almost ﬁve-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to swim, while in children with simple CHD, the ability to swim did not differ signiﬁcantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not signiﬁcant). Figure 1. Prevalence of non-swimmers in children with congenital heart defects (CHD) and in the healthy reference group (in percent). 3. Results Compared to healthy children, the proportion of non- swimmers in children with CHD was significantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD severity: Children with complex CHD had an almost five-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to swim, while in children with simple CHD, the ability to swim did not differ significantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not significant). Figure 1. Prevalence of non-swimmers in children with congenital heart defects (CHD) and in the healthy reference group (in percent). Compared to healthy children, the proportion of non-swimmers in children with CHD was signiﬁcantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD severity: Children with complex CHD had an almost ﬁve-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to swim, while in children with simple CHD, the ability to swim did not differ signiﬁcantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not signiﬁcant). Figure 1. Prevalence of non-swimmers in children with congenital heart defects (CHD) and in the healthy reference group (in percent). Compared to healthy children, the proportion of non- swimmers in children with CHD was significantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD severity: Children with complex CHD had an almost five-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to swim, while in children with simple CHD, the ability to swim did not differ significantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not significant). Figure 1. Prevalence of non-swimmers in children with congenital heart defects (CHD) and in the healthy reference group (in percent). Compared to healthy children, the proportion of non-swimmers in children with CHD was signiﬁcantly higher (16% versus 4.3%; p < 0.001) and was dependent on CHD severity: Children with complex CHD had an almost ﬁve-fold increased risk (20.4%), children with moderate CHD a more than four-fold increased risk (18%) of being unable to swim, while in children with simple CHD, the ability to swim did not differ signiﬁcantly from their healthy reference group (5.6% vs. 4.3% non-swimmers (p = not signiﬁcant). 4. Discussion Integration in the sense of inclusion, i.e., disabled people taking part in community life on equal terms, is a substantial component of the health care of children with chronic diseases including those with CHD. As sports activities are an integral part of successful integration, therefore, all patients with CHD can and should participate in physical activity and exercise. Restrictions should be avoided, except for patients with speciﬁc lesions or complications, who may require counselling regarding precautions and speciﬁc recommen- dations [1]. Swimming is a type of sport with positive effects on cardiorespiratory function and health, that can be recommended to most patients with CHD, and beyond being a competitive sport it is also an important leisure activity for social well-being. However, according to this nationwide survey, more than 20 percent of patients with complex CHD are non-swimmers, thus not taking part in this important inclusion activity, and on the other side theoretically carrying the risk of drowning accidents. There are obviously various potential reasons for this concerning situation. Usually, the swimming learning starts at the age of 4–6 years. However, this is an age, where patients with complex CHD such as Fontan patients frequently get involved to major surgeries or catheter-based interventions and therefore, especially at this point of life, things such as swim-learning must commonly be postponed, or even will be skipped completely. Concomitant handicaps due to syndromal disorders or neurological sequelae of previous interventions may also affect swim education in some patients. Additionally, misperceptions regarding the risks of swimming, as well as the overprotection of parents and physicians might also be potential contributing factors [8,9,14]. Recently, further obstacles arose due to the current global energy crisis with resulting increased energy costs more and more public indoor swimming pools have been closed or have markedly increased entrance fees, reducing the availability of access to swimming waters for children and adolescents. In addition, the lack of staff partly due to the COVID-19 pandemic results in a reduction of swimming lessons in elementary and secondary schools. As an exemplary approach to providing relief, in 2022, the German Federal Association for children with CHD (Bundesverband Herzkranke Kinder e.V., BVHK) offered children with CHD family weekends with swimming lessons especially for children with CHD at various locations in Germany [17]. 4. Discussion Most of these children are particularly dependent on very individual, competent support because every heart defect is different and every child with CHD has different limitations [1,18]. This is a very pragmatic approach and might improve inclusion and integration, as the acquisition of swimming skills might help to address the imbalance in opportunities available to children with a disability to participate in sports and physical activity with their healthy peers, in addition, might reduce or even avoid drowning in this sensitive patient group in the future. The presented study has some limitations: Editing of the questionnaire was performed by the patients/families themselves. Therefore, data can theoretically be prone to bias, including recall bias and misjudgement as swimming skills might be over- or underesti- mated. Furthermore, the results might be affected by the social, educational, and economic situation of the children and their families. The strength of this study, however, is the large-scaled sample size and the representa- tive reference group. 3. Results Of those, who were speciﬁed as swimmers, the vast majority acquired their swimming skills between 4 to 12 years of age. Only 1.2% of CHD patients and 0.4% of healthy peers learnt swimming beyond the age of 12 years. Children 2023, 10, 988 5 of 7 5. Conclusions According to our results, one in ﬁve complex CHD patients are non-swimmers, a situation that is concerning in regard to inclusion and integration, as well as the prevention of drowning accidents. Implementation of swim learning interventions for children with CHD would be a reasonable approach. Children 2023, 10, 988 6 of 7 Author Contributions: Conceptualization, C.A., P.H., C.N. and J.S.; methodology, P.H. and C.N.; software, P.H.; validation, P.H. and C.N.; formal analysis, D.T. and P.H.; investigation, C.A., P.H. and J.S.; resources, P.H. and C.N.; data curation, P.H. and C.N.; writing—original draft preparation, C.A.; writing—review and editing, P.H., U.M.B., D.T., C.N. and J.S.; visualization, C.A.; project administration, U.M.B.; funding acquisition, C.A., P.H., U.M.B. and J.S. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the non-proﬁt organization “Fördergemeinschaft Deutsche Kinderherzzentren” (grant number W-B-009/2020). Funding: This study was supported by the non-proﬁt organization “Fördergemeinschaft Deutsche Kinderherzzentren” (grant number W-B-009/2020). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Charité University Medicine Berlin (protocol code 2/247/20, date of approval 9 December 2020). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Charité University Medicine Berlin (protocol code 2/247/20, date of approval 9 December 2020). Informed Consent Statement: Informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Data Availability Statement: Additional data can be obtained from the authors. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Physical activity, sports participation and aerobi have undergone surgery for congenital heart defects. Acta Paediatr. 2009, 98, 1475–1482. 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https://openalex.org/W4381309705	https://www.researchsquare.com/article/rs-2745368/latest.pdf	English	null	The treatment with sGC stimulator improves survival of hypertensive rats in response to volume-overload induced by aorto-caval fistula	Naunyn-Schmiedeberg's archives of pharmacology	2,023	cc-by	16,243	The treatment with sGC stimulator improves survival of hypertensive rats with volume-overload induced heart failure and cardio-renal syndrome Olga Gawrys ( olga.gawrys@ikem.cz ) The treatment with sGC stimulator improves survival of hypertensive rats with volume-overload induced heart failure and cardio-renal syndrome Olga Gawrys ( olga gawrys@ikem cz ) survival of hypertensive rats with volume-over induced heart failure and cardio-renal syndrom Olga Gawrys (  olga.gawrys@ikem.cz ) Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Zuzana Husková Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Petra Škaroupková Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Zuzana Honetschlägerová Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Zdeňka Vaňourková Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Soňa Kikerlová Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Vojtěch Melenovský Department of Cardiology, Institute for Clinical and Experimental Medicine Barbara Szeiffová Bačová Centre of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research Matúš Sykora Centre of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research Miloš Táborský Department of Internal Medicine I, Cardiology, University Hospital Olomouc and Palacký Un Luděk Červenka Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Olga Gawrys (  olga.gawrys@ikem.cz ) Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Zuzana Husková Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Petra Škaroupková Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Zuzana Honetschlägerová Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Zdeňka Vaňourková Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Soňa Kikerlová Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Vojtěch Melenovský Department of Cardiology, Institute for Clinical and Experimental Medicine Barbara Szeiffová Bačová Centre of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research Matúš Sykora Centre of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research Miloš Táborský Department of Internal Medicine I, Cardiology, University Hospital Olomouc and Palacký University Luděk Červenka Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Research Article Keywords: heart failure, ACF, sGC stimulator, BAY41-8543, cGMP, vericiguat Posted Date: March 30th, 2023 DOI: https://doi org/10 21203/rs 3 rs 2745368/v1 Olga Gawrys (  olga.gawrys@ikem.cz ) Barbara Szeiffová Bačová Centre of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research Matúš Sykora Centre of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research Miloš Táborský Department of Internal Medicine I, Cardiology, University Hospital Olomouc and Palacký University Luděk Červenka Center for Experimental Medicine, Institute for Clinical and Experimental Medicine Research Article Page 1/40 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. Version of Record: A version of this preprint was published at Naunyn-Schmiedeberg's Archives of Pharmacology on June 20th, 2023. See the published version at https://doi.org/10.1007/s00210-023- 02561-y. Page 2/40 Abstract Purpose: Heart Failure (HF) has been declared as global pandemic and current therapies are still ineffective, especially in patients that develop concurrent cardio-renal syndrome. Considerable attention has been focused on the nitric oxide (NO)/ soluble guanylyl cyclase (sGC)/ cyclic guanosine monophosphate (cGMP) pathway. In the current study we aimed to investigate the effectiveness of sGC stimulator (BAY41-8543) with the same mode of action as vericiguat, for the treatment of heart failure (HF) with cardio-renal syndrome. Methods: As a model we chose heterozygous Ren-2 transgenic rats (TGR), with high-output heart failure, induced by aorto-caval fistula (ACF).The rats were subjected into three experimental protocols to evaluate short term effects of the treatment, impact on blood pressure and finally the long term survival lasting 210 days. As control groups we used hypertensive sham TGR and normotensive sham HanSD rats. Results: We have shown that the sGC stimulator effectively increased the survival of rats with HF in comparison to untreated animals. After 60 days of sGC stimulator treatment the survival was still 50% compared to 8 % in the untreated rats. One week treatment with sGC stimulator increased the excretion of cGMP in ACF TGR (109±28 nnmol/12h), but the ACE inhibitor decreased it (-63±21 nnmol/12h). Moreover, sGC stimulator caused a decrease in SBP, but this effect was only temporary (day 0: 117±3; day 2: 108±1; day 14: 124±2 mmHg). Conclusion: These results support the concept that sGC stimulators represent a valuable class of drugs to battle heart failure especially with cardio-renal syndrome. 1. Introduction Heart failure (HF) represents a major health and socioeconomic burden with considerable morbidity and mortality. It is estimated that it affects around 23 million people globally (Murphy et al. 2020). HF can be categorised into three main groups based on the ejection fraction (EF), i.e. heart failure with reduced EF (HFrEF), heart failure with mid-range or mildly reduced EF (HFmrEF), and heart failure with preserved ejection fraction (HFpEF) (Simmonds et al. 2020). Over the recent decades a significant improvement has been made in the management and treatment of HF. Current guideline-directed medical therapy (GDMT) includes combination of β-blockers, angiotensin-converting enzyme inhibitors (ACEi), angiotensin receptor–neprilysin inhibitors (ARNI), angiotensin receptor blocker (ARB) with an addition of a mineralocorticoid receptor antagonists, diuretics and more recently sodium-glucose cotransporter 2 (SGLT2) inhibitors (McDonagh et al. 2021; Heidenreich et al. 2022). The prognosis and life expectancy is especially dreadful for patients that develop concurrent impairment of renal hemodynamic and sodium excretory function, so called cardio-renal syndrome (Rangaswami et al. 2019; McCullough et al. 2022). It is now commonly acknowledged that the reciprocal interaction between cardiac and renal function plays critical role in the progression of HF (Mullens et al. 2017; Ciccarelli et al. 2021; McCullough et al. 2022). Current GDMT are still ineffective in the treatment of HF, especially with cardio-renal syndrome, and the Page 3/40 Page 3/40 morbidity and mortality still remain very high, with a 5-year survival rate of 25% after first hospitalization (Berliner et al. 2020; Murphy et al. 2020). Hence new treatment strategies are still urgently needed to improve the morbidity and mortality. Lately particular attention has been focused on the nitric oxide (NO)/ soluble guanylyl cyclase (sGC)/ cyclic guanosine monophosphate (cGMP) pathway. In the classical cascade NO binds to sGC, which leads to a conversion of guanosine triphosphate (GTP) into cGMP (Singh et al. 2018). In turn, this secondary messenger, cGMP, acts on various effector molecules including protein kinases (PK) or phosphodiesterases (PDE), initiating a number of downstream effects, such as vasodilation and myocardial relaxation. It was proved to exhibit anti-inflammatory, anti-proliferatory, anti-fibrosis and renal protective properties, making this pathway one of the most essential signalling cascades within the cardiovascular and renal systems. Its disruption can lead to development of serious disorders or progression of already existing diseases, such as heart failure, pulmonary hypertension or chronic kidney disease (Farah et al. 2018; Sandner et al. 2021a; Xia et al. 2022). 1. Introduction So far several drug classes targeting NO-sGC-cGMP pathway have been developed, such as NO donors (e.g. nitroglycerine) or PDE5 inhibitors (e.g. sildenafil) (Roberto B. Evora et al. 2012) and more recently sGC stimulators and sGC activators were introduced (Cordwin et al. 2021). The sGC activators (e.g. cinaciguat) activate both the oxidized and heme-free sGC, while sGC stimulators increase cGMP production independently of NO by activating the reduced heme moiety (Cordwin et al. 2021). sGC stimulators, such as riociguat and vericiguat, exhibit also synergistic activity with NO, because they sensitize sGC to low levels of endogenous NO by stabilizing NO–sGC binding (Stasch et al. 2001; Liu et al. 2021). Vericiguat was recently approved for the treatment of heart failure with reduced ejection fraction based on positive outcome of a large clinical trial in patients with HFrEF (Armstrong et al. 2020). However, the exact mode of action of sGC stimulators and how these beneficial effects in HF are mediated are not fully understood yet and particularly it is unknown if it will be effective in cardio-renal syndrome. In the current work, we therefore, treated hypertensive, heterozygous Ren-2 transgenic rats (TGR), with high-output heart failure, induced by creating an aorto-caval fistula (ACF) with the sGC stimulator (BAY41- 8543), which exhibits the same mode of action as riociguat and vericiguat (Sandner et al. 2021b). ACF TGR model is a well-established model of volume-overload heart failure and it is routinely used in our laboratory for many years (Abassi et al. 2011; Honetschlägerová et al. 2021; Kala et al. 2021, 2023), because it represents a model of HF accompanied with development of cardio-renal syndrome. In addition, we also investigated an optimized dose of an ACE inhibitor (trandolapril) as the standard therapy for HF as positive control. Animals Page 4/40 Page 4/40 The studies were performed in accordance with guidelines and practices established by the Animal Care and Use Committee of the Institute for Clinical and Experimental Medicine (Prague) approved by the Ministry of Health of the Czech Republic (decision number MZDR 12482/2021-5/OVZ), which accords with the European Union Directive 63/2010 and ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All animals used in the present study were bred at the Center of Experimental Medicine of this Institute (IKEM), from stock animals supplied by the Max Delbrück Center for Molecular Medicine (Berlin, Germany), which is accredited by the Czech Association for Accreditation of Laboratory Animal Care. Heterozygous TGR [transgenic rats, strain name TGR(mRen2)27] harboring the mouse Ren-2 renin gene have been recently generated as a model for the study of primary hypertension. They were generated by breeding male homozygous TGR with female homozygous Hannover-Sprague Dawley (HanSD) rats. Age- matched HanSD rats served as transgene-negative normotensive controls. The animals were kept on a 12-hour/12-hour light/dark cycle and had free access to tap water throughout the whole observation. Male TGR rats at the initial age of 8 weeks were used for experiments. At this age TGR are already in the sustained phase of hypertension with systolic blood pressure (SBP) comparable with hypertensive patients (SBP around 180 mmHg) and with substantial activation of endogenous renin angiotensin system (RAAS), as demonstrated in previous studies including ours. HanSD and TGR rats were randomly assigned to experimental groups to make sure that the animals from a single litter does not prevail in any group. Heart failure model and exclusion criteria Eight-weeks-old male TGR rats were anesthetized with an intraperitoneal injection of ketamine/midazolam mixture (Calypsol, Gedeon Richter, Hungary, 160 mg/kg and Dormicum, Roche, France, 160 mg/kg). Chronic HF due to volume overload was then induced by creating an aorto-caval fistula (ACF) using a needle technique. This procedure is routinely performed in our laboratory and detailed description was reported repeatedly in our previous studies (Honetschlägerová et al. 2021; Kala et al. 2021, 2023). Sham-operated rats underwent an identical procedure, but without creating ACF. The animals in which the ACF procedure was not successful (based on visual verification of vena cava inferior in the end of each observation) were excluded from the experiment. Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pre Ten days before the ACF creation, telemetry probes were implanted into femoral artery of TGR rats (HanSD rats were omitted in this part of the study) under ketamine/midazolam anaesthesia (as above). HD-S10 radiotelemetric probes (Data Science International, St. Paul, Minnesota, USA) were used for direct BP measurements as described previously (Sporková et al. 2014; Husková et al. 2016). After 10 days period of recovery, rats underwent either sham operation or ACF creation as described above. A week after the appropriate treatment was initiated and rats were monitored for two weeks. Series 3: Effects of the long-term treatment with sGC stimulator and ACEi on the survival rate, morphometric and histopathological scores Series 3: Effects of the long-term treatment with sGC stimulator and ACEi on the survival rate, morphometric and histopathological scores After confirmation that the selected dose of sGC stimulator is effective, the long-term protocol was performed. All rats underwent the same ACF creation or sham operation as described above in Series 1. After two weeks (week labelled 0), after exclusion of acute death cases, the rats were randomly divided into the following experimental groups and the follow-up period of 210 days was performed. Because of the severity of the ACF procedure and high mortality (especially in TGR rats) high initial n values were used in these groups (n = 30). In sham operated animals and HanSD rats the initial number of animals was 10 (calculated by statistical power analysis method). After 210 days of the observation, the surviving animals were decapitated and organs were weighted and collected for morphometric and histopathological analysis (left and right ventricle of the heart, kidney). Detailed experimental design Detailed experimental design of all 3 series is presented on FIGURE 1. Detailed experimental design of all 3 series is presented on FIGURE 1. Series 1: Dose selection and target engagement. Effect of the short-term treatment with sGC stimulator and ACEi Series 1: Dose selection and target engagement. Effect of the short-term treatment with sGC stimulator and ACEi Two weeks before the start of the treatment rats underwent sham-operation or ACF creation procedure. After two weeks (week labelled 0) and after exclusion of acute death cases, the rats were randomly divided into the following experimental groups and selected treatment regimens were applied for one Page 5/40 week. On days − 1, 2, 4 and 7 rats were placed in metabolic cages for 12h urine collection. Also blood was collected on days − 5 and 8. After a week of the treatment, all rats were decapitated and organs were weighted and collected for further biochemical evaluation. week. On days − 1, 2, 4 and 7 rats were placed in metabolic cages for 12h urine collection. Also blood was collected on days − 5 and 8. After a week of the treatment, all rats were decapitated and organs were weighted and collected for further biochemical evaluation. cGMP was measured in the urine and in the renal tissue. Noradrenaline and angiotensin II were measured in the plasma and the kidneys (collected after decapitation). Albuminuria, natriuresis and daily excretion rate of nitric oxide (NO) metabolites (NOx: nitrate and nitrite; indirect marker of NO production) were evaluated. Additionally, to confirm that the selected dose was effective we have measured the concentration of BAY41-8543 in plasma. Experimental groups 1. Sham-operated HanSD rats treated with placebo (sham HanSD) 2. Sham-operated HanSD rats treated with BAY41-8543 (sham HanSD + sGCstim) 3. Sham-operated TGR treated with placebo (sham TGR) 4. Sham-operated TGR treated with BAY41-8543 (sham TGR + sGCstim) 1. Sham-operated HanSD rats treated with placebo (sham HanSD) 4. Sham-operated TGR treated with BAY41-8543 (sham TGR + sGCstim) Page 6/40 Analytical procedures and chemicals The sGC stimulator BAY41-8543 (2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5(4- morpholinyl) -4,6-pyrimidinediamine) was kindly provided by Bayer AG, Pharmaceuticals and is a typical member of the sGC stimulator drug class (Stasch et al. 2002a, b). Standard pellet diet containing BAY41- 8543 in the dose of 3 mg kg− 1day− 1 was prepared (Albert Weber, Prague, Czech Republic). Nutrients (content in 1kg): NL 200g; Fiber 48g; Fat 30g; Vitamin A 24.000 IU; Vitamin D3 2.000 IU; Copper (Cu) 30 mg; NaCl content in the final mix 0.4%. The dose of BAY41-8543 was selected based on the previous study (Stasch et al. 2002b; Sandner et al. 2021b) and current research. As an ACE inhibitor (ACEi) we used trandolapril (Gopten; Abbott, Prague, Czech Republic), which was administered in drinking water. For the best effectiveness and safety, we implemented a titration protocol, which was previously developed and validated in our laboratory. During the first week of administration, the animals received an increasing dose (changed every two days) starting from 0.5 mg/L up to 2 mg/L, which corresponds to a final dose of 0.25 mg kg− 1 day− 1. In our previous studies and here we demonstrated that this titration regimen and selected doses of trandolapril, provided maximal blockade of the renin angiotensin system (RAAS) and were well tolerated both by rats with ACF-induced heart failure and by sham-operated animals. Page 6/40 5. ACF TGR treated with placebo (ACF TGR) 6. ACF TGR treated with BAY41-8543 (ACF TGR + sGCstim) 7. ACF TGR treated with trandolapril (ACF TGR + ACEi) 8. ACF TGR treated with trandolapril and BAY41-8543 (ACF TGR + ACEi + sGCstim) 5. ACF TGR treated with placebo (ACF TGR) 6. ACF TGR treated with BAY41-8543 (ACF TGR + sGCstim) 7. ACF TGR treated with trandolapril (ACF TGR + ACEi) 8. ACF TGR treated with trandolapril and BAY41-8543 (ACF TGR + ACEi + sGCstim) 5. ACF TGR treated with placebo (ACF TGR) 6. ACF TGR treated with BAY41-8543 (ACF TGR + sGCstim) 7. ACF TGR treated with trandolapril (ACF TGR + ACEi) The numbers of rats used in each series is described in the appropriate result section. SDS-PAGE and Western Blotting According to our previous studies (Szeiffová Bačova et al. 2016; Sykora et al. 2023), approximately 100 mg of frozen left ventricular heart tissue was homogenized in lysis buffer [50 mmol/L Tris-HCl, 250 sucrose, 1.0 mmol/L EGTA, 1.0 mmol/L dithiothreitol, 1.0 mmol/L phenylmethylsulfonyl fluoride and 0.5 sodium orthovanadate (pH 7.4)] and mixed with Laemmli sample buffer. Loading samples were separated in 10% SDS-PAGE (Mini-Protean TetraCell, Bio-Rad, Hercules, CA, USA) and transferred to a nitrocellulose membrane (0.2 µm pore size, Advantec, Tokyo, Japan). Membranes were subsequently incubated for 4 h with 5% low-fat milk, overnight with primary antibodies and for 1 h with a horseradish peroxidase-linked secondary antibody (Table 1). Between individual steps were membranes washed in TBS-T. Protein were visualized by enhanced chemiluminescence method and quantitated by densitometric analysis using Carestream Molecular Imaging Software (version 5.0, Carestream Health, New Haven, CT, USA). sitometric analysis using Carestream Molecular Imaging Software (version 5.0, Carestream Health, w Haven, CT, USA). Page 7/40 Table 1 Antibodies used for Western blot analysis and for immunofluorescence methods Antibody Dilution Host Type Supplier/# Catalogue anti-Cx43 1:5000 Rabbit Polyclonal Sigma-Aldrich, St.Louis, MO, USA, #C6219 anti-phospho- ser368-Cx43 1:1000 Rabbit Polyclonal Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101660 anti-PKC-epsilon 1:2000 Rabbit Polyclonal Santa Cruz Biotechnology, Dallas, TX, USA, #sc-214 anti-GAPDH 1:1000 Rabbit Polyclonal Santa Cruz Biotechnology, Dallas, TX, USA#sc-25778 Anti-Rabbit 1:2000 - - Cell Signaling Technology, Danvers, MA, USA, #7074S anti-Cx43 1:500 Mouse Monoclonal CHEMICON International,CA, USA, #MAB 3068 Anti-Mouse, FITC 1:500 Goat Polyclonal Jackson Immuno Research Labs, West Grove, Pennsylvania, USA, #115-095-062 Immunofluorescence detection of Cx43 and Quantitative Image Analysis Table 1 mmunofluorescence detection of Cx43 and Quantitative Image Analysis Immunodetection of Cx43 distribution was performed as described previously (Benova et al. 2013). Briefly, 10 µm thick left ventricular cryosections were washed in phosphate buffer saline (PBS), fixed in ice-cold methanol, permeabilized in 0.3% Triton X-100 in PBS, and blocked with the solution of 1% bovine serum albumin in PBS. Tissue sections were incubated overnight with primary antibody followed by 2- hour incubation with secondary antibody (Table 1). Between individual steps were membranes washed in PBS. Finally, tissues sections were mounted in the Fluoromount-G™ Mounting Medium (00-4958-02, Invitrogen™, Massachusetts, USA) and analysed by Zeiss Apotome 2 microscope (Carl Zeiss, Jena, Germany). Approximately ten randomly acquired images from every tissue were captured and analysed. SDS-PAGE and Western Blotting Immunofluorescence signals were analysed and defined as a number of pixels with the protein signal intensity exceeding a threshold of 30 on the 0–255 Gy scale. The total number of Cx43 positive pixels was expressed as a total integral optical density per area (IOD) (Image-Pro Plus) (Sykora et al. 2019). Evaluation of glomerulosclerosis index (GSI) and tubulointerstitial injury (TSI) The kidneys were fixed in 4% formaldehyde, dehydrated and embedded in paraffin. The sections stained with hematoxylin-eosin and PAS (periodic acid, for Schiff reaction) were examined and evaluated in a blind-test fashion. The calculation of glomerulosclerosis index (GSI) and kidney cortical tubulointerstitial injury (TSI) was described in detail in previous studies (Nakano et al. 2008; Gawrys et al. 2018; Honetschlagerová et al. 2021; Kala et al. 2023). The maximum score for GSI is 4 and for TSI is 3. Described methods are commonly employed in our laboratory for many years and standardly used for evaluating the degree of kidney damage in almost all our studies (Kujal et al. 2014; Sedláková et al. 2017; Honetschlagerová et al. 2021). The kidneys were fixed in 4% formaldehyde, dehydrated and embedded in paraffin. The sections stained with hematoxylin-eosin and PAS (periodic acid, for Schiff reaction) were examined and evaluated in a blind-test fashion. The calculation of glomerulosclerosis index (GSI) and kidney cortical tubulointerstitial injury (TSI) was described in detail in previous studies (Nakano et al. 2008; Gawrys et al. 2018; H hl á l 2021 K l l 2023) Th i f GSI i 4 d f TSI i 3 injury (TSI) was described in detail in previous studies (Nakano et al. 2008; Gawrys et al. 2018; Honetschlagerová et al. 2021; Kala et al. 2023). The maximum score for GSI is 4 and for TSI is 3. Described methods are commonly employed in our laboratory for many years and standardly used for evaluating the degree of kidney damage in almost all our studies (Kujal et al. 2014; Sedláková et al. 2017; Honetschlagerová et al. 2021). Described methods are commonly employed in our laboratory for many years and standardly used for evaluating the degree of kidney damage in almost all our studies (Kujal et al. 2014; Sedláková et al. 2017; Honetschlagerová et al. 2021). Plasma and tissue angiotensin II (ANG II) concentrations were measured by a competitive radioimmunoassay, using the commercially available RIA kit (ED29051, IBL Int., Hamburg, Germany). Plasma creatinine was measured by FUJI DRI-CHEM analyzer using appropriate slides for creatinine CRE- P III (FUJIFILM Corp., Tokyo, Japan). Urine creatinine was determined using Liquick Cor-CREATININE kit that is based on modified Jaffe´s method, without deproteinization (PZ CORMAY S.A., Poland). Nitrate/nitrite levels were measured by a colorimetric assay (780001, Cayman Chemical, Ann Arbor, MI, USA). Evaluation of hydroxyproline content Measurement of hydroxyproline is a useful method to determine collagen content in the samples. Briefly, left ventricle tissue was dried, hydrolyzed in 6 M HCl and oxidated by chloramine T in the acetate-citrate buffer (pH 6.0). This reaction was stopped by pipetting Ehrlich’s reagent solution. Final concentration of hydroxyproline was subsequently measured spectrophotometrically at 550 nm and expressed in mg per total weight of the left ventricle (Pelouch et al. 1993; Reddy and Enwemeka 1996). Histology and enzyme histochemistry of myocardial tissue Histology and enzyme histochemistry of myocardial tissue Page 8/40 Page 8/40 According to Lojda (Lojda and Gutmann 1976) with modifications (Andelova et al. 2022), 10 µm thick left ventricular myocardial tissue cryosections were used for histological hematoxylin–eosin staining and enzyme histochemical demonstration of alkaline phosphatase (AP). For structural changes characterization, haematoxylin–eosin staining was used. Dried cryosection were fixed in 4% buffered formaldehyde, stained with hematoxylin - eosin solutions, poured with Canada balsam and covered with a coverslip. For measurement of capillary related activity of AP (E.C.3.1.3.1), cryosections were incubated in the mixture of solution (1.2 mM L-Leucine 4-methoxy-β-naptylamide hydrochloride; 5% dimethylformamide; 2.4 mM Fast blue BB; 0.1 M Na2 HPO4 x 2H2O; 1 M KH2PO4), poured with gelatin and covered with a coverslip. Staining areas were observed and captured by light microscope (Zeiss Apotome 2 microscope Carl Zeiss, Jena, Germany). For quantitative analysis, ten randomly selected areas of positive signal from every tissue were analysed. The intensity of staining corresponding to the activity of AP, was defined as an area, with a number of pixels, with a code lower than 128 on the "0-255 RGB colour scale”. The total number of positive pixels was expressed as a total integral optical density per area (IOD) (Image-Pro Plus) (Andelova et al. 2022). Evaluation of glomerulosclerosis index (GSI) and tubulointerstitial injury (TSI) Commercially available ELISA kits were used to measure: renal nitrotyrosine (ab113848; Abcam, Cambridge, UK); renal cGMP (ADI-900-013; Enzo, Farmingdale, NY, USA) and plasma cGMP (581021; Cayman Chem., Ann Arbor, MI, USA); plasma and renal noradrenalin (RE59261; IBL Int., Hamburg, Germany); urine 8-isoprostane (516351; Cayman Chem., Ann Arbor, MI, USA). Sodium and potassium in plasma and urine were measured by BWB-XP flame photometer (BWB Technologies Ltd., Berkshire, UK). Page 9/40 Page 9/40 Detailed protocols of plasma and tissue preparation are described in our previous studies (Husková et al. 2006, 2007, 2016; Červenka et al. 2015a; Kratky et al. 2018; Gawrys et al. 2020). Detailed protocols of plasma and tissue preparation are described in our previous studies (Husková et al. 2006, 2007, 2016; Červenka et al. 2015a; Kratky et al. 2018; Gawrys et al. 2020). Data and Statistical Analysis All values are expressed as means ± SEM. Graph-Pad Prism software (Graph Pad Software, San Diego, California, USA) was used for statistical analysis of the data. Comparison of survival curves was performed by log-rank (Mantel-Cox) test. Multiple-group comparisons were performed by multiple t test, Wilcoxon´s signed-rank test, one-way or two-way analysis of variance followed by recommended post hoc test as appropriate. Values exceeding the 95% probability limits (P < 0.05) were considered statistically significant. The significance levels are indicated on figures with asterisks: P > 0.05 (NS); P ≤ 0.05; P ≤ 0.01; P ≤ 0.001; *P ≤ 0.0001. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al. 2018). Series 1: Dose selection and target engagement. Effect of the short-term treatment with sGC stimulator and ACEi (FIGURE 2 and 3; Table 2) 3. Results Series 1: Dose selection and target engagement. 3. Results Effect of the short-term treatment with sGC stimulator and ACEi (FIGURE 2 and 3; Table 2) Page 10/40 Table 2 Table 2 Body weight (BW), plasma and urine parameters collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats treated for one week with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined (n 8 13 in each group); Body weight (BW), plasma and urine parameters collected from heterozygous Ren 2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats treated for one week with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined (n = 8–13 in each group); sham TGR sham TGR + sGCstim ACF TGR ACF TGR + sGCstim ACF TGR + ACEi ACF TGR + sGCstim + ACEi sham HanSD sham HanSD + sGCstim a b c d e f g h BW (g) 449 ± 13 441 ± 6 408 ± 5 429 ± 10 415 ± 9 425 ± 16 455 ± 9 470 ± 10 UNa V (mmol/12h) 1.7 ± 0.1 2.0 ± 0.2 0.7 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 1.2 ± 0.1 c,d 1.0 ± 0.1 0.6 ± 0.1 UK V (mmol/12h) 2.4 ± 0.2 2.0 ± 0.1 1.8 ± 0.1 1.7 ± 0.1 1.9 ± 0.1 2.1 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 UNOx (µmol/12h) 5.2 ± 0.1 4.2 ± 0.4 e,g, 5.2 ± 0.5 4.3 ± 0.4 e,g, # 7.4 ± 0.6 h 4.6 ± 0.6 e,g 7.3 ± 0.4 3.4 ± 0.4 c,g U 8- isoprostane (ng/12h) 8.5 ± 1.2 12.7 ± 1.1 15.8 ± 1.8 a 15.3 ± 1.3 12.5 ± 1.7 12.0 ± 1.8 7.8 ± 0.6 c,d 9.9 ± 1.2 Plasma Na (mmol/L) 137 ± 4 143 ± 5 139 ± 3 137 ± 4 136 ± 2 131 ± 2 134 ± 3 132 ± 2 Plasma K (mmol/L) 4.4 ± 0.2 4.3 ± 0.1 4.8 ± 0.1 4.5 ± 0.1 5.1 ± 0.2 4.6 ± 0.1 4.4 ± 0.1 4.0 ± 0.1 Plasma Cr (µmol/L) 15 ± 1 14 ± 1 18 ± 1 16 ± 1 17 ± 1 21 ± 2 18 ± 2 13 ± 1 Plasma NE (ng/ml) 2005 ± 420 3586 ± 856 2989 ± 192 2095 ± 478 3174 ± 1110 2899 ± 740 1560 ± 579 631 ± 93 Renal NE (ng/g prot.) 211 ± 21 161 ± 12 d 278 ± 20 264 ± 39 286 ± 59 338 ± 51 293 ± 22 239 ± 17 Renal Nitrotyrosine (ng/mg prot.) 26.5 ± 2.3 18.5 ± 1.1 19.7 ± 1.6 17.1 ± 2.4 18.5 ± 2.7 15.1 ± 2.0 a 20.7 ± 3.5 16.3 ± 2.0 BW – body weight; UNa V – urinary sodium excretion; UK V – urinary potassium excretion; UNOx – urinary excretion of nitric oxide metabolites (nitrate/nitrite); Cr – creatinine; NE – noradrenaline; Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * p < 0.05 sham TGR + sGCstim. 3. Results vs untreated ACF TGR by unpaired t test; The first series of experiments was aimed to evaluate the short term effects of treatment with sGC stimulator to support dose selection and demonstrate target engagement. As a marker of target engagement for the sGC stimulator treatment, cGMP excretion in urine was measured. As depicted in FIGURE 2 the excretion of cGMP increased in all groups treated with sGC stimulator (BAY41-8543). The treatment with ACEi decreased the cGMP levels in urine which could not be compensated by sGC stimulator treatment. Interestingly, before the treatment the basal values of urinary cGMP were significantly higher in all groups with heart failure after ACF creation (148.5 ± 7.6 versus 49.6 ± 4.2 nmol/12h in sham operated TGR, # P < 0.0001 unpaired T-Test; FIGURE 2A). Body weight, plasma (sodium, potassium, creatinine, noradrenaline), urine parameters (sodium, potassium, nitric oxide metabolites and 8-isoprostane excretion) and renal levels of noradrenaline and nitrotyrosine collected after one week treatment are presented in Table 2. Urinary excretion of nitric oxide metabolites (NOx: nitrate and nitrite) was measured as an indirect marker of NO production. In all the groups which received sGC stimulator, there was a significant or tendency to decrease the NOx excretion, especially pronounced in sham TGR and sham HanSD rats. The treatment with ACEi increased the excretion of NOx, but after combined therapy with sGC stimulator it was diminished. Plasma and renal levels of ANG II are presented on FIGURE 3 (panel A and B, respectively). Renal ANG II were significantly higher in both sham TGR and ACF TGR in comparison to control sham HanSD rats (81.6 ± 6.2 and 71.0 ± 7.6 versus 38.5 ± 2.4 fmol/g, respectively; # p < 0.05 one way ANOVA; FIGURE 3). The treatment with sGC stimulator significantly increased ANG II in sham HanSD rats, both plasma (17.7 ± 2.6 versus 11.3 ± 0.6 fmol/ml in untreated sham HanSD; p < 0.05 one way ANOVA) and kidney (81.8 ± 6.6 versus 38.5 ± 2.4 fmol/g in untreated sham HanSD; p < 0.05 one way ANOVA); and in ACF TGR rats in the kidney (115.9 ± 18.1 versus 71.0 ± 7.6 fmol/g in untreated TGR rats; p < 0.05 one way ANOVA). 3. Results vs untreated sham TGR by unpaired t test; # p < 0.05 ACF TGR + sGCstim. vs untreated ACF TGR by unpaired t test; BW – body weight; UNa V – urinary sodium excretion; UK V – urinary potassium excretion; UNOx – urinary excretion of nitric oxide metabolites (nitrate/nitrite); Cr – creatinine; NE – noradrenaline; Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * p < 0.05 sham TGR + sGCstim. vs untreated sham TGR by unpaired t test; # p < 0.05 ACF TGR + sGCstim. vs untreated ACF TGR by unpaired t test; Page 11/40 Page 11/40 sham TGR sham TGR + sGCstim ACF TGR ACF TGR + sGCstim ACF TGR + ACEi ACF TGR + sGCstim + ACEi sham HanSD sham HanSD + sGCstim Plasma BAY41-8543 (µg/L) - 29.7 ± 4.5 - 30.8 ± 5.6 - 19.7 ± 2.0 - 26.7 ± 3.4 BW – body weight; UNa V – urinary sodium excretion; UK V – urinary potassium excretion; UNOx – urinary excretion of nitric oxide metabolites (nitrate/nitrite); Cr – creatinine; NE – noradrenaline; Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * p < 0.05 sham TGR + sGCstim. vs untreated sham TGR by unpaired t test; # p < 0.05 ACF TGR + sGCstim. vs untreated ACF TGR by unpaired t test; BW – body weight; UNa V – urinary sodium excretion; UK V – urinary potassium excretion; UNOx – urinary excretion of nitric oxide metabolites (nitrate/nitrite); Cr – creatinine; NE – noradrenaline; Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * p < 0.05 sham TGR + sGCstim. vs untreated sham TGR by unpaired t test; # p < 0.05 ACF TGR + sGCstim. vs untreated ACF TGR by unpaired t test; * p < 0.05 sham TGR + sGCstim. vs untreated sham TGR by unpaired t test; # p < 0.05 ACF TGR + sGCstim. Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pressure (FIGURE 4 and 5) Blood pressure (BP) was measured by telemetry starting four days before ACF creation. The creation of ACF per se caused a significant decrease in mean arterial blood pressure (MBP, FIGURE 4), which is a common phenomenon observed in this model (-52 ± 2 mmHg in all ACF operated groups) (Červenka et al. 2015b). In sham operated animals, the temporary decrease in blood pressure was also observed, probably as a result of anaesthesia and fluid loss during the operation. The treatment with sGC stimulator in sham operated TGR animals caused a significant MBP decrease in comparison to untreated sham TGR (day 0: 140 ± 7 versus 130 ± 2 mmHg after 14 days of treatment). In addition, all applied treatment regimens decreased blood pressure in comparison to untreated ACF TGR (FIGURE 4) including the treatment with sGC stimulator, however the decrease in this groups was only transient (day 0: 94 ± 4 versus 86 ± 2 mmHg after 2 days of treatment; p = 0.03 paired T-test). The course of MBP changes was different between sGC stimulator group and ACEi treated groups, but there were no significant differences between both ACEi treated groups (ACF TGR + ACEi versus ACF TGR + sGCstim + ACEi, NS). In order to better illustrate and evaluate the magnitude of antihypertensive potency of each administered drug, only ACF groups are presented on FIGURE 5 (without sham-operated animals) and the values before the treatment are omitted (before and after ACF creation). The comparison of BP changes within each treated group (compared to baseline values obtained before the implementation of treatments) revealed that the most potent in lowering systolic blood pressure was the combined treatment with ACEi and sGC stimulator (SBP on day 0: 118 ± 5 versus 98 ± 3 mmHg on day 14). In addition, ACEi given alone was clearly antihypertensive, however the statistical analysis did not reveal such strong effect on SBP (SBP on day 0: 119 ± 4 versus 106 ± 2 mmHg on day 14). However, when DBP values were analysed both ACEi treated groups (alone and combined with sGC stimulator) were similarly effective. sGC stimulator caused a decrease in BP (both SBP and DBP) especially pronounced in the beginning, but this effect was only transient and less significant than for other treatment regimens (SBP day 0: 117 ± 3; day 2: 108 ± 1; day 14: 124 ± 2 mmHg). 3. Results Also some increasing tendency was observed in plasma ANG II after sGC stimulator administration to sham TGR (21.8 ± 3.9 versus 16.4 ± 3.6 fmol/ml in untreated sham TGR; NS) and to ACF TGR (21.6 ± 2.3 versus Page 12/40 13.3 ± 1.7 fmol/ml in untreated ACF TGR; p < 0.05 by unpaired T-test). Treatment with ACEi inhibitor decreased the renal level of ANG II, alone and also when combined with sGC stimulator. 13.3 ± 1.7 fmol/ml in untreated ACF TGR; p < 0.05 by unpaired T-test). Treatment with ACEi inhibitor decreased the renal level of ANG II, alone and also when combined with sGC stimulator. To confirm that the selected dose is effective and the rats eat the proper amount of the food mixed with the sGC stimulator, we monitored the food intake and we measured the concentration of BAY41-8543 in plasma in the end of the experiment. There were no differences between groups in the food intake and the plasma levels of sGC stimulator were appropriately elevated in all treated groups (Table 2). Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pressure (FIGURE 4 and 5) Series 3: Effects of the long-term treatment with sGC stimulator and ACEi on survival the rate, morphometric and histopathological scores (FIGURES 6–10, Table 3) Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pressure (FIGURE 4 and 5) The detailed post hoc analysis of changes in each day revealed that already on the 14th day the values of BP (both systolic and diastolic) between untreated ACF TGR and ACF TGR treated with sGC stimulator were no different. Page 13/40 Page 13/40 Page 13/40 Series 3: Effects of the long-term treatment with sGC stimulator and ACEi on survival the rate, morphometric and histopathological scores (FIGURES 6–10, Table 3) Series 3: Effects of the long-term treatment with sGC stimulator and ACEi on survival the rate, morphometric and histopathological scores (FIGURES 6–10, Table 3) Series 3: Effects of the long-term treatment with sGC stimulator and ACEi on survival the rate, morphometric and histopathological scores (FIGURES 6–10, Table 3) Page 14/40 Table 3 Table 3 Morphometric and histological parameters measured in material collected from rats that survived until the end of the observation. Heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats were treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi) alone or combined for 7 months Morphometric and histological parameters measured in material collected from rats that survived until the end of the observation. Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pressure (FIGURE 4 and 5) Heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats were treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined for 7 months sham TGR (n = 8) sham TGR + sGCstim (n = 9) ACF TGR + sGCstim (n = 2) ACF TGR + ACEi (n = 20) ACF TGR + sGCstim + ACEi (n = 12) sham HanSD (n = 9) sham HanSD + sGCstim (n = 10) a b c d e f g BW (g) 765 ± 13 f,g 770 ± 19 f,g 695 ± 43 723 ± 13 f,g 746 ± 25 549 ± 10 592 ± 13 # Heart W/tibia (mg/mm) 45.6 ± 1.2 f,g 41.7 ± 1.2 f, * 67.4 ± 0.3 65.6 ± 2.4 a,b,f,g 58.0 ± 3.5 b,f,g 33.7 ± 0.7 37.1 ± 0.5 # Lungs W /tibia (mg/mm) 46.8 ± 1.3 53.1 ± 2.6 56.0 ± 2.7 57.1 ± 2.1 a,f 59.1 ± 2.9 a,f,g 44.1 ± 1.2 48.6 ± 0.8 # Liver W/tibia (g/cm) 5.6 ± 0.2 f,g 5.5 ± 0.2 f,g 4.9 ± 0.6 4.7 ± 0.2 b,f 4.8 ± 0.2 f 3.8 ± 0.1 4.5 ± 0.1 # Right kidney W/tibia (mg/mm) 49.8 ± 2.1 f,g 47.6 ± 2.0 41.0 ± 8.6 37.5 ± 0.6 a,g 41.9 ± 1.0 a,b 39.1 ± 1.1 44.4 ± 1.7 # Left kidney W/tibia (mg/mm) 53.1 ± 2.6 f,g 50.6 ± 2.4 40.0 ± 3.9 37.6 ± 0.8 a,b,g 42.8 ± 1.7 a,b 39.5 ± 1.0 45.3 ± 2.0 # GSI 0.43 ± 0.13f 0.23 ± 0.01 0.09 ± 0.04 0.10 ± 0.01a,g 0.07 ± 0.01a,g 0.11 ± 0.02 0.29 ± 0.06 # TSI 0.26 ± 0.06 0.23 ± 0.06 0.20 ± 0.00 0.06 ± 0.01 a,b 0.08 ± 0.02 a,b 0.13 ± 0.03 0.14 ± 0.02 BW – body weight; W – weight; GSI – glomerulosclerosis index; TSI – tubulointerstitial injury; Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * sham TGR + sGCstim vs sham TGR; # sham HanSD + sGCstim vs sham HanSD by unpaired t test. Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pressure (FIGURE 4 and 5) Please note that in ACF TGR + sGCstim group only 2 rats survived until the d h hi l d d f h i i l l i b h l d Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * sham TGR + sGCstim vs sham TGR; # sham HanSD + sGCstim vs sham HanSD by unpaired t test. Please note that in ACF TGR + sGCstim group only 2 rats survived until the end, hence this group was excluded from the statistical analysis, but the values are presented Superscript letter given next to the value (a-g) indicate statistically significant difference versus group indicated in the second row of the table (under the group’s name) by one way ANOVA with Tukey's multiple comparisons test; * sham TGR + sGCstim vs sham TGR; # sham HanSD + sGCstim vs sham HanSD by unpaired t test. Please note that in ACF TGR + sGCstim group only 2 rats survived until the end, hence this group was excluded from the statistical analysis, but the values are presented Page 15/40 The model of ACF-induced HF in TGR is characterized by high mortality rates due to very early onset of decompensation of HF as compared with the course of ACF-induced HF in HanSD rats, as reported in previous studies including ours (Melenovsky et al. 2012; Červenka et al. 2015b; Kratky et al. 2021; Kala et al. 2023). The survival rates are presented on FIGURE 6. All normotensive HanSD rats survived until the end of 210 days observation (100% survival in placebo and in the sGC stimulator treated group); in sham-operated TGR rats (both placebo and sGCstim) the survival was 90% in the end (1 rat died in each group). The sham-operated groups (HanSD and TGR) were omitted for clarity from FIGURE 4. The untreated TGR rats with heart failure (ACF TGR) rapidly started to die in the first few weeks of the observation. This was not attenuated in the initial phase by the sGC stimulator treatment, whereas it was almost stopped from the beginning by the optimized dose of the ACEi. However, in the further progression of the disease phenotype, the stand alone treatment with sGC stimulator significantly improved survival in comparison to untreated rats (ACF TGR). Series 2: Effect of the treatment with sGC stimulator and ACEi on blood pressure (FIGURE 4 and 5) After 60 days of sGC stimulator treatment the survival was still 50% compared to 8% in the untreated rats. After day 140 the effectiveness of the treatment with the sGC stimulator was then fading out also, however being still significantly better compared to placebo. The best effectiveness on mortality in this experiment was achieved with the optimized ACE inhibitor administered alone (ACF TGR + ACEi); the survival in this group was 90% at the end of the experiment. Surprisingly, the effectiveness of the combined therapy with ACEi and sGC stimulator (ACF TGR + sGCstim) was lower than stand-alone ACEi treatment with 44% at the end of the study. Organ weights and renal histopathology scores (Table 3) At the end of the study (after 210 days), the surviving animals were decapitated and organs were weighted and collected for further analysis. Noteworthy the placebo treated rats with heart failure and the sGC stimulator-treated rats were excluded from the statistical analysis due to the low animal numbers; therefore, this analysis was conducted mainly to compare the effects of the treatments in sham operated animals (both TGR and HanSD) and between ACEi administered alone and combined treatment with ACEi and BAY41-8543. Out of all ACF rats the lowest heart weight (expressed as a ratio to tibia length) as indicator for heart hypertrophy was in the group treated with the combined treatment with sGCstim and ACEi (58.0 ± 3.5 mg/mm) which was not significantly different from untreated sham TGR (45.6 ± 1.2 mg/mm). Interestingly, the heart hypertrophy in ACF-TGR treated with the combined therapy tended to be lower compared to the ACEi inhibitor treatment alone (58.0 ± 3.5 mg/mm versus 65.6 ± 2.4 mg/mm, respectively; P = 0.0777 by unpaired T-test; NS). Treatment with sGC stimulator also decreased heart weight in sham TGR (41.7 ± 1.2 mg/mm versus 45.6 ± 1.2 mg/mm in untreated animals; P < 0.05 by unpaired T-test; Table 3). Except the obvious differences between sham-operated animals versus rats with ACF and differences between healthy HanSD rats and hypertensive TGR, there were no major changes in lung, liver and kidney Page 16/40 Page 16/40 weights between applied treatment regimens. However, in normotensive HanSD rats the treatment with sGC stimulator significantly increased almost all of the measured parameters, when compared to untreated HanSD rats (Table 3, # by T-test). Since in this group the increase in body weight (BW) was also the highest we calculated the ratio of organ weights’ to BW which was however, not statistically significant, with the exception of liver weight (sham HanSD + sGCstim: 33.0 ± 0.06 versus 30.3 ± 0.05 mg/g in untreated sham HanSD; P = 0.003 by unpaired t-Test). To evaluate the degree of the kidney damage the glomerulosclerosis index (GSI) and kidney cortical tubulointerstitial injury (TSI) were measured (Table 3). The inflammatory cell infiltration, interstitial fibrosis and tubular dilatation were evaluated. Protein levels of Cx43 and PKCε assessed by western blot method in left ventricle (FIGURE 7) To evaluate the intercellular communication the protein levels of Cx43 and PKCε were assessed. Structural remodelling, such as hypertrophy, excessive accumulation of extracellular matrix (ECM) and myocardial fibrosis perturb Cx43 channels mediated intercellular communication in addition to down- regulation of Cx43 due to disease aetiology. The protein levels of the Cx43 phosphorylated at serine 368 characterized by reduced channel conductivity, were significantly supressed in the left ventricle of hypertensive sham TGR and normotensive sham HanSD + sGCstim (63 ± 6 and 60 ± 3 respectively versus 100 ± 5% in untreated sham HanSD; P < 0.0001 by one way ANOVA). The protein levels of total Cx43 were significantly decreased in sham HanSD + sGCstim rats (76 ± 4 versus 98 ± 3% in untreated sham HanSD; p = 0.02 by one way ANOVA). On the contrary, protein abundance of total Cx43 and PKCε, which directly phosphorylate Cx43, was significantly increased in hypertensive sham TGR treated with sGC stimulator (total Cx43: 133 ± 5 versus 86 ± 7 and PKCε: 195 ± 10 versus 87 ± 6% in untreated sham TGR, respectively; P < 0.0001 by one way ANOVA). Organ weights and renal histopathology scores (Table 3) Noteworthy all values for GSI and TSI were relatively low, indicating no severe renal damage in all groups (it did not exceed the value of 0.5 in any group, which is considered as a borderline between healthy and renal damage), irrespective of the treatment. However, some subtle changes and significant differences were observed. ACEi administered alone and combined with sGC stimulator decreased GSI (0.10 ± 0.01 and 0.07 ± 0.01, respectively) to the greatest extend and to similar level as in untreated sham HanSD rats (0.11 ± 0.02). Also ACEi (alone and combined with sGC stimulator) decreased TSI when compared to untreated sham TGR and sham TGR treated with sGC stimulator (Table 3). The treatment with sGC stimulator tended to decrease GSI in sham TGR (0.23 ± 0.01 versus 0.43 ± 0.13 in untreated sham TGR; NS), but significantly increased it in HanSD rats (0.29 ± 0.06 versus 0.11 ± 0.02 in untreated sham HanSD; P = 0.017 by unpaired T-test). These changes were not reflected in tubulointerstitial injury score, since TSI was not changed in both sham TGR and sham HanSD (Table 3). Myocardial capillary density (FIGURE 10) Activity of alkaline phosphatase (AP) reflects function and myocardial density of the arterial part of capillaries. AP activity was significantly normalized in sham TGR rats after sGC stimulator treatment (73 ± 6 versus 117 ± 11% in untreated sham TGR; P < 0.001 by one way ANOVA). No other differences were detected between other groups. Haematoxylin and eosin (HE) histological staining of left ventricle and hydroxyproline assay (FIGURE 9) To evaluate the myocardial remodelling and the level of fibrosis HE staining of left ventricle and hydroxyproline assay was performed. HE staining of left ventricle showed that, compared to sham HanSD rats, the left ventricular tissue of sham TGR and ACF TGR rats exhibited enlarged cardiomyocytes and focal areas infiltrated with polymorphonuclears, mainly in sham TGR (FIGURE 9A). Hydroxyproline content (FIGURE 9B) in the left ventricular tissue was the highest in untreated sham TGR rats (33.5 ± 2.4 mg/g versus 26.9 ± 3.3 in untreated sham HanSD; p < 0.05 by one way ANOVA). It seems that all applied treatments diminished the hydroxyproline content, since the values were not different than in healthy normotensive HanSD rats (FIGURE 9B). Myocardial Topology and Quantification of Cx43 (FIGURE 8) In healthy normotensive rats (sham HanSD) Cx43 was detected mainly at the gap junction plaques of the intercalated discs into end-to-end pattern. However, in the hypertensive sham TGR the lateral distribution Page 17/40 Page 17/40 of Cx43 (side-to-side pattern) was more pronounced. A quantitative image analysis of Cx43 showed a significant decrease of Cx43 in hypertensive sham TGR and normotensive sham HanSD treated with sGC stimulator, compared to normotensive sham HanSD rats. of Cx43 (side-to-side pattern) was more pronounced. A quantitative image analysis of Cx43 showed a significant decrease of Cx43 in hypertensive sham TGR and normotensive sham HanSD treated with sGC stimulator, compared to normotensive sham HanSD rats. 4. Discussion The main objective of the present study was to evaluate the effectiveness of sGC stimulator for the treatment of HF in ACF TGR, which is a model of high-output HF associated with development of cardio- renal syndrome. We have shown that BAY41-8543, which exhibits the same mode of action as the sGC stimulator vericiguat, effectively increased the survival of rats with HF with cardio-renal syndrome in comparison to untreated animals. The discovery of orally active sGC stimulators and sGC activators was a breakthrough in the pharmacology of NO/sGC/cGMP field and seems to have an extensive therapeutic potential. Hence, there is still a great need for pre-clinical and clinical research to fully understand the mode of action and to explore their beneficial activity in cardiovascular and cardio-renal diseases in special patient populations (Cordwin et al. 2021; Sandner et al. 2021b). In our current study, we employed the rat model of volume overload induced by creation of the aorto-caval fistula, which mimics human HF and t is recommended for pre-clinical research (Riehle and Bauersachs 2019; Kala et al. 2023). The ACF was created in ren-2 transgenic rats, in which the hypertension and endogenous activation of the renin angiotensin system (RAAS) are combined, consequently progressing the development of HF with cardio-renal syndrome (Sobieraj et al. 2021). Page 18/40 The first task in our study was to determine if the selected dose of BAY41-8543 (3 mg kg− 1 day− 1) is effective in our model of HF, specifically if it increases the production of secondary messenger cGMP. Therefore, we measured cGMP excretion in urine which was increased after sGC stimulator administration already after one week of treatment. Since the sGC stimulator was administered in the food, we measured the food intake and the concentration of BAY41-8543 in plasma in the end of the short term experiment. We did not record any differences in the food intake between groups and the plasma levels of sGC stimulator were in accordance to previous studies (Stasch et al. 2015). These data imply that we were able to reach sufficient exposure and target engagement with our sGC stimulator dose. In the next step we verified that our sGC stimulator does not significantly impact blood pressure. Indeed, the treatment with only sGC stimulator proved to have only transient and minor impact on blood pressure in ACF TGR rats. 4. Discussion After having identified the optimal dose, that lead to target engagement but not to significant BP reduction, we conducted a long term study to evaluate potential beneficial effects of the sGC stimulator BAY 41-8543 in our rat HF model. We have shown that sGC stimulator significantly improved the survival of ACF TGR rats in comparison to untreated animals. This is in line with recent clinical data from the VICTORIA phase 3 pivotal clinical trial which demonstrated that the vericiguat significantly reduced the incidence of the primary outcome of death from cardiovascular causes or first hospitalization for heart failure in comparison to placebo group. The beneficial activity of vericiguat over placebo in the clinical trial appeared after 3 months of treatment and was sustained until the end of 10.8 months follow-up period (Armstrong et al. 2020). In our study the considerable improvement was already visible after three weeks of the treatment (the mortality in untreated rats was already 75% by the 23rd day versus only 34% in sGC stimulator-treated group). The beneficial activity of sGC stimulator persisted for another 2 months (by the 59th day of treatment 48% of the rats were still alive), however after that period the effectiveness of the treatment started slowly to fade out. Currently we cannot exclude that this is related to the ACF- procedure and volume overload since preclinical studies in other chronic diseases models have shown long-term prevention (Follmann et al. 2013; Stasch et al. 2015). Interestingly, we have observed that the optimized dose of the ACEi completely prevented the mortality of the rats, but the efficacy of the ACEi in the combination with the sGC stimulator was substantially reduced. This unanticipated outcome raises some difficulties in interpretation and we cannot provide a plausible explanation. One of the possible hypothesis includes the excessive antihypertensive potency of both drugs in this particular model of HF. As already previously mentioned, the ACF creation causes a significant decrease in blood pressure (around 50 mmHg), therefore our first concept was that the combined treatment causes adverse hypotension, where the values of BP decrease below autoregulatory levels. Low BP is a common symptom in subjects with HFrEF (around 10–15%) and it has been repeatedly demonstrated that blood pressure lower than 90 mmHg is a marker of poor outcome in acute HF (Cautela et al. 2020). 4. Discussion Considering the high risk of negative outcomes in patients with HFrEF, current guideline-directed medical therapy recommends the titration of ACEi up to the maximally tolerated dosage (Ouwerkerk et al. 2017; Sharma et al. 2022). Based on the clinical evidence and our previous Page 19/40 Page 19/40 studies, we have developed a fully effective titration protocol of trandolapril (ACEi), which is very well tolerated by ACF TGR, as shown by almost 100% long term survival in the current study. studies, we have developed a fully effective titration protocol of trandolapril (ACEi), which is very well tolerated by ACF TGR, as shown by almost 100% long term survival in the current study. sGC stimulators (including BAY41-8543, vericiguat and riociguat) may cause symptomatic hypotension, but it is modest and dose-dependent (Stasch et al. 2002b; Sharkovska et al. 2010; Lam et al. 2021). Moreover, it was shown that even among the population of patients predisposed to hypotension, the efficiency of vericiguat persisted regardless of baseline SBP (Lam et al. 2021). However, we cannot exclude that in this particular model of HF (ACF TGR rats), the effects of the combined treatment (ACEi + sGCstim) depends on the used sGC stimulator dose and/or the sensitivity of this model per se (model specific effect). This is supported by recent findings in ACF TGR model in which the ACEi effects were also blunted by adding an ETA antagonist (Kala et al. 2023). The clinical importance of NO/sGC/cGMP signalling in cardiovascular and cardio-renal diseases including HF is very obvious, however a better understanding of the underlying mechanisms is necessary for the optimal HF treatment and prevention and selection of patients profiting best from sGC stimulator therapies (Numata and Takimoto 2022). Hence the second goal of our study was to further investigate the NO/sGC/cGMP pathway in pathological as well as in physiological states and to further elucidate the possible mechanisms of sGC stimulators. We therefore, have measured a variety of cardiovascular and renal biomarkers and found several interesting findings. The basal urinary levels of cGMP (before treatment) were significantly higher in all groups in which HF was induced by ACF creation (FIGURE 2A). This seems in contrast to data showing impaired cGMP signalling in heart failure (Blanton 2020). However, it seems that NO/sGC/cGMP pathway could be initially activated to counteract the development of HF after ACF creation. 4. Discussion In fact we have found a number of beneficial effects of sGC stimulator treatment in hypertensive sham TGR group (without ACF creation) exerted both on the renal and cardiovascular systems. The glomerulosclerosis index (GSI) tended to be lower after the sGC stimulator treatment. Furthermore the analysis of the heart collected after the survival experiment (after 210 days) revealed a great deal of positive activity of sGC stimulator in sham TGR in comparison to untreated hypertensive animals. Hypertension is linked with an increased occurrence of severe arrhythmias and development and progression of heart failure (Egan Benova et al. 2016). The key factors facilitating such life-threatening events are myocardial structural remodelling, fibrosis and altered topology and disorders of connexin-43 (Cx43) channels. It has been established that down-regulation of Cx43 as well as its abnormal topology contribute to the arrhythmic substrate in failing human heart promoting occurrence of life-threatening arrhythmias, hence the increase in Cx43 appears to be a mechanism to avoid lethal arrhythmia (Danik et al. 2004). We observed a number of positive effects resulted from the long term treatment with sGC stimulator of hypertensive sham TGR, i.e. decreased heart hypertrophy, significantly up-regulated Cx43 and PKCε in the left ventricle. Moreover, the long term treatment with sGC stimulator tended to decrease hydroxyproline (marker of fibrosis)(Díez 2007) and significantly decreased the activity of alkaline phosphatase (marker of myocardial capillary density) (Schultz-Hector et al. 1993). Certainly observed beneficial effects of BAY41-8543 treatment can be partially escribed to antihypertensive effectiveness of the sGC stimulator, however the magnitude of the BP decrease, which was only modest suggests also blood-pressure independent beneficial mechanisms. Taken together, the number of positive effects of the treatment with sGC stimulator in hypertensive TGR rats (without ACF), exerted on the kidney (lower GSI) and heart (diminished hypertrophy, up-regulated Cx43 and PKCε, decreased fibrosis) suggest a great potential of this class of the drug in prevention and/or treatment of persistent hypertension and related disorders. As an additional control group we evaluated the effectiveness of sGC stimulator in normotensive HanSD rats and surprisingly more detailed analysis of the collected data, on the contrary to hypertensive TGR, revealed mildly unfavourable consequences of the long term treatment. The GSI was significantly elevated and Cx43 was down-regulated. 4. Discussion We have also measured the urinary excretion of nitric oxide metabolites (NOx: nitrate and nitrite), which is considered as a marker of NO production (Tsikas 2004). Interestingly in all groups treated with sGC stimulator the excretion of NOx was decreased (Table 2). This, at first unexpected result, might be explained by the second mode of action sGC stimulators. As mentioned above, sGC stimulators in addition to stimulating sGC independently of NO, they also sensitize sGC to low levels of endogenous NO by stabilizing NO–sGC binding (Stasch et al. 2001; Liu et al. 2021). Due to this activity, endogenous NO is utilised to a greater extent, therefore less of it might be detected in the urine. Hence, in this particular case the measured excretion of NOx in urine does not reflect the changes in the actual production of NO and the results should be interpreted cautiously as previously suggested (Süto et al. 1995). Moreover, this could be a hint that the application of sGC stimulators could decrease the need for endogenous NO- production which also leads to less NO-derived radicals and oxidative stress. Page 20/40 Furthermore, we found that the treatment with sGC stimulator increased or tended to increase the ANG II levels, which also might be in compliance with increased activation of NO/sGC/cGMP signalling pathway mentioned above (both by NO-independent stimulation of sGC stimulator and by endogenous NO). The reciprocal interplay between RAAS and NO/sGC/cGMP is complex and still not fully understood (Persson 2003; Krishnan et al. 2018). cGMP, a secondary messenger in NO/sGC/cGMP cascade, was shown to Page 20/40 exhibit both stimulatory and inhibitory action on renin secretion and the results of many studies are contradictory (Kurtz and Wagner 1998; Persson 2003; Curnow et al. 2020). Based on the results of our studies, i.e. increased activity of RAAS after sGC stimulator treatment, we could hypothesise rather stimulatory activity on renin secretion after short term treatment. The increase in cGMP resulting in beneficial effects, such as vasodilation or anti-inflammatory activity, could be counterbalanced by the succeeding activation of RAAS. Noteworthy, this phenomenon was only observed in ACF TGR and sham HanSD rats treated with sGC stimulator, but not in sham TGR, in which the treatment with BAY41-8543 did not affect ANG II levels to such extend (only tendency). 4. Discussion Also, as already mentioned above, the plasma and renal levels of ANG II were elevated in sham HanSD + sGCstim group (measured after one week of treatment), which might be an actual cause of these adverse effects. Notwithstanding, we would like to emphasize that the Page 21/40 Page 21/40 survival of the animals in this group was 100% after 210 days and the rats did not bare any signs of any disorders. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This study was supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES, Project No. LX22NPO5104) - funded by the European Union - Next Generation EU. This study was also supported by the Ministry of Health of the Czech Republic (grant number 20-02-00052) and by the Slovak Research and Development Agency (under the contract no. 21- 0410). Authors Contributions The authors declare that all data were generated in-house and that no paper mill was used. LČ, VM, OG conceived and designed study; OG, ZHu, PŠ, ZHo, ZV, SK, BS and MS performed research; OG, ZHu, PŠ, ZHo, VM, BS, MS and LČ analysed data; OG and LČ wrote the paper. All authors read and approved the manuscript. Ethics approval This study were performed in accordance with guidelines and practices established by the Animal Care and Use Committee of the Institute for Clinical and Experimental Medicine (Prague) approved by the Ministry of Health of the Czech Republic, which accords with the European Union Directive 63/2010 and ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). 5. Summary And Conclusions The main goal of the current study was to evaluate the effectiveness of sGC stimulator (BAY 41-8543) for the treatment of HF due to volume overload combined with cardio-renal syndrome (ACF TGR). We have shown that the sGC stimulator effectively increased the survival of ACF TGR in comparison to untreated animals. Taken together, we show that sGC stimulators represent a valuable class of drugs to treat heart failure and renal dysfunction. Data availability Page 22/40 The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgment We would like to thank Peter Sandner, PhD from Bayer AG (Wuppertal, Germany) for providing the sGC stimulator and invaluable help with writing and reviewing the manuscript. References 1. Abassi Z, Goltsman I, Karram T, et al (2011) Aortocaval fistula in rat: A unique model of volume- overload congestive heart failure and cardiac hypertrophy. J Biomed Biotechnol 2011:. https://doi.org/10.1155/2011/729497 2. 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Sykora M, Kratky V, Kopkan L, Tribulova N (2023) Anti-Fibrotic Potential of Angiotensin (1–7) in Hemodynamically Overloaded Rat Heart. Int J Mol Sci 24:3490. https://doi.org/10.3390/ijms24043490. 68. Szeiffová Bačova B, Egan Beňová T, Viczenczová C, et al (2016) Cardiac connexin-43 and PKC signaling in rats with altered thyroid status without and with omega-3 fatty acids intake. Figures Page 28/40 Figure 1 The experimental design of the whole study depicting the time sequence and experimental manoeuvres; MC – metabolic cage with 12h urine collection; B – blood collection from the tail vein. Figure 1 The experimental design of the whole study depicting the time sequence and experimental manoeuvres; MC – metabolic cage with 12h urine collection; B – blood collection from the tail vein. Page 29/40 Figure 2 Urinary excretion of cGMP A: throughout the whole observation and B: the diffe values (day -1) and the end (day 7) in heterozygous Ren-2 transgenic rats (TGR) Page 30/40 Figure 2 Urinary excretion of cGMP A: throughout the whole observation and B: the difference (Δ) be values (day -1) and the end (day 7) in heterozygous Ren-2 transgenic rats (TGR) rats with ao fistula (ACF) or without (sham) and normotensive sham HanSD rats treated with sGC stimu 8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined; P>0.05 (N P≤0.01; * P≤0.001; **** P≤0.0001; by 2way ANOVA with Dunnett's (within each group) Figure 2 Figure 2 Urinary excretion of cGMP A: throughout the whole observation and B: the difference (Δ) between basal values (day -1) and the end (day 7) in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats treated with sGC stimulator (BAY41- 8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined; P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; by 2way ANOVA with Dunnett's (within each group) and Tukey’s Page 30/40 (between groups) multiple comparisons tests; # P≤ 0.0001 basal values after ACF versus sham operation (day-1) by unpaired T-test; Figure 3 Angiotensin II (ANG II) A: in plasma and B: in renal tissue in heterozygous Ren-2 tran Figure 3 Angiotensin II (ANG II) A: in plasma and B: in renal tissue in heterozygous Ren-2 transgenic rats (TGR) Figure 3 Figure 3 Angiotensin II (ANG II) A: in plasma and B: in renal tissue in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats after 7 day Angiotensin II (ANG II) A: in plasma and B: in renal tissue in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats after 7 day Page 31/40 Page 31/40 treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001 by one way ANOVA with Tukey’s multiple comparisons tests; # p<0.05 versus sham HanSD by one way ANOVA with Tukey’s multiple comparisons tests; treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001 by one way ANOVA with Tukey’s multiple comparisons tests; # p<0.05 versus sham HanSD by one way ANOVA with Tukey’s multiple comparisons tests; Figure 4 Time course of mean arterial blood pressure (MBP) throughout the whole observation in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. * P<0.05 by 2way ANOVA with Bonferroni's multiple comparisons test; # P<0.05 versus all other groups 2way ANOVA with Tukey's multiple comparisons tests; Figure 4 Time course of mean arterial blood pressure (MBP) throughout the whole observation in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. * P<0.05 by 2way ANOVA with Bonferroni's multiple comparisons test; # P<0.05 versus all other groups 2way ANOVA with Tukey's multiple comparisons tests; Page 32/40 Page 33/40 Figure 5 Time course of (A) systolic (SBP) and (B) diastolic (DBP) blood pressure throughout 2-week observation in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. * P<0.05 versus baseline values on day 0 within each group; # values different between all other groups; $ P<0.05 values different between BAY41-8543 and both ACEi treated groups of ACF TGR by 2way ANOVA Figure 5 Time course of (A) systolic (SBP) and (B) diastolic (DBP) blood pressure throughout 2-week observation in heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. * P<0.05 versus baseline values on day 0 within each group; # values different between all other groups; $ P<0.05 values different between BAY41-8543 and both ACEi treated groups of ACF TGR by 2way ANOVA Page 33/40 Page 33/40 with Dunnet’s (within each group) and Tukey's (between groups) multiple comparisons tests. The BP values before the treatment and sham-operated groups are omitted for clarity; with Dunnet’s (within each group) and Tukey's (between groups) multiple comparisons tests. The BP values before the treatment and sham-operated groups are omitted for clarity; p g p y; Figure 6 The survival rate in heterozygous Ren-2 transgenic rats (TGR) with aorto-caval fistula (ACF) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. All groups significantly differ between each other (by log-rank Mantel-Cox test); P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; Control normotensive and sham operated TGR groups were omitted for clarity (the survival in these groups was 90-100% in the end). Figure 6 Figure 6 The survival rate in heterozygous Ren-2 transgenic rats (TGR) with aorto-caval fistula (ACF) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. All groups significantly differ between each other (by log-rank Mantel-Cox test); P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; Control normotensive and sham operated TGR groups were omitted for clarity (the survival in these groups was 90-100% in the end). The survival rate in heterozygous Ren-2 transgenic rats (TGR) with aorto-caval fistula (ACF) treated with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. All groups significantly differ between each other (by log-rank Mantel-Cox test); P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; Control normotensive and sham operated TGR groups were omitted for clarity (the survival in these groups was 90-100% in the end). Page 34/40 Figure 7 Myocardial protein levels of total Cx43 (A), phosphorylated Cx43 at serine 368 (B), protein kinase C Epsilon (C), and representative western blot images (D) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulato (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Values are normalized to GAPDH and presented as mean ± SEM; Cx43 – connexin 43; PKCε – protein kinase C Epsilon; GAPDH- glyceraldehyde-3-phosphate dehydrogenase; P>0 05 (NS); * P≤0 05; P≤0 01; * Figure 7 Myocardial protein levels of total Cx43 (A), phosphorylated Cx43 at serine 368 (B), protein kinase C Epsilon (C), and representative western blot images (D) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Figure 6 Values are normalized to GAPDH and presented as mean ± SEM; Cx43 – connexin 43; PKCε – protein kinase C Epsilon; GAPDH- glyceraldehyde-3-phosphate dehydrogenase; P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; by one-way ANOVA and Tukey's test multiple comparison test; # versus all oth groups by one-way ANOVA and Tukey's test multiple comparison test; (n=5 in all groups except ACF TG + BAY41-8543 where only 2 rats survived; note that no rats survived in untreated ACF TGR group) Figure 7 Figure 7 Myocardial protein levels of total Cx43 (A), phosphorylated Cx43 at serine 368 (B), protein kinase C Epsilon (C), and representative western blot images (D) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Values are normalized to GAPDH and presented as mean ± SEM; Cx43 – connexin 43; PKCε – protein kinase C Epsilon; GAPDH- glyceraldehyde-3-phosphate dehydrogenase; P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; by one-way ANOVA and Tukey's test multiple comparison test; # versus all other groups by one-way ANOVA and Tukey's test multiple comparison test; (n=5 in all groups except ACF TGR + BAY41-8543 where only 2 rats survived; note that no rats survived in untreated ACF TGR group) Myocardial protein levels of total Cx43 (A), phosphorylated Cx43 at serine 368 (B), protein kinase C Epsilon (C), and representative western blot images (D) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Values are normalized to GAPDH and presented as mean ± SEM; Cx43 – connexin 43; PKCε – protein kinase C Epsilon; GAPDH- glyceraldehyde-3-phosphate dehydrogenase; P>0.05 (NS); * P≤0.05; P≤0.01; * P≤0.001; **** P≤0.0001; by one-way ANOVA and Tukey's test multiple comparison test; # versus all oth groups by one-way ANOVA and Tukey's test multiple comparison test; (n=5 in all groups except ACF TGR + BAY41-8543 where only 2 rats survived; note that no rats survived in untreated ACF TGR group) Page 35/40 8 Figure 8 Figure 8 Immunofluorescence labelling of Cx43 (green) localization (A) and quantitative image analysis of Cx43 (B) represents as total integral optical density per area (IOD) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Double arrows Page 36/40 Page 36/40 show end to end pattern of Cx43 and simple arrows lateral pattern of Cx43. Scale bar represents 200 µm. Values are presented as mean ± SEM; * P≤0.05by one-way ANOVA and Tukey's test multiple comparison test (approximately 10 randomly acquired images from every tissue were captured and analysed in each group); show end to end pattern of Cx43 and simple arrows lateral pattern of Cx43. Scale bar represents 200 µm. Values are presented as mean ± SEM; * P≤0.05by one-way ANOVA and Tukey's test multiple comparison test (approximately 10 randomly acquired images from every tissue were captured and analysed in each group); ure 9 Page 37/40 gure 9 Figure 9 Page 37/40 Page 37/40 Hematoxylin–eosin staining (A) and hydroxyproline content detection in the left heart ventricle (B) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Values are normalized to the weight of tissue used for analysis and presented as mean ± SEM. * P≤0.05 by one-way ANOVA and Tukey's test multiple comparison test (n=5 in all groups except ACF TGR + BAY41-8543 where only 2 rats survived; note that no rats survived in untreated ACF TGR group) Hematoxylin–eosin staining (A) and hydroxyproline content detection in the left heart ventricle (B) measured in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Values are normalized to the weight of tissue used for analysis and presented as mean ± SEM. Figure 8 * P≤0.05 by one-way ANOVA and Tukey's test multiple comparison test (n=5 in all groups except ACF TGR + BAY41-8543 where only 2 rats survived; note that no rats survived in untreated ACF TGR group) Page 38/40 Page 39/40 gure 10 yocardial capillary density based on the histochemical demonstration of the alkaline phosphata tivity (blue) and quantification of its evaluation (B) in left ventricle collected from heterozygous ansgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham anSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), o giotensin-converting enzyme inhibitor (ACEi), alone or combined. Scale bar represents 50µm. Va Figure 10 Figure 10 Myocardial capillary density based on the histochemical demonstration of the alkaline phosphatase (A) activity (blue) and quantification of its evaluation (B) in left ventricle collected from heterozygous Ren-2 transgenic rats (TGR) rats with aorto-caval fistula (ACF) or without (sham) and normotensive sham HanSD rats that survived until the end of 7 months treatment with sGC stimulator (BAY41-8543), or with angiotensin-converting enzyme inhibitor (ACEi), alone or combined. Scale bar represents 50µm. Values Page 39/40 Page 39/40 are presented as mean ± SEM *** P≤0.001by one-way ANOVA and Tukey's test multiple comparison test (approximately 10 randomly acquired images from every tissue were captured and analysed in each group); Page 40/40
https://openalex.org/W2133414910	https://www.maxwellsci.com/announce/RJASET/6-3277-3281.pdf	English	null	Mineral Compositions and Micro-Structural of Epoxy-Repaired Rock Revealed by X-ray Diffraction and Scanning Electron Microscopy	Research journal of applied sciences, engineering and technology	2,013	cc-by	2,684	Corresponding Author: Xingguo Yang, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610065, China This work is licensed under a Creative Commons Attribution 4.0 International License (URL: http://creativecommons.org/licenses/by/4.0/). 3277 INTRODUCTION structural analysis of epoxy-repaired rock. At the Jinping Hydropower Station, epoxy resins are used to repairing the shear zone and faults, to reduce the permeability and increase strength of rock in fault. The mineral proportions and micro structural properties of epoxy-repaired rock are very important to determine the improvement of mechanical characteristics by epoxy resins. The objective of this study is a dopt electronic methods (X-ray diffraction and scanning electron microscopy) to analysis the mineral compositions and micro structural of epoxy-repaired rock. Rock contains lots of cracks, the mechanical properties of rock are influenced by these cracks and the cracks may represent the total extent of the damage (Issa and Debs, 2009; Zhou et al., 2012). Their significance depends on the type of structure, as well as the nature of the cracking. In order to improve their properties, grouting is a special technique developed in recent years with the repairing of cracks in rock. It is a procedure which involves grout injection into voids, fissures and cracks in rock, specifically to reduce permeability, to increase strength and durability (Zhou et al., 2010; Anagnostopoulos et al., 2011). 1Wei Hu, 2Xu Wu, 1Jiawen Zhou, 1Xingguo Yang and 1Minghui Hao 1State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, 610065, China 2Si H d B 7 CO Ltd Si H d G Ltd Ch d 610081 Chi , 2Sino Hydro Bureau 7 CO., Ltd., Sino Hydro Group Ltd., Chengdu, 610081, Chin Abstract: In order to improve the mechanical properties of rock with lots of cracks, this study adopts electronic methods (X-ray diffraction and scanning electron microscopy) to determine the mineral compositions and micro structural characteristics of epoxy-repaired rock. Fractured rocks with low strength or high permeability may not be appropriate for dam foundation. The strength and durability of fractured rock are increased after chemical grouting. Test results show that, some of the cracks and voids are repaired by epoxy particles, resulted in the decreasing rock porosity. The proportion of epoxy resins is about 3.5%-5.5%, showing a discrete distribution and can not connect with each other. Keywords: Epoxy-repaired rock, microstructural, mineral compositions, scanning electron microscopy, X-ray diffraction Published: September 20, 2013 Published: September 20, 2013 Submitted: January 17, 2013 Research Article Mineral Compositions and Micro-Structural of Epoxy-Repaired Rock Revealed by X-ray Diffraction and Scanning Electron Microscopy 1Wei Hu, 2Xu Wu, 1Jiawen Zhou, 1Xingguo Yang and 1Minghui Hao 1State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, 610065, China 2Si H d B 7 CO L d Si H d G L d Ch d 610081 Chi EXPERIMENTAL In this section, the materials for X-ray diffraction and scanning electron microscope experiments are retrieved from the fault zone after chemical grouting. Rock should be pressed into powder for XRD analysis and cut and polished into thin slice for SEM analysis. Finally, a brief introduction for XRD and SEM methods are made. A granular ordinary type of cement in some times cannot be grouted into the fissures of the rock mass, when the crack size is very small (Anagnostopoulos, 2005). The cement particles were blocked under higher grouting pressure during the grouting process (Minh et al., 2007). However, chemical grouting usually can solve this problem. Epoxy resins are widely used in chemical grouting of rock due to their advantageous properties of adhesion, good toughness and superior mechanical properties (Rogers and Daniels, 2002; Hasanpour and Choupani, 2009). Materials: A two component epoxy resin was selected for optimization and several modifications were made to the original composition provided. Figure 1 shows a polarizing microscope photo of the epoxy resin for chemical grouting of faults. Mineral phase quantification based on powder X- Rays Diffraction (XRD) has always been an attractive option (Trimby and Prior, 1999), as it provides reproducible and representative results. The scanning electron microscope (SEM) is a powerful tool for micro Component A is mixture of epoxy resins, component B is amine-hardener, the gravity ratio of Res. J. Appl. Sci. Eng. Technol., 6(17): 3277-3281, 2013 Fig. 4: X-ray diffraction test system Fig. 5: Scanning electron microscopy test system Fig. 1: Polarizing microscope photo of the epoxy resin Fig. 4: X-ray diffraction test system Fig. 4: X-ray diffraction test system Fig. 4: X-ray diffraction test system Fig. 4: X ray diffraction test system Fig. 5: Scanning electron microscopy test system Fig. 1: Polarizing microscope photo of the epoxy resin Fig. 2: Epoxy-repaired rocks Fig. 3: Sample preparation for XRD and SEM tests Fig. 2: Epoxy-repaired rocks Fig. 5: Scanning electron microscopy test system Sample preparation: The epoxy-repaired rock should be pressed into powder for XRD test, the particle diameter is less than 0.001 mm. But the epoxy-repaired rock should be cut and polished into thin slice for SEM test, the length and width are all 1.0 cm, the height should be not larger than the length. Fig. 2: Epoxy-repaired rocks Fig. 2: Epoxy-repaired rocks Fig. EXPERIMENTAL Sci. Eng. Technol., 6(17): 3277-3281, 2013 Table 1: Variation of the porosity before and after grouting Rock type Original (%) Epoxy-repaired (%) W 14.73 9.68 S 18.32 12.16 C 20.5 13.25 Table 2: Mineral compositions of the epoxy-repaired rocks Rock type W S C Proportion (%) SiO2 37.63 40.46 54.32 CaCO3 30.56 28.28 16.13 FeO (OH) 12.65 10.86 6.56 Al2O3 5.64 4.86 6.86 CaO 3.29 3.58 2.65 K2O 2.35 2.65 3.21 FeO 2.16 3.16 1.67 Na2O 1.15 1.57 2.31 Epoxy 3.45 4.36 5.61 Fig. 6: Polarizing microscope photo of the epoxy-repaired rock sample Table 1: Variation of the porosity before and after grouting Rock type Original (%) Epoxy repa Table 1: Variation of the porosity before and after grouting Rock type Original (%) Epoxy-repaired (%) W 14.73 9.68 S 18.32 12.16 C 20.5 13.25 Table 2: Mineral compositions of the epoxy-repaired rocks Rock type W S C Proportion (%) SiO2 37.63 40.46 54.32 CaCO3 30.56 28.28 16.13 FeO (OH) 12.65 10.86 6.56 Al2O3 5.64 4.86 6.86 CaO 3.29 3.58 2.65 K2O 2.35 2.65 3.21 FeO 2.16 3.16 1.67 Na2O 1.15 1.57 2.31 Epoxy 3.45 4.36 5.61 Fig. 6: Polarizing microscope photo of the epoxy-repaired rock sample Quantitative analyses of the minerals present in each sample were made using commercial software based on the full-profile XRD analysis technique. If the internal standard weight percentage is X, will be greater than the weighted amount Y. The mineral proportion in the tested sample is given by the following equation: ( ) X Y W − =1 a (1) (1) where, where, , Wa = The mineral proportion in the tested sample Wa = The mineral proportion in the tested sample X = The mineral amount found by the XRD program Y = The weighed amount of powder sample. Y = The weighed amount of powder sample. Scanning electron microscopy Scanning electron microscopy analyses on extracted micro structural characteristics of epoxy-repaired rock provide the detail morphology and texture of such grains. This is important since anthropogenic magnetic particles can be distinguished from specific morphology. Figure 5 shows the test system for scanning electron microscopy. Selection criteria for grain analyses by SEM were based on the subjective differences observed in the morphology and texture behaviors. The filling situation of epoxy resins in the fractured rock can be determined by SEM method. Fig. EXPERIMENTAL 6: Polarizing microscope photo of the epoxy-repaired rock sample (a) (b) 0 100 200 300 400 500 600 Intensity(Counts) 33-1161> Quartz - SiO2 24-0027> Calcite - CaCO3 81-0464> Goethite - FeO(OH) 19-0814> Muscovite-2M1 - K(Al,V)2(Si,Al)4O10(OH)2 20 30 40 50 60 70 80 2-Theta(¡ã [1#-1.rd] 1#-1 0 100 200 300 400 500 Intensity(Counts) 83-2465> Quartz low - SiO2 86-2334> Calcite - Ca(CO3) 81-0464> Goethite - FeO(OH) 19-0814> Muscovite-2M1 - K(Al,V)2(Si,Al)4O10(OH)2 20 30 40 50 60 70 80 2-Theta(¡ã [2#-3.rd] 2#-3 (a) 0 100 200 300 400 500 600 Intensity(Counts) 33-1161> Quartz - SiO2 24-0027> Calcite - CaCO3 81-0464> Goethite - FeO(OH) 19-0814> Muscovite-2M1 - K(Al,V)2(Si,Al)4O10(OH)2 20 30 40 50 60 70 80 2-Theta(¡ã [1#-1.rd] 1#-1 EXPERIMENTAL 3: Sample preparation for XRD and SEM tests Figure 3 shows the sample preparation for XRD and SEM tests. As shown in Fig. 3, three types of rock powder are corresponding to the different rock type. The left, middle and right ones are rock powder of epoxy-repaired weak weathered green schist, strongly weathered green schist and carbonaceous green schist. There is an obvious difference in rock powder between each rock type. At the lower of Fig. 3, these specimens are thin slice of epoxy-repaired rock for SEM tests. Here we can see the epoxy resin existed in the fractured rocks. Fig. 3: Sample preparation for XRD and SEM tests component A and B is 6:1. The final epoxy resin composition adequately fulfilled the project requirements and is now commercially available. There three types of rock in the fault zone are weak weathered green schist (W), strongly weathered green schist (S) and carbonaceous green schist (C). At the beginning of field chemical grouting process, the physical and mechanical characteristics of epoxy resin should be determined. Uniaxial compressive stress of epoxy resin at the age of 28 day is about 90 MPa, a long enough coagulation time is needed for the construction process. X-ray diffraction method: X-ray diffraction (XRD) is an analysis method that uses X-rays to determine the range of physical and chemical characteristics of materials (Ruan and Ward, 2002; Hestnes and Soensen, 2012). This includes analysis for the types and quantities of different phases in the sample. During the semi-quantitative XRD method, the mineral proportions received by sample analysis are compared to known intensity and information found in a database distributed by the International Centre for Diffraction Data. After the epoxy resin is completely solidified, the chemical grouting process for rock fault is finished. The epoxy-repaired rock is prepared by borehole drilling. Figure 2 shows the epoxy-repaired rocks in the fault zone. Representative portions of each epoxy-repaired rock were more finely ground in a ring-grinder mill and the particle diameter is less than 0.001 mm. The minerals in each case were identified from the diffract grams by reference to the rock Powder Diffraction File. Figure 4 shows the test system for X-ray diffraction. As shown in Fig. 2, the left one is epoxy-repaired weak weathered green schist, the middle one is epoxy- repaired strongly weathered green schist and the right one is epoxy-repaired carbonaceous green schist. 3278 Res. J. Appl. CONCLUSION Fractured rock can be repaired by chemical grouting of epoxy resins, how to determine the improvement of epoxy resin for fractured rock is very important. In this study, XRD and SEM methods are adopted to make certain the chemical grouting effect on rocks. Test results show that, the cracks and voids in rock are partly filled by epoxy resins; the proportion of epoxy resin in rock is about 4.5%. ACKNOWLEDGMENT The authors wish to thank the helpful comments and suggestions from my teachers and colleagues in State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University. This study was supported by the National Natural Science Foundation of China (No. 41102194 and No. 51209156) and the China Postdoctoral Science Foundation (No. 2012T50785). (c) Fig. 7: X-ray diffraction analysis results: (a) weak weathered greenschist; (b) strong weathered greenschist; (c) carbonaceous greenschist Fig. 8: Microstructural characteristics of the epoxy-repaired rock RESULTS AND DISCUSSION In this section, some test results about the mineral proportions and micro structural characteristics of epoxy-repaired rock are illustrated. General: Because of there are lots of cracks and voids existed in the original rock at fault zone, so that the porosity of rock is very large. After the chemical grouting process, the fractured rock is repaired by epoxy resins and then the porosity of rock will be decreased. Table 1 shows the variation of the rock porosity before and after chemical grouting. As shown in Table 1, the porosity of original fractured rock is about 15%-20%, after the chemical grouting by epoxy resins, some of the cracks and voids are repaired by epoxy particles, resulted in the decreasing rock porosity. Its mean that, the quality of fractured rock is improved by the epoxy resins. Figure 6 shows a polarizing microscope photo of the epoxy- repaired rock sample. (a) 0 100 200 300 400 500 Intensity(Counts) 83-2465> Quartz low - SiO2 86-2334> Calcite - Ca(CO3) 81-0464> Goethite - FeO(OH) 19-0814> Muscovite-2M1 - K(Al,V)2(Si,Al)4O10(OH)2 20 30 40 50 60 70 80 2-Theta(¡ã [2#-3.rd] 2#-3 As shown in Fig. 6 (compared with Fig. 1), the cracks and voids in the fractured rock are filled with epoxy resins. Mineral compositions: Several test for mineral compositions of epoxy-repaired rock by X-ray diffraction method are carried out. Figure 7 shows the X-ray diffraction analysis results for mineral proportions of epoxy-repaired rock. (b) 3279 Res. J. Appl. Sci. Eng. Technol., 6(17): 3277-3281, 2013 (c) Fig. 7: X-ray diffraction analysis results: (a) weak weathered greenschist; (b) strong weathered greenschist; (c) carbonaceous greenschist 0 250 500 750 Intensity(Counts) 79-1906> Quartz - SiO2 24-0027> Calcite - CaCO3 06-0263> Muscovite-2M1 - KAl2(Si3Al)O10(OH,F)2 81-0464> Goethite - FeO(OH) 20 30 40 50 60 70 80 2-Theta(¡ã [3#-1.rd] 3#-1 (c) 0 250 500 750 Intensity(Counts) 79-1906> Quartz - SiO2 24-0027> Calcite - CaCO3 06-0263> Muscovite-2M1 - KAl2(Si3Al)O10(OH,F)2 81-0464> Goethite - FeO(OH) 20 30 40 50 60 70 80 2-Theta(¡ã [3#-1.rd] 3#-1 REFERENCES Anagnostopoulos, C.A., 2005. Laboratory study of an injected granular soil with polymer grouts. Tunn. Undergr. Sp. Tech., 20(6): 525-533. Anagnostopoulos, C.A., T. Papaliangas, S. Manolopoulou and T. Dimopoulos, 2011. Physical and mechanical properties of chemically grouted sand. Tunn. Undergr. Sp. Tech., 26(6): 718-724. Fig. 8: Microstructural characteristics of the epoxy-repaired rock Hasanpour, R. and N. Choupani, 2009. Rock fracture characterization using the modified Arcan test specimen. Int. J. Rock Mech. Min. Sci., 46(2): 346-354. As shown in Fig. 7, there is an obvious difference between each mineral particle, so that the mineral proportions of epoxy-repaired rock can be determined by XRD method. Table 2 shows the mineral compositions of the epoxy-repaired rocks. Hestnes, K.H. and B.E. Soensen, 2012. Evaluation of quantitative x-ray diffraction for possible use in the quality control of granitic pegmatite in mineral production. Mineral Eng., 39: 239-247. As shown in Table 2, the proportion of SiO2 is the largest one of mineral, about 40%-50%. The proportion of epoxy resins is about 3.5%-5.5%, compared with Table 1, after chemical grouting, the cracks and voids in the fractured rock are partly repaired. Some smaller cracks and voids can not be filled by epoxy resin particles. Issa, C.A. and P. Debs, 2007. Experimental study of epoxy repairing of cracks in concrete. Constr. Build. Mater., 21(1): 157-163. Minh, H., H. Mutsuyoshi and K. Niitani, 2007. Influence of grouting condition on crack and load- carrying capacity of post-tensioned concrete beam due to chloride-induced corrosion. Constr. Build. Mater., 21(7): 1568-1575. Microstructural characteristics: The micro structural characteristics of epoxy-repaired rock are analysis by the SEM method. Figure 8 shows the typical micro structural characteristics of the epoxy-repaired rock. Rogers, K.D. and P. Daniels, 2002. An X-ray diffraction study of the effects of heat treatment on bone mineral microstructure. Biomaterials, 23(12): 2577-2585. As shown in Fig. 8, there is lots of epoxy resin particles existed at the crack surface, showing a discrete distribution and can not connect with each other. That mean the filling of epoxy resin in the cracks and voids is partly. Ruan, C.D. and C.R. Ward, 2002. Quantitative x-ray powder diffraction analysis of clay minerals in Australian coals using Rietveld methods. Appl. Clay Sci., 21(5-6): 227-240. 3280 Res. J. Appl. Sci. Eng. Technol., 6(17): 3277-3281, 2013 Zhou, J.W., X.G. Yang and H.T. Li, 2012. Numerical solution for mixed mode crack propagation in brittle solids combined with finite element method and failure criteria. REFERENCES Int. J. Mater. Prod. Technol., 45(1-4): 96-107. Trimby, P.W. and D.J. Prior, 1999. Microstructural imaging techniques: A comparison between light and scanning electron microscopy. Tectonophysics, 303(1-4): 71-81. ( ) Zhou, J.W., W.Y. Xu and X.G.Yang, 2010. A microcrack damage model for brittle rocks under uniaxial compression. Mech. Res. Commun., 37(4): 399-405. 3281 3281
https://openalex.org/W4284892790	https://noyam.org/?smd_process_download=1&download_id=7811	English	null	The Foundation and Nature of Traditional African Morality: A Review of Selected Literature.	E-Journal of Humanities, Art and Social Sciences	2,022	cc-by	5,172	ABSTRACT Correspondence Richmond Osei Amoah Email: foseiamoah@yahoo.com A plethora of questions are asked when discussing moral issues concerning traditional African societies.Common among them are, is African morality dependent on religion only or has its foundation solely on the society, and what is the nature of the traditional African morality? These questions when answered would solve the myriad of ethical issues inherent in the African Traditional Religion. This article looks at the foundation and the nature of traditional African morality. The article establishes theocentric, communalistic, anthropocentric, utilitarian and shame-oriented as the nature of the traditional African morality. It fills the gap that both God and humanity are the foundations of African morality. The authors reviewed selected literature from various scholars. The paper also affirms that God and human society are the foundation of traditional African morality. It recommends that the theocentric and anthropocentric elements of African ethics must be brought together to have a true nature and source of the traditional African morality. This article seeks to contribute to the debate on the nature and foundation of traditional African morality. Keywords: African Tradition, morality, religion Keywords: African Tradition, morality, religion © 2022 The Author(s). Published and Maintained by Noyam Publishers. This is an open access article under the CCBY license (http://creativecommons.org/licenses/by/4.0/). The Foundation and Nature of Traditional African Morality: A Review of Selected Literature. Richmond Osei Amoah1 & Augustine Kojo Peprah1 1 Department of Social Sciences, St. Louis College of Education, Kumasi, Ghana. © 2022 The Author(s). Published and Maintained by Noyam Publishers. This is an open access article under the CCBY license (http://creativecommons.org/licenses/by/4.0/). E-Journal of Humanities, Arts and Social Sciences (EHASS) ISSN Online 2720-7722 \| Print 2821-8949 Volume 3 Issue 7– July 2022 pp 279-285 Available online at: https://noyam.org/journals/ehass/ DOI: https://doi.org/10.38159/ehass.2022373 1 Alfred Burdon Ellis, The Tshi-Speaking Peoples of the Gold Coast (London: Frank Cass, 1966), 10-11. 2 Ellis, The Tshi-Speaking Peoples of the Gold Coast, 10-11. 3 Kofi Asare Opoku, West Africa Traditional Religion (Accra: FEP International Private Limited, 1978), 152. , p g p f , 3 Kofi Asare Opoku, West Africa Traditional Religion (Accra: FEP International Private Limited, 1978), 152. Alfred Burdon Ellis, The Tshi-Speaking Peoples of the Gold Coast (London: Frank Cass, 1966), 10-11. Ellis, The Tshi-Speaking Peoples of the Gold Coast, 10-11.i Alfred Burdon Ellis, The Tshi-Speaking Peoples of the Gold Coast (London: Frank Cass, 1966), 10-1 2 Ellis, The Tshi-Speaking Peoples of the Gold Coast, 10-11.i Correspondence Richmond Osei Amoah Email: foseiamoah@yahoo.com Publication History Received 4th May, 2022 Accepted 27th June, 2022 Published online 8th July, 2022 2 Ellis, The Tshi-Speaking Peoples of the Gold Coast, 10-11.i Publication History Received 4th May, 2022 Accepted 27th June, 2022 Published online 8th July, 2022 Alfred Burdon Ellis, The Tshi-Speaking Peoples of the Gold Coast (London: Frank Cass, 1966), 10-11. Elli Th T hi S ki P l f h G ld C 10 11 © 2022 The Author(s). Published and Maintained by Noyam Publishers. Thi i ti l d th CCBY li (htt // ti /li /b /4 0/) 1 Alfred Burdon Ellis, The Tshi-Speaking Peoples of the Gold Coast (London: Frank Cass, 1966), 10-1 Religion as the Foundation of African Morality. Laurenti Magesa contends that those who think human traditions created morality are oblivious to the fact that, their very human life comes from God. “Tradition contains the moral code and prescription for ethical life.”6 He makes his point clearer when he opines that morality which helps to make people conform to the norms of society is geared toward the maintenance and enhancement of life. The spate of immorality and nonconformance to the traditions handed to Africans by their ancestors weaken the life of the community. Good life in a traditional African society is attained through close ties with religion, God, the ancestors and other humans.7 Magesa does not only blame human actions for destabilizing the life of the community in African Traditional Religion but also opposes spirits that can be beseeched by humans to cause harm. In the same way, benign spirits can be called upon to protect and prompt people to do good.8 John S. Mbiti also believes that many African societies believe that God is part of the social life of the community. The moral order in the African societies was given to them by God in order that people would live gladly and cordially with one another in the society. The moral order from God aids humans to work and have knowledge of what is good and evil, what is right and wrong. The customs and institutions like the chieftaincy institutions in African societies that have sustained the morality of Africans from the past to the present were created by people through the moral order given to them by God. He does this to ensure that the life of the individual and the community is protected 9 Opoku also shares the view that in African traditional societies, religion is the basis of morality. Religion in Africa, he continues, is so all-encompassing that religion and morality cannot be detached from one another. Religion to him is the binding force and the source of moral codes that regulate the life of the African society.10 God is believed to be the custodian and the final authority in all matters in the African Traditional Religion. He mentions the Akan of Ghana, the Fon of Benin and the Yoruba of Nigeria as examples of African societies that see God as the final judge when the life of the African on earth is ended. 4 Opoku, West Africa Traditional Religion, 156. 5 Kwame Gyekye, African Cultural Values, (Accra: Sankofa Publishing Co. Ltd., 1998), 41. 6 LaurentiMagesa,African Religion: The Moral Tradition of Abundant Life (Mary knoll: Orbis Books, 1997), 35. 7 Magesa,AfricanReligion,52. 8 Magesa, African Religion,53. 9 John S. Mbiti, An Introduction to African Religion, (Lusaka: Heinemann Educational Books Ltd, 1975), 36. 10 Opoku, West Africa Traditional Religion, 152. 11 Opoku,West Africa Traditional Religion,153. 12 Opoku,West Africa Traditional Religion,156. 13 Opoku,West Africa Traditional Religion,156. 14 E.Bolaji Idowu, Olodumare: God in Yoruba Belief (London: Longmans Group Ltd. 1962), 144- 145. INTRODUCTION The concepts of nature and foundations are key to understanding and appreciating a phenomenon. The morality of the African is not fully understood and appreciated by some European scholars. The authenticity of African morality is also sometimes challenged due to the lack of understanding of what really is the foundation and nature of the traditional African morality. Alfred Burdon Ellis for instance, comments that morality among the Akan tribe of Ghana is sin, and is not linked to religion in any way. He posits that religion and morality merge when humanity is socialized by a higher culture.1 Ellis goes on to mention that in the Akan culture, they limit sin only to offences against the gods and not crimes against humanity like murder and stealing.2i Kofi Asare Opoku refutes the assertions made by Ellis and describes them as wrong and disparaging. Opoku insists that morality and religion are indissoluble when it comes to African ethics, and morality springs from religious considerations.3 He points out specifically that the Akan morality has its basis in Amoah R.O. & Peprah A.K., E-Journal of Humanities, Arts and Social Sciences Vol.3 No.7(2022) pp 269-278 religion.4 African ethicists and philosophers such as Kwame Gyekye also contend that African morality has the society and the welfare of humanity as its basis.5 The paper contributes to the widespread debate that religion is the basis of African morality and elucidates on the true nature of traditional African morality. It fills the vacuum created that African moral beliefs have no infractions against humans but only on the gods. The morality of Africans is fashioned on their interaction with their fellow humans and with the divine. The foundation and nature of traditional African morality are theocentric and anthropocentric. Exploring the foundation and the nature of African ethics contributes to comprehending and valuing traditional African morality. E-Journal of Humanities, Arts and Social Sciences (EHASS) p , f g , 14 E.Bolaji Idowu, Olodumare: God in Yoruba Belief (London: Longmans Group Ltd. 1962), 144- 145. y y f ( g ) Magesa,African Religion: The Moral Tradition of Abundant Life (Mary knoll: Orbis Books, 1997), 35. ricanReligion 52 Humans and Society as the Foundation of African Morality Humans and Society as the Foundation of African Morality In the 17th century, the generally held notion that religion was the foundation of morality and that without religion, morality cannot exist was challenged.16 In 1690 Pierre Bayle17 posited that “religion is neither necessary nor sufficient for morality”.18 Arthur Schopenhauer is one of the Western philosophers who did not believe that morality should be based on religion. Religion according to him makes people behave morally because believers are promised an eternal bliss that would be attained in life after death. Many Christians live morally because they want to be in Heaven. Buddhists, on the other hand, behave ethically due to the promise of Nirvana and Muslims abide by the moral teachings of the Quran and the Hadith because of Jannah which Quran 31:8 describes as a “garden of pleasure”. This motivation for doing what is good on the basis of religion according to Schopenhauer is ultimately founded on “selfish egoism”.19 Schopenhauer also declares that “if any action has as its motive an egoistic aim, it cannot have any moral worth”.20 In one of his writings on ethics, Schopenhauer posited that morality rather has compassion as its foundation and not religion. Compassion is a day-by-day occurrence, free of any clandestine motive. It involves sharing and alleviating the suffering of others.21 In Africa, Kwame Gyekye and Kwasi Wiredu both Ghanaians and Akan are the stalwarts when it comes to African ethics. They made great use of the Akan culture to elucidate their moral concept. The duo belongs to the school of thought that does not ascribe to the idea that African morality should be based on religion. Gyekye observed that because Africans are said to be notoriously religious in every aspect of their life, hence, the popular notion that morality is based on religion. He rejects this popular notion to state emphatically that “the religious basis of African moral values is greatly mistaken.”22 Gyekye tries to explore how Ghanaian morality in particular and African morality, in general, are expressed in maxims, folktales, artistic symbols, institutions and practices of the people.23 Gyekye argues that if Traditional African Religion is not a revealed religion and does not have scriptures and prophets or founders to guide their moral life, how come Africans have elaborate moral codes that have stood the test of time? 15 Idowu, Olodumare: God in Yoruba Belief, 3. 16 Stephen Gaukroger, Objectivity: A Very Short Introduction (Oxford: University Press, 2012), 91. 17 Pierre Bayle (1647-1706) was a Calvinist French Philosopher, Author and Lexicographer, accessed 4/02/2021. http:www.britinnica.com. 18 Gaukroger, Objectivity: A Very Short Introduction, 91. 19 Arthur Schopenhauer, The Basis of Morality, trans, A. B. Bullock (New York: Dover Publications, 2005), 7. 20 Schopenhauer, The Basis of Morality, 7. 21 Schopenhauer, The Basis of Morality, 16. 22 Gyekye, African Cultural Values, 41. 23 Gyekye, African Cultural Values, 41. 24 Gyekye, African Cultural Values, 42. E-Journal of Humanities, Arts and Social Sciences (EHASS) Religion as the Foundation of African Morality. Opoku specifically mentions that the Akan see God as the guardian of law and order and morality flows from him. This is expressed in the Akan maxim Onyame mpjbcne “God does not like evil.”11 Opoku also highlights that among the Akan, the ancestors also have a vested interest in the moral life of the living.12 The gods too are not left out when it comes to shaping the moral life of Africans. They punish and reward their devotees based on their character.13fi E. Bolaji Idowu affirms the stance of Magesa, Mbiti and Opoku that “morality in the traditional African society is basically the fruit of religion and that it is dependent on it.”14 Idowu indicates that the 280 E-Journal of Humanities, Arts and Social Sciences (EHASS) Amoah R.O. & Peprah A.K., E-Journal of Humanities, Arts and Social Sciences Vol.3 No.7(2022) pp 269-278 Yoruba also draw their morality from God, Oludumare who has “the Pure” and “the Perfect King” as his attributes. Oludumare is the judge and the standard of morality. He judges the character of humanity. He is omnipresent and has his eagle’s eyes on the morality of his subjects; no one can escape the judgment of God.15 The assumptions of the African philosophers who ascribe to the widespread view that African morality depends on religion are of the view that African morality comes from God, the divinities and the ancestors. They enlighten people to do what is right, reward the good and punish the evil. This makes religion indispensable in determining morality in Africa. g j y y thur Schopenhauer, The Basis of Morality, trans, A. B. Bullock (New York: Dover Publications, 2005), 7. hopenhauer, The Basis of Morality, 7. 24 Gyekye, African Cultural Values, 42. Humans and Society as the Foundation of African Morality If it consents that African religion is a natural religion, that is created by the people’s own experience and from their world view, then morality too was created by them to regulate the behaviour of people in the society. Moralities of African societies are “formed based on their knowledge of the nature of society, human interactions, aspirations and the value of human life. This means that their morality will not be based on religion, but on the considerations of human welfare”.24 Gyekye again cites some writers of this school of thought and their views to buttress his claim. Godfred Wilson cited by Gyekye has said that among the Nyakyusa of Tanzania and Malawi, ethics is not 281 E-Journal of Humanities, Arts and Social Sciences (EHASS) Amoah R.O. & Peprah A.K., E-Journal of Humanities, Arts and Social Sciences Vol.3 No.7(2022) pp 269-278 related to religion and again moral virtues are not made in religious provisions. In Rwanda, J.J. Marquethas hinted that the morality of the Banyarwandar has no links to the command of God. These comments Gyekye believes are “unequivocal statements to support the stance that religion is not the basis of the morality of at least in some African societies.”25 Kwasi Wiredu also holds the view that religion should not be the foundation of African Morality. He claims “The will of God, not to talk of that of any other extra-human being, is logically incapable of defining the good.”26 Specifically, on the Akan morality, Wiredu says “ in fact, I deny that Akan moral thought is “supernaturalistic” to any extent”27 These bold statements of Wiredu run contrary to the views of Idowu and Magesa who claim that the supernatural, God, the gods and the ancestors have roles to play in determining what is moral in Africa. He also disagrees vehemently with Opoku on the fact that Akan morality is founded on religion.28 Wiredu proposes humanism as the foundation of African morality by analyzing the Akan maxim Onipanahia, “A human being has value.”29 Wiredu by this asserts that to be moral is to value humans and there is no need to appeal to religion to determine the worth of what is moral. Another aspect worth mentioning about Wiredu’s moral theory is his idea of “welfarism” when it comes to African morality. 26 KwasiWiredu, “Moral Foundations of An African Culture”, In KwasiWiredu and Kwame Gyekye, eds, “Person and Community,” Ghanaian Philosophical Studies, 1, Washington DC, The Council for Research in Value and Publishing, (1992):194. p g p 30 Leonard Wayne Sumner, Welfare, Happiness, and Ethics (Oxford: Clarendon Press, 1996), 3. E-Journal of Humanities, Arts and Social Sciences (EHASS) 25 Kwame Gyekye, African Ethics, 2010, accessed 16/02/2021. https:/plato.standford.edu. ( ) 27 KwasiWiredu, Cultural Universals and Particulars: An African Perspective (Indianapolis: Indiana University Press, 1996), 234. 33 Molefe, A Critique of KwasiWiredu’s Humanism and Impartiality, 95. Humans and Society as the Foundation of African Morality On the other hand, those who do not agree that African morality is not based on religion but on the welfare of the society also propose a humanistic or anthropocentric view of African morality. Another issue to be noted is that the widespread view that African morality is based on religion is neither influenced by the advent of Christianity nor Islam. It must be appreciated that both schools of thought have contributed to the African concept of morality. It can therefore be said that morality in African societies has both religious and social foundations. Those who speak for the motion highlight the religious foundation of African morality. They proposed a theistic foundation or theocentric view of African morality. On the other hand, those who do not agree that African morality is not based on religion but on the welfare of the society also propose a humanistic or anthropocentric view of African morality. Another issue to be noted is that the widespread view that African morality is based on religion is neither influenced by the advent of Christianity nor Islam. 31 Wiredu, Cultural Universals and Particulars, 64. Wiredu, Cultural Universals and Particulars,234. 29 Motsamai Molefe, “A Critique of KwasiWiredu’s Humanism and Impartiality”, ActaAcademica, 1,(2016): 94, 32 Wiredu, Cultural Universals and Particulars, 65. 29 Motsamai Molefe, “A Critique of KwasiWiredu’s Humanism and Impartiality”, ActaAcademica, 1,( accessed 6/02/2021.https://www.researchgate.net/publication. p g p 30 Leonard Wayne Sumner, Welfare, Happiness, and Ethics (Oxford: Clarendon Press, 1996), 3. 31 Humans and Society as the Foundation of African Morality Welfarism is the claim that human well-being “is the only value which an ethical theory needs to take seriously, ultimately and for its own sake”.30 Wiredu contends that every good action of an individual should seek the welfare of humans and the society. This he expressed by saying “human well-being is an irreducible presupposition of all morality … every moral endeavour is a certain kind of quest after human well-being”.31 Welfarism according to Wiredu hinges on the fact that “Every Akan moral saying assumes the harmonization of interest as a means. These moral sayings aim at securing human’s welfare as an end. Human’s welfare is the basis of all moral venture.”32 Motsamai Malofe summaries Wiredu’s welfarism that, “actions are right or wrong insofar as they produce or lead to well-being or wrong if they detract from it.”33 Wiredu proposes humanism as the foundation of African morality by analyzing the Akan maxim Onipanahia, “A human being has value.”29 Wiredu by this asserts that to be moral is to value humans and there is no need to appeal to religion to determine the worth of what is moral. Another aspect worth mentioning about Wiredu’s moral theory is his idea of “welfarism” when it comes to African morality. Welfarism is the claim that human well-being “is the only value which an ethical theory needs to take seriously, ultimately and for its own sake”.30 Wiredu contends that every good action of an individual should seek the welfare of humans and the society. This he expressed by saying “human well-being is an irreducible presupposition of all morality … every moral endeavour is a certain kind of quest after human well-being”.31 Welfarism according to Wiredu hinges on the fact that “Every Akan moral saying assumes the harmonization of interest as a means. These moral sayings aim at securing human’s welfare as an end. Human’s welfare is the basis of all moral venture.”32 Motsamai Malofe summaries Wiredu’s welfarism that, “actions are right or wrong insofar as they produce or lead to well-being or wrong if they detract from it.”33 It must be appreciated that both schools of thought have contributed to the African concept of morality. It can therefore be said that morality in African societies has both religious and social foundations. Those who speak for the motion highlight the religious foundation of African morality. They proposed a theistic foundation or theocentric view of African morality. 29 Motsamai Molefe, “A Critique of KwasiWiredu’s Humanism and Impartialit accessed 6/02/2021.https://www.researchgate.net/publication. , “Moral Foundations of An African Culture”, In KwasiWiredu and Kwame Gyekye, eds, “Person and 28 Wiredu, Cultural Universals and Particulars,234. , , 29 Motsamai Molefe, “A Critique of KwasiWiredu’s Humanism and Impartiality”, ActaAcademica, 1,(2016): 94, accessed 6/02/2021 https://www researchgate net/publication The Nature of African Moralityfi The discussions above offer five (5) main characteristics of traditional African morality. They are theocentric, communalistic, anthropocentric, utilitarian and shame-oriented. 33 Molefe, A Critique of KwasiWiredu’s Humanism and Impartiality, 95. 282 E-Journal of Humanities, Arts and Social Sciences (EHASS) Amoah R.O. & Peprah A.K., E-Journal of Humanities, Arts and Social Sciences Vol.3 No.7(2022) pp 269-278 Theocentric: Magesa,34Mbiti,35Opoku36 and Idowu37 underscore the fact that morality in traditional African societies has religion as its foundation. They believe that the Supreme Being, the gods and the ancestors play vital roles in moral development. They give humanity, laws to govern the society to bring about peace in the community. They inspire people to do what is right. They punish and reward people and the community for their moral deeds. The divine cannot be separated from morality in African traditional societies. Communalistic: The morality of the traditional African society as communalistic has been held by Gyekye and others. Gyekye believes that morality in Africa is based on the common good of the society and not that of the individual. “Seeking for the interest of others is central to the meaning and practices of morality in traditional African societies.”38Kollman also says that “African morality and ethics … cannot be conceived outside of the community.”39 Anthropocentric: Benezet Bujo claims fundamentally the morality of the African is anthropocentric.40 Wiredu asserts that humanity is the value and centre of all that is good and moral. He stresses that it is “not God/gods but humans that are relevant to the conceptual foundations of morality in the Akan thought.” 41 This characteristic places value on God as an object of respect and worship and not one whose will is to be obeyed. What must be obeyed are the will and the welfare of the society.42 According to Gyekye, it suffices to say that “Morality in traditional African societies is essentially social, arising out of the relations between individuals. This means that if there is no such thing as human society, there would be no such thing as morality.”43 Utilitarian/Pragmatic: Bujo44 and Wiredu45 use the term “utilitarian” to qualify traditional African ethics. The latter even goes further that among the Akan, the value and adulation of the gods depend on their usefulness to humanity. 34 Magesa,African Religion, 1997, 52. 35 Mbiti, An Introduction to African Religion, 36. 36 Opoku, West Africa Traditional Religion, 152. 37 Idowu, Olodumare: God in Yoruba Belief, 3. 38 Gyekye, African Cultural Values, 4. 39 P. Kollman, “Tribalism and Justice”, African Christian Studies, 4(2)(1988): 59. 40 Benezet Bujo, African Christian Morality at the Age of Inculturation (Nairobi: St. Paul, 1990), 49. 41 KwesiWiredu, The Moral Foundations of African Culture. (In Coetzee, P.H. & Roux, A.P.J., eds. Philosophy from Africa. Halfway House: International Thompson Publishing (S.A.) 1998. 307. 42 E-Journal of Humanities, Arts and Social Sciences (EHASS) 46 Segun Gbadegesin, Individuality, Community and the Moral Order. (In Coetzee, P.H. & Roux, A.P.J., Africa. Halfway House: International Thompson Publishing (SA). 1998), 305.f 47 Ruth Lienhard. “A “Good Conscience”, Difference between Honour and Justice Orientation.”Missiolo 139 f ( )( ) Benezet Bujo, African Christian Morality at the Age of Inculturation (Nairobi: St. Paul, 1990), 49. , African Cultural Values, 4. man, “Tribalism and Justice”, African Christian Studies, 4(2)(1988): 59. j f y g f ( ) 41 KwesiWiredu, The Moral Foundations of African Culture. (In Coetzee, P.H. & Roux, A.P.J., eds. Philosophy from Africa. Halfway House: International Thompson Publishing (S.A.) 1998. 307. 34 Magesa,African Religion, 1997, 52. 35 Mbiti, An Introduction to African Religion, 36. 36 Opoku, West Africa Traditional Religion, 152. 37 Idowu, Olodumare: God in Yoruba Belief, 3. 38 Gyekye, African Cultural Values, 4. 39 P. Kollman, “Tribalism and Justice”, African Christian Studies, 4(2)(1988): 59. 40 Benezet Bujo, African Christian Morality at the Age of Inculturation (Nairobi: St. Paul, 1990), 49. 41 KwesiWiredu, The Moral Foundations of African Culture. (In Coetzee, P.H. & Roux, A.P.J., eds. Philosophy from Afric Halfway House: International Thompson Publishing (S.A.) 1998. 307. 42 Wiredu, Cultural Universals and Particulars, 65. 43 Gyekye, African Cultural Values, 41. 44 Bujo, African Christian Morality, 50. 45 Wiredu, The Moral Foundations of African Culture 307. 46 Segun Gbadegesin, Individuality, Community and the Moral Order. (In Coetzee, P.H. & Roux, A.P.J., eds. Philosophy fro Africa. Halfway House: International Thompson Publishing (SA). 1998), 305. 47 Ruth Lienhard. “A “Good Conscience”, Difference between Honour and Justice Orientation.”Missiology, 29(2) (2001): 1 139 48 Gyekye, African Cultural Values, 101. 40 Benezet Bujo, African Christian Morality at the Age of Inculturation (Nairobi: St. Paul, 1990), 49. 41 KwesiWiredu, The Moral Foundations of African Culture. (In Coetzee, P.H. & Roux, A.P.J., eds. Phil Halfway House: International Thompson Publishing (S.A.) 1998. 307. 43 Gyekye, African Cultural Values, 41. KwesiWiredu, The Moral Foundations of African Culture. (In Coetzee, P.H. & R Halfway House: International Thompson Publishing (S.A.) 1998. 307. The Nature of African Moralityfi Segun Gbadegsin also intimates that morality in Africa is acts of humans that solve the problems of the world.46 Shame-oriented: Ruth Lienhard argues that an individual’s orientation in life contributes greatly to eliciting feelings of guilt and shame. When a bad act is committed shame is experienced by the individual who is honour oriented. In the same way, a transgressor will feel guilty if he is justice-oriented. In relation to the problem of sin and reconciliation, when one transgresses, the person is restored to his relationship with the divine and the community through the expression of shame and guilt associated with sin. This links reconciliation and sin very well in African society. Justice must be served to restore the guilt and honour of the penitent.47 Moral transgressions in traditional African societies evoke feelings of being dirty and ugly.48 42 Wiredu, Cultural Universals and Particulars, 65. 43 Gyekye, African Cultural Values, 41. 44 Bujo, African Christian Morality, 50. 48 Gyekye, African Cultural Values, 101. E-Journal of Humanities, Arts and Social Sciences (EHASS) 283 Amoah R.O. & Peprah A.K., E-Journal of Humanities, Arts and Social Sciences Vol.3 No.7(2022) pp 269-278 The penitent and the community are dishonoured and covered with shame due to sin.49 CONCLUSION The debate whether African morality is based on religion or on society would never cease. The two schools of thought should be annexed to conceptualize the true nature of African morality. The humanist view of African morality tries to imply that one can be moral without being religious. This view also corroborates the fact that there are nonreligious sources of African morality like proverbs, arts and symbols. It is proposed that the two should be put together to say the foundations of African morality are both from religion and society. Kwame Gyekye even concedes that if African morality is gleaned from their social interactions and not based on religion, “religion cannot be completely removed from the area of moral practice”50 This affirms the conclusion that African morality is both a creation of God and humans and because God created the human society, it can be ascribed to him. Hence the widespread view that African morality depends on religion. 50 Kwame Gyekye, African Cultural Values. 42. 49 Mbiti, An Introduction to African Religion, 177. E-Journal of Humanities, Arts and Social Sciences (EHASS) BIBLIOGRAPHY Alfred BurdonEllis, The Tshi-Speaking Peoples of the Gold Coast, London: Frank Cass, 1966. Asare-Danso, Seth, African Traditional Religion, Cape Coast: Beret Outlook Press 2019. Bujo, Benezet, African Christian Morality at the Age of Inculturation, Nairobi: St. Paul, 1990. Gaukroger, Stephen, Objectivity: A Very Short Introduction, Oxford: University Press, 2012. Gbadegesin, Segun “Individuality, Community and the Moral Order”. (In Coetzee, P.H. & Roux, A.P.J., ed Alfred BurdonEllis, The Tshi-Speaking Peoples of the Gold Coast, London: Frank Cass, 1966. Asare-Danso, Seth, African Traditional Religion, Cape Coast: Beret Outlook Press 2019. Bujo, Benezet, African Christian Morality at the Age of Inculturation, Nairobi: St. Paul, 1990. Gaukroger, Stephen, Objectivity: A Very Short Introduction, Oxford: University Press, 2012. Gbadegesin, Segun “Individuality, Community and the Moral Order”. (In Coetzee, P.H. & Roux, A.P.J., ed , p g p f , , Asare-Danso, Seth, African Traditional Religion, Cape Coast: Beret Outlook Press 2019. Bujo, Benezet, African Christian Morality at the Age of Inculturation, Nairobi: St. Paul, 1990. Gaukroger, Stephen, Objectivity: A Very Short Introduction, Oxford: University Press, 2012. roger, Stephen, Objectivity: A Very Short Introduction, Oxford: University Press, 2012. Gbadegesin, Segun “Individuality, Community and the Moral Order”. (In Coetzee, P.H. & Roux, A.P.J., eds. Philosophy from Africa. Halfway House: International Thompson Publishing (SA). (1 Gyekye, Kwame, African Ethics, 2010. Accessed 16/02/2021. https:/plato.standford.edu. p y f f y p g ( ) ( Gyekye, Kwame, African Ethics, 2010. Accessed 16/02/2021. https:/plato.standford.edu. ____. African Cultural Values, Accra: Sankofa Publishing Co. Ltd., 1998. ______. African Cultural Values, Accra: Sankofa Publishing Co. Ltd., 1998. Idowu, E., Bolaji, Olodumare: God in Yoruba Belief, London: Longmans Group Ltd. 1962. Kollman P “Tribalism and Justice” African Christian Studies 4(2)(1988):49-70 ______. African Cultural Values, Accra: Sankofa Publishing Co. Ltd., 1998. Idowu, E., Bolaji, Olodumare: God in Yoruba Belief, London: Longmans Group Ltd. 1962. ll b l d f h d ( )( ) Idowu, E., Bolaji, Olodumare: God in Yoruba Belief, London: Longmans Group Ltd. 1962. Kollman, P.,“Tribalism and Justice”, African Christian Studies, 4(2)(1988):49-70. Mbiti, S. John, An Introduction to African Religion, Lusaka: Heinemann Educational Books Ltd., 1975. Megasa La renti Af i R li i Th M l T diti f Ab d t Lif Mar Knoll: Orbis Books 19 Mbiti, S. John, An Introduction to African Religion, Lusaka: Heinemann Educational Books Ltd., 1975. E-Journal of Humanities, Arts and Social Sciences (EHASS) BIBLIOGRAPHY Megasa, Laurenti, African Religion: The Moral Tradition of Abundant Life, Mary Knoll: Orbis Books, 199 Molefe, Motsamai “A Critique of KwasiWiredu’s Humanism and Impartiality”, Acta Academica, 2016.1, 9. 91-110. Accessed 6/02/2021.https://www.researchgate.net/publication. Opoku, K., Asare West Africa Traditional Religion, Accra: FEP International Private Limited, 1978.f Opoku, K., Asare West Africa Traditional Religion, Accra: FEP International Private Limited, 1978. Lienhard, Ruth, “A “Good Conscience”, Difference between Honour and Justice Orientation.” Missiology, 29(2)(2001):131-141. Schopenhauer, Arthur, The Basis of Morality, trans, A. B. Bullock, New York: Dover Publications, 2005. Sumner, L., Wayne, Welfare, Happiness, and Ethics, Oxford: Clarendon Press, 1996. Wiredu, Kwasi, Cultural Universals and Particulars: An African Perspective, Indianapolis: Indiana University Press, 1996. Wiredu, Kwasi “Moral Foundations of An African Culture”, In KwasiWiredu and Kwame Gyekye, eds, “Person and Community,” Ghanaian Philosophical Studies, 1, Washington DC, The Council for Research in Value and Publishing, (1992):194. Wiredu, Kwasi, “The Moral Foundations of African Culture”. (In Coetzee, P.H. & Roux, A.P.J., eds. Philosophy from Africa. Halfway House: International Thompson Publishing (S.A.) (1998). 306- 316. 284 E-Journal of Humanities, Arts and Social Sciences (EHASS) Amoah R.O. & Peprah A.K., E-Journal of Humanities, Arts and Social Sciences Vol.3 No.7(2022) pp 269-278 ABOUT AUTHORS ABOUT AUTHORS Richmond Osei Amoah and Augustine Kojo Peprah are priests of the Catholic Archdiocese of Kumasi and tutors at the Department of Social Science, St. Louis College of Education, Kumasi-Ghana. Their areas of research are Religious and Moral Education. 285
https://openalex.org/W4290988567	https://www.frontiersin.org/articles/10.3389/fcvm.2022.981972/pdf	English	null	Myocardial disturbances of intermediary metabolism in Barth syndrome	Frontiers in cardiovascular medicine	2,022	cc-by	6,817	TYPE Mini Review PUBLISHED 10 August 2022 DOI 10.3389/fcvm.2022.981972 TYPE Mini Review PUBLISHED 10 August 2022 DOI 10.3389/fcvm.2022.981972 TYPE Mini Review PUBLISHED 10 August 2022 DOI 10.3389/fcvm.2022.981972 Myocardial disturbances of intermediary metabolism in Barth syndrome OPEN ACCESS EDITED BY Diederik Wouter Dimitri Kuster, Amsterdam University Medical Center, Netherlands REVIEWED BY Vasco Sequeira, University Hospital Würzburg, Germany CORRESPONDENCE John R. Ussher jussher@ualberta.ca SPECIALTY SECTION This article was submitted to Cardiovascular Metabolism, a section of the journal Frontiers in Cardiovascular Medicine RECEIVED 29 June 2022 ACCEPTED 25 July 2022 PUBLISHED 10 August 2022 CITATION Greenwell AA, Tabatabaei Dakhili SA and Ussher JR (2022) Myocardial disturbances of intermediary metabolism in Barth syndrome. Front. Cardiovasc. Med. 9:981972. doi: 10.3389/fcvm.2022.981972 Amanda A. Greenwell1,2, Seyed Amirhossein Tabatabaei Dakhili1,2 and John R. Ussher1,2 1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada, 2Women and Children’s Health Research Institute, University of Alberta, Edmonton, AB, Canada 1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada, 2Women and Children’s Health Research Institute, University of Alberta, Edmonton, AB, Canada Barth Syndrome (BTHS) is a rare X-linked mitochondrial disorder due to mutations in the gene TAFAZZIN, which leads to immature cardiolipin (CL) remodeling and is characterized by the development of cardiomyopathy. The immature CL remodeling in BTHS results in electron transport chain respiratory defects and destabilization of supercomplexes, thereby impairing ATP production. Thus, BTHS-related cardiomyopathy appears to share metabolic characteristics of the failing heart being an “engine out of fuel.” As CL associates with numerous mitochondrial enzymes involved in ATP production, BTHS is also characterized by several defects in intermediary energy metabolism. Herein we will describe the primary disturbances in intermediary energy metabolism relating to the heart’s major fuel sources, fatty acids, carbohydrates, ketones, and amino acids. In addition, we will interrogate whether these disturbances represent potential metabolic targets for alleviating BTHS-related cardiomyopathy. Greenwell AA, Tabatabaei Dakhili SA and Ussher JR (2022) Myocardial disturbances of intermediary metabolism in Barth syndrome. Front. Cardiovasc. Med. 9:981972. doi: 10.3389/fcvm.2022.981972 © 2022 Greenwell, Tabatabaei Dakhili and Ussher. 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. KEYWORDS Barth syndrome (BTHS), cardiomyopathy, cardiac energetics, glucose oxidation, fatty acid oxidation, ketone oxidation Barth syndrome (BTHS), cardiomyopathy, cardiac energetics, glucose oxidation, fatty acid oxidation, ketone oxidation Introduction Of interest, CL has been shown to be essential for the assembly of ETC supercomplexes, and supercomplexes were shown to be destabilized in hearts of Tafazzin knockdown (TazKD) mice secondary to doxycycline-mediated induction of a short-hairpin RNA against Tafazzin (21). Analysis of cardiac mitochondria isolated from cardiac-speciﬁc Tafazzin deﬁcient mice prior to the development of cardiomyopathy, also revealed structural remodeling of the respiratory chain with a shift from high molecular weight supercomplexes in the ETC toward lower molecular weight forms such as heterooligomers and individual complexes (22). Furthermore, complex III activity is decreased in cardiac mitochondria isolated from TazKD mice (15, 23, 24), whereas complex V (F1F0-ATP synthase) activity is decreased in both TazKD mice and inducible pluripotent stem cell-derived cardiomyocytes from BTHS patients (15, 25). Although the detrimental consequences of TAFAZZIN deﬁciency on the structural integrity and optimal functioning of the ETC are well- deﬁned, several studies have identiﬁed speciﬁc derangements in upstream intermediary energy metabolism pathways that we will herein interrogate (15–17). Such observations illustrate that mechanisms beyond respiratory chain dysfunction may contribute to the cardiac energy deﬁcit in BTHS-related cardiomyopathy, while representing possible metabolic targets for intervention. Introduction Cardiomyopathy is the most common clinical feature and primary driver of disease outcomes in Barth syndrome (BTHS; Online Mendelian Inheritance in Man [OMIM] 302060), a rare X-linked mitochondrial disorder ﬁrst described by Dr. Peter Barth and colleagues in 1983 (1, 2). Despite the broad variability in clinical presentation, which can include features such as neutropenia, skeletal myopathy, exercise intolerance, 3-methylglutaconic aciduria, and pre-pubertal growth retardation, cardiomyopathy manifests in approximately 90% of males with BTHS (3, 4). However, the severity and speciﬁc cardiomyopathic phenotype can vary both between individuals and throughout disease progression, with no deﬁnitive genotype-phenotype correlations having yet been identiﬁed. Dilated cardiomyopathy is most common, however other forms including restrictive (5), hypertrophic, and hypertrophic-dilated cardiomyopathy have also been reported (2, 6). Frontiers in Cardiovascular Medicine frontiersin.org 01 10.3389/fcvm.2022.981972 Greenwell et al. BTHS is caused by mutations in the TAFAZZIN gene, formerly notated as TAZ, on chromosome Xq28, which encodes for the protein tafazzin, a phospholipid transacylase responsible for cardiolipin (CL) remodeling (3, 7). CL is a unique, dimeric, tetra-acyl phospholipid found almost exclusively in the inner mitochondrial membrane (IMM) with essential roles in the maintenance of mitochondrial morphology, and in fundamental mitochondrial functions including ﬁssion-fusion dynamics, mitophagy, apoptosis, and energy metabolism (8–10). Impaired cardiac ATP production is a principal contributor to the development and progression of heart failure (11, 12), which appears to also be present in BTHS. Indeed, the use of 31P magnetic resonance spectroscopy to measure the phosphocreatine to ATP ratio (PCr/ATP), an index of cardiac high-energy phosphate metabolism and energy status, was reduced in young adults (18–36 years of age) and children/adolescents with BTHS compared to healthy participants (13, 14). Nonetheless, the molecular mechanisms linking TAFAZZIN deﬁciency to perturbed myocardial energetics are presently not well-characterized. Importantly, evidence that impairments in oxidative metabolism are substrate speciﬁc in BTHS may indicate a primary defect in upstream intermediary metabolism pathways (15–17). Accordingly, the focus of this mini-review will be to characterize the alterations in intermediary metabolism that accompany TAFAZZIN deﬁciency, and to discuss the potential of targeting these pathways as a therapeutic approach to mitigate the development and progression of BTHS-related cardiomyopathy (Figure 1). proteome demonstrated a reduction in complex 1 (following normalization to citrate synthase) in left ventricular (LV) tissue from individuals with BTHS compared to non-failing control heart samples (20). Frontiers in Cardiovascular Medicine Greenwell et al. Mitochondrial respiratory abnormalities in Barth syndrome Fatty acids are the predominant fuel source for the heart and an impaired capacity to eﬀectively utilize fatty acids can contribute to an energy deﬁcit and subsequent cardiac dysfunction (12, 26, 27), which appears to be present in BTHS. Myocardial fatty acid extraction and uptake following positron emission tomography (PET) imaging with [1–11C]palmitate were signiﬁcantly reduced in young adults with BTHS compared to healthy, age-matched controls, though myocardial fatty acid β-oxidation remained similar (14). Furthermore, cardiac mitochondria isolated from TazKD mice at 2 months or 4–6 months of age, and from 2- month-old mice with a cardiac-speciﬁc Tafazzin deﬁciency demonstrated a signiﬁcant repression of state 3 (ADP- stimulated) respiration rates supported by palmitoylcarnitine and malate (15, 16, 22). A decrease in palmitoylcarnitine supported state 3 respiration was also observed in permeabilized cardiac ﬁbers with intact mitochondrial matrices from 4– 6-month-old TazKD mice with preserved cardiac function, as determined by high-resolution respirometry (16). Protein expression of fatty acid binding protein and acyl-CoA synthetase, which mediate intracellular fatty acid transport and Notably, TAFAZZIN deﬁciency is associated with an impairment of mitochondrial respiratory capacity and ATP production, which to date, has largely been attributed to destabilization of the IMM electron transport chain (ETC) (18). Normal production of ATP in healthy mitochondria involves the oxidation of fatty acids, glucose, ketones, and amino acids to produce acetyl-CoA, which enters the Krebs cycle resulting in the generation of reducing equivalents (i.e., NADH and FADH2) by its 4 dehydrogenase enzymes. As these reducing equivalents deliver electrons to complex I (NADH) and II (FADH2) of the ETC, an electrochemical gradient across the IMM is created as electrons are passed along the ETC through a series of redox reactions, which drives the production of ATP by ATP synthase (complex V) (Figure 1). In a Tafazzin deﬁcient mouse model that relies on cre- mediated deletion of the Tafazzin gene containing loxP sites ﬂanking exons 5–10, label-free quantitative proteomics revealed reductions in the relative abundances of all ETC complexes (complexes I-V) (19). Moreover, liquid chromatography (LC)-mass spectrometry (MS)/MS analysis of the cardiac Frontiers in Cardiovascular Medicine frontiersin.org 02 Greenwell et al. 10.3389/fcvm.2022.981972 FIGURE 1 Myocardial metabolic disturbances in Barth syndrome. Clinical and preclinical studies have identiﬁed multiple mechanisms that may contribute to impaired cardiac ATP production in the context of TAFAZZIN deﬁciency. Mitochondrial respiratory abnormalities in Barth syndrome Although the results of the CARDIOMAN trial are not yet published, a summary of the initial results of this single center, double- blinded, randomized, placebo-controlled crossover study of 11 participants with BTHS who were administered bezaﬁbrate treatment for 15-weeks, was released by the Barth Syndrome Foundation. Assessment of cardiac function by ultrasound echocardiography revealed a signiﬁcant improvement in heart strain at rest but not during peak exercise, and a trend toward improved heart chamber size. However, given that neither traditional cardiac parameters nor quality of life was changed signiﬁcantly, these ﬁndings of cardiac improvement may not be clinically relevant (https://www.barthsyndrome.org/research/ clinicaltrials/cardioman.html, accessed June 03, 2022). Taken together, these bezaﬁbrate-mediated cardiac improvements in preclinical and clinical studies are encouraging and suggest that stimulating myocardial fatty acid β-oxidation may have utility in BTHS. Nonetheless, it has been suggested by others that systemic activation of PPARα with ﬁbrates actually decreases myocardial fatty acid β-oxidation due to the stimulation of hepatic fatty acid β-oxidation and subsequent reduction in circulating lipids (32). Hence, further studies are still required to delineate whether the speciﬁc enhancement of cardiac fatty acid β-oxidation is indeed eﬀective in alleviating BTHS-related cardiomyopathy. carnitine was not reduced, providing further evidence against a speciﬁc defect in mitochondrial fatty acid uptake (16). Alternatively, impaired tafazzin-mediated CL remodeling in BTHS may result in a selective block in long-chain fatty acid β-oxidation. Protein levels of very long-chain acyl CoA dehydrogenase (VLCAD) were decreased in LV tissue from male BTHS subjects compared to age-matched, non-failing male subjects and male subjects diagnosed with idiopathic dilated cardiomyopathy, as assessed by both immunoblotting and LC-MS/MS based proteomic proﬁling (20). Likewise, VLCAD protein expression was also decreased in cardiac mitochondria from 4–6-month-old TazKD mice compared to their WT littermates, as determined via LC-MS/MS based proteomic proﬁling (16). This is consistent with evidence of disrupted interactions between VLCAD and ETC supercomplexes in isolated cardiac mitochondria from 3-month-old TazKD mice (21). Of interest, protein expression of the alpha subunit of the trifunctional protein complex (TFPα) of fatty acid β-oxidation is elevated in heart tissue from BTHS subjects (20). As TFPα also possesses monolysocardiolipin (MLCL) acyltransferase activity, further investigation is required to determine whether the accumulation of MLCL in BTHS may impair the β-oxidation function of TFPα by shifting toward MLCL reacylation. Mitochondrial respiratory abnormalities in Barth syndrome Further evidence for a general defect in the enzymatic machinery of fatty acid β-oxidation in BTHS is also seen by the decline in medium-chain acyl-CoA dehydrogenase protein expression and a reduction in octanoylcarnitine-supported respiration in cardiac mitochondria from TazKD mice (16). While the above described studies allude to impaired myocardial fatty acid β-oxidation in BTHS, there are limitations with assessing intermediary metabolism in isolated mitochondria where key cellular regulators (i.e., malonyl CoA) may be removed, and measures of protein expression do not necessarily reﬂect ﬂux (28). Indeed, palmitate oxidation rates measured using [9, 10-3H]palmitate were elevated in the isolated working hearts of 8–10-week-old TazKD mice compared to their WT littermates (17). The reported increase in palmitate oxidation was not associated with enhanced gene or protein expression of β-oxidation enzymes and was surprisingly associated with an increase in myocardial triacylglycerol content, as well as a reduction in mRNA expression of the fatty acid transporter, cluster of diﬀerentiation 36. Reasons that may explain the elevated palmitate oxidation in the isolated working heart are unclear, though substrate oxidation rates in the working heart operate at a much higher workload than that of isolated mitochondria, and these perfusions were performed in the presence of insulin. Frontiers in Cardiovascular Medicine Mitochondrial respiratory abnormalities in Barth syndrome In addition to direct impairment of the mitochondrial electron transport chain, speciﬁc defects have also been identiﬁed relating to the intermediary metabolism of fatty acids, carbohydrates, ketones, and amino acids. ANT, adenine nucleotide translocase; BCAA, branched-chain amino acid, BDH1, β-hydroxybutyrate dehydrogenase; βOHB, β-hydroxybutyrate; CI-IV, complex 1–4; GLUT4, glucose transporter type 4; IR, insulin receptor; LCFA, long-chain fatty acids; LDH, lactate FIGURE 1 Myocardial metabolic disturbances in Barth syndrome. Clinical and preclinical studies have identiﬁed multiple mechanisms that may contribute to impaired cardiac ATP production in the context of TAFAZZIN deﬁciency. In addition to direct impairment of the mitochondrial electron transport chain, speciﬁc defects have also been identiﬁed relating to the intermediary metabolism of fatty acids, carbohydrates, ketones, and amino acids. ANT, adenine nucleotide translocase; BCAA, branched-chain amino acid, BDH1, β-hydroxybutyrate dehydrogenase; βOHB, β-hydroxybutyrate; CI-IV, complex 1–4; GLUT4, glucose transporter type 4; IR, insulin receptor; LCFA, long-chain fatty acids; LDH, lactate dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; MCFA, medium-chain fatty acids; MCU, mitochondrial calcium uniporter; MDH, malate dehydrogenase; P, phosphate group; PDH, pyruvate dehydrogenase; SCOT, succinyl-CoA:3-ketoacid CoA transferase; SLC, solute carrier; TFP, mitochondrial trifunctional protein; VLCAD, very long-chain acyl-CoA dehydrogenase. suggests that BTHS-related defects in myocardial fatty acid metabolism may be downstream of mitochondrial fatty acid uptake. Supporting this premise, respiratory capacity of cardiac mitochondria from TazKD mice was decreased compared to wild-type (WT) littermates using palmitoylcarnitine, which bypasses CPT-1, whereas respiration with palmitoyl CoA plus fatty acid esteriﬁcation to CoA, respectively, were similar in LV tissue from male patients with BTHS and non-failing hearts (20). Furthermore, similar enzymatic activities of carnitine palmitoyltransferase I and 2 (CPT-1 and CPT-2) were observed in LV tissue homogenates from male patients with BTHS-related cardiomyopathy compared to non-failing controls (20). This Frontiers in Cardiovascular Medicine frontiersin.org 03 10.3389/fcvm.2022.981972 Greenwell et al. BTHS, the pan-peroxisome proliferator activated receptor (PPAR) agonist, bezaﬁbrate, is currently being investigated in the CARDIOlipin MANipulation (CARDIOMAN) trial (29). PPAR agonists, in particular PPARα, promote fatty acid β-oxidation via transcriptional upregulation of genes encoding for enzymes involved in fatty acid β-oxidation (30). Intriguingly, treatment of 3-month-old TazKD mice with bezaﬁbrate for 4-months prevented the development of dilated cardiomyopathy and systolic dysfunction, which was associated with increased expression of genes involved in multiple energy metabolism pathways (31). Perturbations in myocardial carbohydrate metabolism in Barth syndrome This impairment in normal Ca2+ handling was associated with increased myoﬁlament Ca2+ sensitivity and decreased cross-bridge cycling velocity, thereby contributing to diastolic dysfunction in TazKD mice and consistent with observations that a cardiac-speciﬁc deﬁciency of PDH promotes diastolic dysfunction (40). bioanalyzer, neonatal cardiac myocytes isolated from TazKD mice demonstrated an ∼40% decrease in oxygen consumption coincident with an increase in extracellular acidiﬁcation rates, suggestive of uncoupled glucose metabolism due to elevated glycolysis and decreased glucose oxidation (23). Furthermore, ADP-stimulated respiration in isolated cardiac subsarcolemmal and intermyoﬁbrillar mitochondria, as well as permeabilized cardiac muscle ﬁbers, was signiﬁcantly decreased with pyruvate and malate as substrates in 4–6-month-old TazKD mice vs. their WT littermates (16). Conversely, no impairment in ADP-stimulated respiration with pyruvate as a substrate was observed in isolated cardiac mitochondria from 2-month- old TazKD mice vs. their WT littermates (15). In addition, C2C12 mouse myoblasts subjected to CRISPR/Cas9-mediated knockout of Tafazzin demonstrated decreased incorporation of [U-13C]glucose into acetyl-CoA (37). This reduction was associated with increased inhibitory phosphorylation of PDH at serine 293, resulting in an approximate 50% decrease in enzymatic activity that could be rescued by incubation with exogenous CL. Deﬁciency of mature CL may also blunt Ca2+-dependent PDH dephosphorylation by reducing the abundance and stability of the mitochondrial calcium uptake protein 1, the primary regulator of the mitochondrial calcium uniporter (MCU), thereby impairing mitochondrial Ca2+ uptake and PDH activation (38). A cardiac-speciﬁc downregulation of the MCU pore-forming MCUa subunit has also been observed in 10-week-old TazKD mice, which abrogates Ca2+ uptake in isolated cardiac mitochondria (39) and could further explain impairments in PDH activity in BTHS. This impairment in normal Ca2+ handling was associated with increased myoﬁlament Ca2+ sensitivity and decreased cross-bridge cycling velocity, thereby contributing to diastolic dysfunction in TazKD mice and consistent with observations that a cardiac-speciﬁc deﬁciency of PDH promotes diastolic dysfunction (40). fraction, uptake and utilization were signiﬁcantly elevated compared to healthy age-matched controls (14). However, given that myocardial glucose oxidation rates were not directly assessed in these subjects, it is plausible that the elevated myocardial glycolytic rates observed in BTHS may explain the observed increase in myocardial glucose utilization. As the stimulation of glucose oxidation in the heart has been shown to improve cardiac function in ischemic heart disease, heart failure, and diabetic cardiomyopathy (33, 35, 42–44), this may be an exciting metabolic approach to alleviate BTHS- related cardiomyopathy. Perturbations in myocardial carbohydrate metabolism in Barth syndrome Surprisingly, treatment of 6-week-old TazKD mice for 6-weeks with dichloroacetate (DCA; 70 mM in the drinking water), a pyruvate analog that inhibits PDH kinase to prevent inhibitory phosphorylation of PDH, did not alleviate their hypertrophic cardiomyopathy phenotype despite an enhancement of PDH activity (45). Reasons that DCA may have been devoid of beneﬁt in TazKD mice may be due to the possibility that despite PDH activity and glucose oxidation being elevated, the immature CL remodeling and destabilized ETC supercomplexes would still be present, resulting in no net improvement in myocardial ATP production. Despite the failure of DCA to alleviate hypertrophic cardiomyopathy in TazKD mice, further investigation is needed to determine whether the pharmacological optimization of glucose oxidation may still have clinical utility in BTHS. On the contrary, it is also possible that any metabolic intervention to improve myocardial oxidative metabolism in BTHS will fail unless the CL-induced defects in ETC function are also resolved. Perturbations in myocardial carbohydrate metabolism in Barth syndrome Numerous forms of cardiovascular disease (e.g., ischemic heart disease, heart failure, diabetic cardiomyopathy) are characterized by an impairment in myocardial glucose oxidation, often due to defects in pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation (33–35). This metabolic perturbation also appears to be present in BTHS-related cardiomyopathy, as circulating lactate levels are elevated in individuals with BTHS (36), consistent with uncoupled glucose metabolism as glycolytically-derived pyruvate is shunted to lactate vs. being oxidized via PDH. Further evidence for a glucose oxidation defect in BTHS has been reported in several studies utilizing the TazKD mouse model and Tafazzin deﬁcient cell lines. Utilizing a XF24 Seahorse Despite these discrepancies in myocardial fatty acid β- oxidation in BTHS, current dogma in the ﬁeld posits that stimulating myocardial fatty acid β-oxidation may be a novel approach to improve cardiac function in heart failure via increasing ATP production to support contraction (12). Supporting such a strategy as a therapeutic approach in Frontiers in Cardiovascular Medicine frontiersin.org 04 10.3389/fcvm.2022.981972 Greenwell et al. bioanalyzer, neonatal cardiac myocytes isolated from TazKD mice demonstrated an ∼40% decrease in oxygen consumption coincident with an increase in extracellular acidiﬁcation rates, suggestive of uncoupled glucose metabolism due to elevated glycolysis and decreased glucose oxidation (23). Furthermore, ADP-stimulated respiration in isolated cardiac subsarcolemmal and intermyoﬁbrillar mitochondria, as well as permeabilized cardiac muscle ﬁbers, was signiﬁcantly decreased with pyruvate and malate as substrates in 4–6-month-old TazKD mice vs. their WT littermates (16). Conversely, no impairment in ADP-stimulated respiration with pyruvate as a substrate was observed in isolated cardiac mitochondria from 2-month- old TazKD mice vs. their WT littermates (15). In addition, C2C12 mouse myoblasts subjected to CRISPR/Cas9-mediated knockout of Tafazzin demonstrated decreased incorporation of [U-13C]glucose into acetyl-CoA (37). This reduction was associated with increased inhibitory phosphorylation of PDH at serine 293, resulting in an approximate 50% decrease in enzymatic activity that could be rescued by incubation with exogenous CL. Deﬁciency of mature CL may also blunt Ca2+-dependent PDH dephosphorylation by reducing the abundance and stability of the mitochondrial calcium uptake protein 1, the primary regulator of the mitochondrial calcium uniporter (MCU), thereby impairing mitochondrial Ca2+ uptake and PDH activation (38). A cardiac-speciﬁc downregulation of the MCU pore-forming MCUa subunit has also been observed in 10-week-old TazKD mice, which abrogates Ca2+ uptake in isolated cardiac mitochondria (39) and could further explain impairments in PDH activity in BTHS. Summary, conclusions & future directions Although amino acids contribute minimally to cardiac ATP production under normal physiological conditions (54), combined glutamate and malate-supported state 3 respiration was upregulated by 45–68% in cardiac mitochondria isolated from TazKD mice 4–6 months of age compared to their WT littermates (16). Because TAFAZZIN deﬁciency may reduce CoA availability, it was postulated that the increased reliance on glutamate metabolism manifests since glutamate can be oxidized through the malate-aspartate shuttle, which generates NADH through CoA-independent reactions. The upregulated glutamate oxidation in cardiac mitochondria from TazKD mice was associated with increased protein expression of malate dehydrogenase, a key enzyme of the malate-aspartate shuttle (55). Likewise, a 25% increase in glutamate-stimulated state 3 respiration was also reported in isolated cardiac mitochondria from 2-month-old TazKD mice (15). This particular study also reported a 6-fold increase in glutamate-stimulated activity of the CL-regulated adenine nucleotide translocase in TazKD mouse cardiac mitochondria, thus suggesting that aberrant CL remodeling may inﬂuence substrate selectivity of the adenine nucleotide translocase and overall ETC ﬂux. The development of multiple murine models of BTHS has greatly advanced our understanding of the metabolic perturbations present in BTHS, where it is clear that not only defects in ETC respiratory function, but also intermediary metabolism characterize this rare genetic disorder. As optimizing cardiac intermediary energy metabolism is being extensively pursued as a pharmacological strategy to improve cardiac function in heart failure, such an approach may also have clinical utility for managing BTHS-related cardiomyopathy. Unfortunately, the majority of studies that have tried to speciﬁcally optimize cardiac intermediary metabolism have yet to yield any major success. It does remain possible that any intervention that stimulates cardiac intermediary metabolism in BTHS in the absence of correcting the ETC respiratory defects, would not succeed since augmented oxidation of fuel (i.e., glucose) would not translate into increased ATP production to support cardiac function. As such, in order for metabolic therapy to yield beneﬁt in BTHS it may need to be coupled with interventions that stabilize ETC respiratory function. Indeed, elamipretide is a cell-permeable, aromatic- cationic mitochondria-targeting tetrapeptide that localizes to the IMM, where it selectively associates with CL (59, 60) and is currently being tested in phase 2 studies for the treatment of BTHS (5). Perturbations in myocardial ketone metabolism in Barth syndrome Supporting this perspective, a higher whole-body leucine rate of appearance per kg of fat-free mass, a measure of proteolysis, demonstrated a trend to being associated with worsened LV function as determined by a lower LV global strain in adolescents and young adults with BTHS (58). While there appears to be several disturbances in myocardial amino acid metabolism present in BTHS, further investigation will be required to determine whether such disturbances are a viable metabolic target to alleviate BTHS-related cardiomyopathy. perfusate βOHB concentrations (0.8 mM) was a limiting factor in the aforementioned study, and that ketone oxidation rates are elevated in vivo in TazKD mice. It has been proposed that the salutary actions of sodium- glucose cotransporter-2 inhibitors in heart failure with reduced or preserved ejection fraction in the presence or absence of diabetes may be due in part to increasing circulating ketone levels and subsequent myocardial ketone oxidation (49– 51). Accordingly, interventions that optimize cardiac ketone oxidation may represent a potential target to alleviate BTHS- related cardiomyopathy. Moreover, such a strategy may prove to be a more viable metabolic target, due to the enzymes of ketone oxidation not being as reliant on interactions with CL for their activity, as they are primarily localized to the mitochondrial matrix (52, 53). Perturbations in myocardial ketone metabolism in Barth syndrome Observations that the failing heart increases its reliance on ketones as an oxidative fuel source (26, 46) has led to increased investigation of potential perturbations in myocardial ketone metabolism during the pathology of cardiovascular disease. Whether a similar shift in myocardial substrate utilization occurs in BTHS remains enigmatic, though metabolomics studies in 23 individuals with BTHS demonstrated a 1.8-fold increase in circulating β-hydroxybutyrate (βOHB) levels in comparison to 15 age-matched individuals not known to have an inborn error of metabolism (47). An increase in myocardial βOHB content was also observed in LV samples from BTHS subjects compared to age-matched non-failing control heart samples (20). Furthermore, an ∼4-fold increase in myocardial protein expression of the ketone oxidation enzyme, βOHB dehydrogenase 1 (BDH1), was observed in 10-week-old TazKD mice vs. their WT littermates. Interestingly, this increased BDH1 expression did not translate into an increase in βOHB oxidation rates assessed during isolated working perfusions. As myocardial ketone oxidation rates are highly dependent on ketone delivery to the heart (48), it is plausible that identical BTHS-related impairments in myocardial glucose oxidation using [U-14C]glucose have also been observed in aerobically perfused isolated working hearts from 8–10-week-old TazKD mice, which was also associated with impaired PDH activity compared to their WT littermates (17). However, this reduction in myocardial PDH activity was independent of alterations in phosphorylation or acetylation. Of interest, the insulin- stimulated enhancement of glucose oxidation rates was also blunted in the TazKD isolated working heart suggestive of cardiac insulin resistance, which coincided with a reduction in the gene expression of glucose transporter 4. Evidence of increased and decreased circulating glucose and insulin levels, respectively, and of reduced pancreatic islet insulin secretion in TazKD mice provide further support for the paradigm of impaired insulin action in the context of Tafazzin deﬁciency (17, 41). PET imaging studies with [1-11C]glucose in individuals with BTHS (age 18–36 years) contrast the majority of ﬁndings in preclinical models of BTHS, as myocardial glucose extraction Frontiers in Cardiovascular Medicine frontiersin.org 05 10.3389/fcvm.2022.981972 Greenwell et al. acids involved in Krebs Cycle anaplerosis, including arginine, ornithine and citrulline (58). Nonetheless, an increased reliance on amino acid metabolism to support energetic demands could potentially result in increased proteolysis of skeletal and cardiac muscle, which may contribute to the decreased lean mass and cardiac dysfunction characteristic of the BTHS phenotype (3). Frontiers in Cardiovascular Medicine References 1. Barth PG, Scholte HR, Berden JA, Van der Klei-Van Moorsel JM, Luyt- Houwen IE, Van ’t Veer-Korthof ET, et al. An X-linked mitochondrial disease aﬀecting cardiac muscle, skeletal muscle and neutrophil leucocytes. J Neurol Sci. (1983) 62:327–35. doi: 10.1016/0022-510X(83)90209-5 14. Cade WT, Laforest R, Bohnert KL, Reeds DN, Bittel AJ, de Las Fuentes L, et al. Myocardial glucose and fatty acid metabolism is altered and associated with lower cardiac function in young adults with Barth syndrome. J Nucl Cardiol. (2021) 28:1649–59. doi: 10.1007/s12350-019-01933-3 15. Kiebish MA, Yang K, Liu X, Mancuso DJ, Guan S, Zhao Z, et al. Dysfunctional cardiac mitochondrial bioenergetic, lipidomic, and signaling in a murine model of Barth syndrome. J Lipid Res. (2013) 54:1312–25. doi: 10.1194/jlr.M034728 2. Taylor C, Rao ES, Pierre G, Chronopoulou E, Hornby B, Heyman A, et al. Clinical presentation and natural history of Barth syndrome: an overview. J Inherit Metab Dis. 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JRU is a Tier 2 Canada Research Chair (Pharmacotherapy of Energy Metabolism in Obesity). 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. Acknowledgments The ﬁgure in this review was created with BioRender.com. Summary, conclusions & future directions Although improvements in primary and secondary outcome measures were not observed in subjects with BTHS after 12 weeks during part 1 of the study, during the 36 week extension of part 2, exercise capacity, patient fatigue, 6-min walk test distances, and cardiac stroke volumes demonstrated improvement. Taken together, it will be imperative for future studies to assess whether elamipretide in combination with a metabolic therapy like DCA, which has been safely used in humans, may be a more eﬀective approach to alleviate BTHS- related cardiomyopathy. Circulating branched-chain amino acids (BCAAs) are positively associated with cardiovascular disease (26, 56), and there appears to be both transcriptional and proteomic downregulation of pathways involved in BCAA metabolism in hearts from TazKD mice at 2–3 months of age (15, 21). A decrease in cardiac BCAA metabolism, depending on where the restriction is, may lead to increased levels of myocardial BCAAs and their correspondent keto acids, the latter of which are proposed to explain how BCAA metabolism promotes cardiac insulin resistance and cardiac hypertrophy (57). 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https://openalex.org/W2298156313	https://www.frontiersin.org/articles/10.3389/fpubh.2016.00045/pdf	English	null	Are School Feeding Programs Prepared to Be Inclusive of Children with Disabilities?	Frontiers in public health	2,016	cc-by	2,740	THE LAST 10% According to the World Health Organization and the World Bank “approximately 93 million chil- dren in the world – 1 in 20 children aged 14 or younger – have a moderate or severe disability of some kind. The majority of them live in the undeveloped world, are not enrolled in school, and have very poor access to the most basic health and nutrition opportunities. In low- and middle-income countries, children with disabilities are more likely to be out of school than any other group of children” (1). Girls with disabilities are particularly disadvantaged and most likely to be excluded from education (2). Edited by: David Alan Ervin, The Resource Exchange, USA Edited by: David Alan Ervin, The Resource Exchange, USA Due to poor data and a lack of knowledge about their needs, school health and nutrition pro- grammers are challenged to respond effectively. However, lack of visibility is not the only barrier to children with disabilities receiving school health and nutrition. Structural challenges do exist for children and adults with disabilities to access health and education services, most noticeably, inadequate infrastructure (2).hi Reviewed by: Ellen M. Jensby, Alliance, USA Ashley Aubree Williams, New Heights Behavioral Health, A Service of The Resource Exchange, USA The ratification of the International Convention on the Rights of Persons with Disabilities has had significant impact in most countries. It has brought up a number of new challenges for public policies to be inclusive of children with disabilities in existing and new programs. Such a challenge can be summarized in one insight: it is now clear that the development and sustainability goals of the world cannot be achieved without the full and effective inclusion of the most vulnerable children, many of whom are children with disabilities. Correspondence: Sergio Meresman sergio.meresman@gmail.com Sergio Meresman1 and Lesley Drake2 1 Inter American Institute on Disability and Inclusive Development, Montevideo, Uruguay, 2 The Partnership for Child Development, London, UK Keywords: disabled children, school health services, nutritional status, equity, disability Opinion published: 17 March 2016 doi: 10.3389/fpubh.2016.00045 Opinion published: 17 March 2016 doi: 10.3389/fpubh.2016.00045 MALNUTRITION AND DISABILITIES Specialty section: This article was submitted to Child Health and Human Development, a section of the journal Frontiers in Public Health The intersection between nutrition and disability has been largely overlooked in both programming and research. This is a significant gap in knowledge and policy making, given that over one billion people are undernourished and more than one billion live with a disability (1). Received: 09 November 2015 Accepted: 07 March 2016 Published: 17 March 2016 Malnutrition is found worldwide and is directly or indirectly linked to major causes of death and disability. Deafness, for instance, is most often the result of malaria, typhoid, and meningitis, which are highly prevalent in many undeveloped regions of the world. Other diseases, such as foodborne or waterborne diarrhea, often result in a disability. In the developing world, malnourishment and disability severely limit life opportunities. Both are global development priorities as well as crucial equity and human rights issues. In spite of this, the two issues are rarely linked. Are School Feeding programs prepared to Be inclusive of Children with Disabilities? Sergio Meresman1* and Lesley Drake2 PRIMARY BARRIERS Several barriers prevent children with disabilities from receiving nutrition outreach efforts. Some are structural: persons with disabilities face numerous challenges to access health and social services; little available data makes them invisible as a population group; and, consequently, most education and health initiatives are not disability inclusive. Regular health and education person- nel are often unprepared to communicate effectively with children with disabilities, and health and nutrition education programs often fail to consider their learning needs (4). Within the Focusing Resources on Effective School Health (FRESH) framework, inclusion means having proactive policies and plans to ensure learning for all children, particularly those who are at risk of being left out or excluded from school (5). The question of whether school feeding programs and services are prepared to be inclusive of children with disabilities mandates that all these children will be included and accounted for. t Other barriers could be more “systemic” (related to the func- tional characteristics of programs and responsible institutions) and might take various forms: • Dining halls and other important premises such as washrooms and toilets are often not accessible to those with mobility impairments.1 1 “From 78 schools in Kenya, only in 5 I have seen accessible toilets” (Personal communication, Department of Disability Mainstreaming, Ministry of Labor and Social Affairs, South Africa) 2 “Quite often, feeling that you are unable to help a child with a disability or been just intimidated by a potential difficulty, results in children been sent back to their homes at the school lunch time” (Personal communication, School Feeding staff at the Partnership for Child Development Office in Kenya) DELIVERING SCHOOL FEEDING TO CHILDREN WITH DISABILITIES • Because of a lack of experience with persons with disabilities, some teachers as well as other school personnel and health staff are unaware of the codes of interaction that are needed to work effectively and comfortably with children with disabilities2 (4). There are two main ways of delivering school feeding programs to children with disabilities: • Through regular school feeding in mainstreaming education. • Including special education institutions in school feeding. • Through regular school feeding in mainstreaming education. • Including special education institutions in school feeding. f • Some children might require assistance in feeding. School per- sonnel responsible for preparing meals may not have the skills or awareness of the specific needs of children with disabilities in terms of hygiene, eating, or swallowing. There is often disagreement over whether children with dis- abilities are more effectively reached through “special education” or regular, universal programs. The alternative approach is to make educational resources more accessible to all persons, with and without disabilities. While a significant percentage of children remain in specialized institutions, growing consensus indicates that including children with disabilities in the mainstreaming sector is both cost effective and socially effective and leads to overall improvements in educational outcomes. However, in most cases, this debate is futile because both strategies are valid and necessary. Furthermore, they can easily be combined. The com- bination of “mainstreaming” disability in all nutrition projects and having disability-specific interventions available is called the “twin-track” approach (4). • Special dietary restrictions might be required. • Even when nutrition topics are systematically included in school curricula and health topics associated with nutrition are included in cross-curricular activities, curricular adapta- tions, or teaching resources to make these contents accessible and affable to children with disabilities are barely accessible.t f • Discrimination is often present due to stigma. For instance, children without disabilities could be given priority by school personnel based on the belief that their safety and well-being are of greater value than those of children with disabilities. • Some children might be ostracized and face negative attitudes in the same way they face physical barriers. Fostering inclusion will always require overcoming these obstacles and confront- ing long-standing taboos created by prejudice. Citation: Meresman S and Drake L (2016) Are School Feeding Programs Prepared to Be Inclusive of Children with Disabilities? Front. Public Health 4:45. doi: 10.3389/fpubh.2016.00045 Although research and experience have largely proven the relationships among nutritional status, cognition, school participation, and academic achievement (3), the nutritional needs of children with disabilities are rarely considered in the design of school feeding programs. Inclusive School March 2016 \| Volume 4 \| Article 45 1 Frontiers in Public Health \| www.frontiersin.org Are School Feeding Programs Inclusive? Meresman and Drake Nutrition programs are intended to significantly help children with disabilities by promoting their educational access, retention, and learning outcomes. Nutrition programs are intended to significantly help children with disabilities by promoting their educational access, retention, and learning outcomes. children who have visual impairments, hearing limitations, reduced mobility, or experience difficulties learning in “regular” classrooms. Inclusive education is an important strategy for the most vulnerable groups, for example, “children who don’t speak the common classroom language or belong to a different religion or caste, and children who may be at risk of dropping out because they are sick, hungry, or not excelling academically” (3). The term can also include girls who are pregnant, children affected by HIV/ AIDS, and all girls and boys who are not enrolled in school, espe- cially children who are employed in order to help their families survive.f 1 “From 78 schools in Kenya, only in 5 I have seen accessible toilets” (Personal communication, Department of Disability Mainstreaming, Ministry of Labor and Social Affairs, South Africa)t CHECK LIST The following check list is proposed to conduct a rapid assessment of the conditions for participation of children with disabilities at school level. It seeks to ensure that all key components for inclu- sion are in place. External Alliances, Partnerships, Support p pp • Support from itinerant teachers and local inclusive education resource centers • Support from disability organizations • Support from itinerant teachers and local inclusive education resource centers • Support from disability organizations Support from disability organizations • Support from parents and parents’ organizations • Support from parents and parents’ organizations • Support from parents and parents’ organizations • Participation of parents of children with disabilities in parents’ organizations WHAT IS “INCLUSIVE”? Implementing inclusive school feeding programs is not necessar- ily complex. As described in Partnership for Child Development, three principles were identified that need to be addressed (4). Over the years, the term “inclusive education” has been used to refer to “including children with disabilities,” for example, 1. Include children with disabilities in the planning process: Improved consultation will help to make this group visible and increase the likelihood that programs are sensitive to their needs. 2. Ensure that accessibility is guaranteed throughout the delivery plan: 2 “Quite often, feeling that you are unable to help a child with a disability or been just intimidated by a potential difficulty, results in children been sent back to their homes at the school lunch time” (Personal communication, School Feeding staff at the Partnership for Child Development Office in Kenya) p Addressing issues of physical access to the places where feeding services are delivered and providing nutritional information March 2016 \| Volume 4 \| Article 45 Frontiers in Public Health \| www.frontiersin.org 2 Are School Feeding Programs Inclusive? Meresman and Drake and education in a way that is accommodated to the children’s needs and functional styles is essential: and education in a way that is accommodated to the children’s needs and functional styles is essential: and education in a way that is accommodated to the children’s needs and functional styles is essential: CONCLUSION The recommended course of action in situations involving substitutions for special dietary needs is to encourage school personnel to work closely with families and provide supervision of the appropriate professionals to ensure that schools make reasonable accommodations to allow for the child’s participation in the meal service (6). In most cases, special dietary needs can be met reasonably through a diet order that meets the program standards and is appropriate for the child. School feeding programs urgently need to go through inclusive- ness assessments as well as engage in consultation with families and disability organizations to ensure that their rights are fulfilled and their participation contributes to closing the equity gap. This is essential as children and adolescents with disabilities constitute a significant proportion of the population, especially in the most deprived regions of the world (3). The goal of development and education for all must incorporate the full and effective inclusion of the most vulnerable children, many of whom are children with disabilities. AUTHOR CONTRIBUTIONS Prof. SM has been working in community and school-based health and inclusion for many years. In this article, his expertise benefited from the extensive experience brought over by Dr. LD who directs the Partnership for Child Development and is a worldwide expert on school nutrition. Dr. LD and her PCD team provided the programmatic framework and the financial support that allowed Prof. SM to explore the inclusion of children with disabilities in existing school feeding programs. Prof. SM led the process of consulting several specialists on the ground and proposed the question around which the topic is explored. The result is this opinion article that proposed a platform for future research. Inclusion • Number of teachers trained on inclusion. • Number of teachers trained on inclusion. • Number of teachers with experience on inclusion. • Adaptations to assessments/tests. • Assistive devices and teaching resources for blind/low vision. • Training: pre- and in-service training is needed to improve the knowledge of staff to appropriately respond to the needs of children with disabilities. • Assistive devices and teaching resources for those with hearing impairments. p • Teaching resources for intellectual disabilities. 3. Promote family and community awareness and participation: Engaging families and community organizations such as local disability organizations, churches, special education centers, and rehabilitation programs is of great support to the effective and sustainable implementation of inclusive school feeding programs. Such an approach will have mutual benefits for school programs and school health and nutrition teams, as well as parent organizations, which often have little informa- tion and awareness about school feeding programs and other available resources. • Teaching assistants available. Attendance and education in a way that is accommodated to the children’s needs and functional styles is essential: Attendance • Are there children with disabilities in school? • Are there children with disabilities in school? • Accessibility to the physical environment: the school com- pound, dining hall, wash rooms, and toilets (see standards on physical accessibility). • Have there been children with disabilities in the recent years that have dropped out? • Have schools located and contacted all children with disabili- ties in the area? • Accessibility to nutrition information and education: Providing access to nutrition information and communica- tion is crucial for children with disabilities to decrease their risk of infections and improve their health and nutrition- related decisions. Materials and campaigns, including printed materials, media-based campaigns, and digital contents offered over the internet, must be designed to effec- tively reach children with different types of disabilities and take into account their diverse functional characteristics (4). Accessibility • Is the school compound accessible? • Is the school compound accessible? • Is there accessible transportation to come to the school? • Are school meals accessible? Have adaptations been made to respond to needs of children with disabilities? • Are health activities accessible? Have adaptations been made to respond to needs of children with disabilities? • Is the drinking water accessible to all children? • Are latrines/toilets and handwashing facilities accessible to all children? • Is the playground accessible to all children? • Is the playground accessible to all children? March 2016 \| Volume 4 \| Article 45 Frontiers in Public Health \| www.frontiersin.org 3 Are School Feeding Programs Inclusive? Meresman and Drake Frontiers in Public Health \| www.frontiersin.org REFERENCES 6. Fiore S. Accommodating Special Dietary Needs in School Nutrition Programs. Connecticut State Department of Education’s (2001). Available from: http:// www.sde.ct.gov/sde/lib/sde/pdf/deps/nutrition/SDN/ASDsnp.pdf 6. Fiore S. Accommodating Special Dietary Needs in School Nutrition Programs. Connecticut State Department of Education’s (2001). Available from: http:// www.sde.ct.gov/sde/lib/sde/pdf/deps/nutrition/SDN/ASDsnp.pdf 1. World Health Organization and World Bank. World Report on Disability. Geneva: World Health Organization (2011). 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. g 2. Meresman S. The State of the World’s Children. UNICEF (2013) 3. Inclusive School Health and Nutrition Programs. A Roadmap for Mainstreaming Disability into the FRESH Agenda, FRESH (2015). Available from: www.school- sandhealth.org Copyright © 2016 Meresman and Drake. 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. 4. Inclusive School Feeding Programs. A Contribution to Reaching the Last 10%. PCD Working Paper Series N2. London: The Partnership for Child Development (2015).f 5. Focusing Resources on Effective School Health. A FRESH Approach for Achieving Education for All (2006). Available from: http://www.unicef.org/ lifeskills/files/FreshDocument.pdf March 2016 \| Volume 4 \| Article 45 Frontiers in Public Health \| www.frontiersin.org
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https://openalex.org/W2087768472	https://jiasociety.biomedcentral.com/track/pdf/10.1186/1758-2652-11-S1-P178	English	null	Re-evaluation of resistance algorithms for lopinavir/ritonavir in the TITAN trial	Journal of the International AIDS Society	2,008	cc-by	586	Methods Baseline genotype data were classified using seven geno- typic resistance algorithms: IAS-USA LPV mutations (cut- off = 6), Abbott 2007 (King) mutation list (cut-off = 3), ANRS mutations (cut-off = 6), FDA mutations (cut-off = 3), Stanford, REGA and IAS-USA major PI mutations. Effi- cacy in the TITAN trial (HIV-1 RNA <50 at week 48) was correlated with the number of mutations from each list, to show the "efficacy advantage cut-off level": the number of mutations from each list associated with a difference in efficacy between treatment arms of at least 12%. The line- arity of the correlation between mutation count and effi- cacy of LPV/r was analysed in TITAN, with sensitivity analysis for the French LPV ATU, BMS-045 and RESIST tri- als. The analysis identified more sensitive cut-off levels for several lopinavir genotypic algorithms, below those cur- rently used, at which there is significant efficacy advantage for treatment with darunavir/ritonavir versus lopinavir/ ritonavir in the TITAN trial. These new cut-off levels also detect a higher percentage of patients with virological fail- ure than previous cut-off levels. Background viously reported genotypic cut-off levels where there was at least 12% lower efficacy for LPV/r versus darunavir/ ritonavir. These "efficacy advantage cut-off levels" were: IAS-USA LPV mutations, cut-off = 3; Abbott 2007, cut-off = 2; ANRS LPV cut-off = 3; FDA LPV mutations, cut-off = 2 and major IAS-USA PI mutations, cut-off = 1, Stanford algorithm, cut-off = low level LPV resistance; REGA algo- rithm, cut-off = Intermediate level LPV resistance. There were linear falls in HIV-1 RNA suppression rates with ris- ing mutation counts in the TITAN, French LPV ATU, BMS- 045 and RESIST trials. viously reported genotypic cut-off levels where there was at least 12% lower efficacy for LPV/r versus darunavir/ ritonavir. These "efficacy advantage cut-off levels" were: IAS-USA LPV mutations, cut-off = 3; Abbott 2007, cut-off = 2; ANRS LPV cut-off = 3; FDA LPV mutations, cut-off = 2 and major IAS-USA PI mutations, cut-off = 1, Stanford algorithm, cut-off = low level LPV resistance; REGA algo- rithm, cut-off = Intermediate level LPV resistance. There were linear falls in HIV-1 RNA suppression rates with ris- ing mutation counts in the TITAN, French LPV ATU, BMS- 045 and RESIST trials. g Genotypic algorithms used to predict the clinical efficacy of lopinavir/ritonavir (LPV/r) have included a range of mutation lists and efficacy end-points. HIV clinical trials are normally powered to detect a difference between treat- ment arms of 10–12% for the end-point of HIV-1 RNA suppression <50 copies/mL. The TITAN trial (TMC114- C214) evaluated LPV/r vs. darunavir/ritonavir in treat- ment-experienced patients with HIV-1 RNA >1000 copies/ mL. Baseline genotypic resistance to darunavir was rare in the TITAN trial. Published: 10 November 2008 Journal of the International AIDS Society 2008, 11(Suppl 1):P178 doi:10.1186/1758-2652-11-S1-P178 This abstract is available from: http://www.jiasociety.org/content/11/S1/P178 © 2008 Calvez et al; licensee BioMed Central Ltd. Journal of the International AIDS Society BioMed Central Open Access Open Acce Poster presentation Re-evaluation of resistance algorithms for lopinavir/ritonavir in the TITAN trial V Calvez1, AM Hill2 and AG Marcelin1 V Calvez1, AM Hill2 and AG Marcelin1 from Ninth International Congress on Drug Therapy in HIV Infection Glasgow, UK. 9–13 November 2008 Page 1 of 1 (page number not for citation purposes) Summary of results y In TITAN, the concordance between baseline lopinavir resistance, defined by the mutation scores, ranged from 79–95%. Multivariate analysis identified lower than pre- Page 1 of 1 (page number not for citation purposes) Page 1 of 1 (page number not for citation purposes)
https://openalex.org/W2641153533	https://europepmc.org/articles/pmc5482057?pdf=render	English	null	Self-assembled three dimensional network designs for soft electronics	Nature communications	2,017	cc-by	10,576	1 Frederick Seitz Materials Research Laboratory, Department of Materials Science and Engineering, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA. 2 Department of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, South Korea. 3 Departments of Civil and Environmental Engineering, Mechanical Engineering, and Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208, USA. 4 Department of Electrical Engineering and Computer Science, Northwestern University, Evanston, Illinois 60208, USA. 5 Department of NanoEngineering, University of California at San Diego, La Jolla, California 92093, USA. 6 Department of Engineering Mechanics, Center for Mechanics and Materials, AML, Tsinghua University, Beijing 100084, China. 7 Institute of High Performance Computing, Singapore 138632, Singapore. 8 School of Electrical and Electronic Engineering, Yonsei University, Seoul 03722, Republic of Korea. 9 Department of Electronics Convergence Engineering, Kwangwoon University, Seoul 01897, Republic of Korea. 10 Department of Materials Science and Engineering, Pusan National University, Busan 46241, Republic of Korea. 11 Department of Electrical, Computer and Energy Engineering, University of Colorado, Boulder, Colorado 80309, USA. 12 School of Electrical Engineering and Computer Science, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea. 13 Departments of Materials Science and Engineering, Biomedical Engineering, Chemistry, Mechanical Engineering, Electrical Engineering and Computer Science, Neurological Surgery, Center for Bio-Integrated Electronics, Simpson Querrey Institute for BioNanotechnology, McCormick School of Engineering and Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.H. (email: y-huang@northwestern.edu) or to Y.Z. (email: yihuizhang@tsinghua.edu.cn) or to J.A.R. (email: jrogers@northwestern.edu). ARTICLE Received 17 Jan 2017 \| Accepted 11 May 2017 \| Published 21 Jun 2017 DOI: NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 1 Frederick Seitz Materials Research Laboratory, Department of Materials Science and Engineering, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA. 2 Department of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, South Korea. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 R apid advances in the development of precision chem/ biosensors, low-power radio communication systems, efﬁcient energy harvesting/storage devices, high-capacity memory technologies and miniaturized electronic/optoelectronic components create opportunities for qualitatively expanding the ways that microsystem technologies can be integrated with the human body for treating disease states and monitoring health status1–5. Realizing this potential requires not only advances in the components but also in the strategies for their collective integration into systems that offer stable, long-term operation at intimate biotic/abiotic interfaces1,3,4. Promising research in this direction focuses on development of materials and mechanics concepts to enable system-level properties that are mechanically and geometrically matched to those of the soft tissues of the human body6–25. Although previously reported approaches provide signiﬁcant utility in this context12,14,26–42, they all rely on planar, two-dimensional (2D) layouts of the functional elements and electrical interconnects, where the 2D geometries and/or materials deﬁne the essential physical characteristics. The work reported here pursues a different strategy, in which system architectures adopt engineered, three-dimensional (3D) designs to provide properties that circumvent intrinsic limita- tions associated with traditional, 2D counterparts. Speciﬁcally, combined experimental and theoretical results demonstrate that open networks of 3D microscale helical interconnects offer nearly ideal mechanics for soft electronic systems that embed chip-scale components, by virtue of model, spring-like behaviours similar to those in man-made43–45 (for example, coil spring) and biological46–50 (for example, tendrils) analogues. Furthermore, scalable methods for forming the required 3D mesostructures together with systematic approaches for deﬁning mechanically and electrically optimized layouts allow immediate application to complex systems. Wireless sensor platforms capable of physio- logical status monitoring in soft, skin-mounted formats provide demonstration examples. These ﬁndings could ﬁnd broad utility in many classes of soft microsystems technologies. Although previous studies establish the utility of planar serpentine interconnects in soft electronics, the existence of sharp, localized stress concentrations that follow from their 2D formats and their physical coupling to the substrate limit performance for systems that require low modulus, elastic mechanics in compact designs. By comparison, 3D helical microstructures avoid this unfavourable mechanics due to smoothly varying, uniform distributions of deformation-induced stresses that follow directly from their 3D layouts. The result enables exceptionally high levels of stretchability and mechanical robustness, without the propensity for localized crack formation or fracture, as supported by results in Fig. 1c,d and Supplementary Fig. 2. Results h Mechanics of 3D helical coils. The assembly approach builds on recently introduced concepts in deterministically controlled buck- ling processes51,52, in which initial 2D structures spontaneously transform into desired 3D shapes. Figure 1a presents ﬁnite element analyses (FEA, see Methods section for details) for the formation of an extended, spiral network of 3D helical microstructures from a corresponding 2D precursor that takes the form of a collection of ﬁlamentary serpentine ribbons (widths: 50mm; multilayer construction: 2.5 mm PI/1.0 mm Au/5 nm Cr/2.5 mm PI). Each unit cell consists of two identical arcs with central angle y0 and radius r0. The two ends include small discs that form strong covalent siloxane bonds to an underlying elastomeric silicone substrate in a state of biaxial prestrain; other regions adhere only through weak van der Waals interactions. Compressive forces induced by releasing the prestrain cause the 2D precursor to geometrically transform, through a coordinated collection of in-plane and out-of-plane translational and rotational motions, into an engineered 3D conﬁguration via controlled buckling deformations51,52. Speciﬁ- cally, each unit cell transforms into a single turn of a corresponding 3D helical microcoil whose pitch (p) depends on the serpentine geometry and the magnitude of the prestrain, epre, according to p ¼ 4r0 sin(y0/2)/(1 þ epre). The overall shape of coil follows mainly from epre and y0 (Supplementary Fig. 1). Optical images of a representative structure appear in Fig. 1b. The key dimensions match those from FEA. For example, the experimental and FEA results for the pitch, height of the microcoils are 1,150±40mm, 530±50 mm and 1,170, 520 mm, respectively. Fnon-uniformity ¼ R 1 0 scross-section Mises S ð Þ Mean scross-section Mises ð Þ j jdS Mean scross-section Mises ð Þ , where S denotes Fn Mean sMises ð Þ the natural arc coordinate normalized by the total trace length of entire interconnect. This dimensionless factor also highlights the qualitative differences (for example, 0.15 versus 1.06 for 50% stretch) between behaviours of the 3D helical and 2D serpentine microstructures. Even when compared to fractal 2D designs, in which microﬂuidic enclosures afford mechanical decoupling from the substrate14 or to bar-type 2D serpentines, in which planar, scissor-like mechanics dominates53, the 3D helical geometry is superior due to the spring-like responses and associated uniformity in the stress distribution (see Supplementary Figs 3–7 for details). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 For purposes of quantitative comparison, consider a 2D serpentine interconnect with the same material composition (PI/Au/PI) and key geometric parameters (Supplementary Fig. 2a), including the width (w), the thickness (tmetal and tPI), the span (S) and the amplitude (A), as a corresponding 3D helix. Here the number of unit cells in the 2D serpentine is selected such that its total trace length (ltotal) is approximately the same as that of the 3D counterpart (10.68mm, y0 ¼ 180o and r0¼ 425 mm). The limit of elastic stretchability, deﬁned as the maximum overall dimensional change below which structural deformations can recover completely, corresponds to the point at which the constituent materials undergo plastic deforma- tion at the locations of highest Mises stress. Results for analogous 2D and 3D systems appear in Supplementary Fig. 2b. Due to the absence of stress concentrations, the elastic stretchability of the 3D helices signiﬁcantly exceeds that of the 2D serpentines. The enhancement corresponds to a factor of B3 for epre¼ 50%, and this factor increases continuously with epre until it reaches B9.5 for epre ¼ 300% (Supplementary Fig. 2b). R a b e p These improvements follow from qualitatively distinct deforma- tion mechanisms in 3D compared to 2D layouts. Figure 1c illustrates the nearly ideal, spring-like mechanics that characterize responses in 3D helices. Here, deformations are almost completely decoupled from those of elastomeric substrate and the cross- sectional maximum Mises stress (scross-section Mises ) are spatially uniform, except for small regions near the bonding sites. Quantitative results from FEA appear in Fig. 1e, which shows the maximum Mises stress for each cross section along the natural coordinate normal- ized by the arc length. Deformations of the 2D serpentine lead to sharp, unavoidable stress concentrations at the arc regions, as illustrated by the results in Fig. 1f. The ratio of the peak stress [Peak scross-section Mises ] to its mean value [Mean scross-section Mises ] serves as a metric for the magnitude of this concentration. This ratio is only B1.2 for the 3D helices under both applied strains of 25 and 50%, which is nearly an order of magnitude smaller than the ratio (B9.8) for the 2D serpentines. The non-uniformity in the stress distribution can be characterized by the average absolute deviation relative to the mean value, as given by R 1 cross-section S ð Þ M cross-section ð Þ j jdS ARTICLE ARTICLE NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications Self-assembled three dimensional network designs for soft electronics Kyung-In Jang1,2,, Kan Li3,, Ha Uk Chung1,4,*, Sheng Xu5, Han Na Jung1, Yiyuan Yang1, Jean Won Kwak1, Han Hee Jung2, Juwon Song2, Ce Yang6, Ao Wang3,6, Zhuangjian Liu7, Jong Yoon Lee1,2, Bong Hoon Kim1, Jae-Hwan Kim1, Jungyup Lee1, Yongjoon Yu1, Bum Jun Kim1, Hokyung Jang1, Ki Jun Yu8, Jeonghyun Kim9, Jung Woo Lee10, Jae-Woong Jeong11, Young Min Song12, Yonggang Huang3, Yihui Zhang6 & John A. Rogers13 Low modulus, compliant systems of sensors, circuits and radios designed to intimately interface with the soft tissues of the human body are of growing interest, due to their emerging applications in continuous, clinical-quality health monitors and advanced, bioelectronic therapeutics. Although recent research establishes various materials and mechanics concepts for such technologies, all existing approaches involve simple, two-dimensional (2D) layouts in the constituent micro-components and interconnects. Here we introduce concepts in three-dimensional (3D) architectures that bypass important engi- neering constraints and performance limitations set by traditional, 2D designs. Speciﬁcally, open-mesh, 3D interconnect networks of helical microcoils formed by deterministic compressive buckling establish the basis for systems that can offer exceptional low modulus, elastic mechanics, in compact geometries, with active components and sophisticated levels of functionality. Coupled mechanical and electrical design approaches enable layout optimi- zation, assembly processes and encapsulation schemes to yield 3D conﬁgurations that satisfy requirements in demanding, complex systems, such as wireless, skin-compatible electronic sensors. 1 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 a f x y z x y z d c x y z x y z 200 MPa 0 200 MPa 0 b 200 MPa 0 Pre-stretched Strain released 0% uniaxial strain 50% uniaxial strain 0% uniaxial strain 50% uniaxial strain a x y z x y z d c x y z x y z 200 MPa 0 0 200 MPa 0 Pre-stretched Strain released 0% uniaxial strain 50% uniaxial strain 0% unia 50% uni b b a d x y z 200 MPa 0 0% uniaxial strain 50% uniaxial strain d c f e 200 100 0 Maximum mises stress (MPa) 300 = 25% = 50% = 0% 0 0.2 0.4 0.6 1 Normalized position along length 0.8 e e f 200 100 0 Maximum mises stress (MPa) 300 = 25% = 50% = 0% 0 0.2 0.4 0.6 1 Normalized position along length 0.8 = 50% = 25% = 0% 0 0.4 0.6 1 Normalized position along length 0.8 200 100 0 Maximum mises stress (MPa) 300 0.2 Maximum mises Normalized position along length Figure 1 \| Assembly of conductive 3D helical coils and their mechanical properties. (a) Process for assembly illustrat Figure 1 \| Assembly of conductive 3D helical coils and their mechanical properties. (a) Process for assembly illustrated by ﬁnite element analysis (FEA). A 2D ﬁlamentary serpentine structure bonded at selected locations to an underling, bi-axially stretched (epre) soft elastomeric substrate (pre-streched). Corresponding 3D helical coils formed by relaxation of the substrate to its initial, unstretched state (strain released). The colour represents the magnitude of Mises stress in the metal layer. (b) Angled and cross-sectional optical images of an experimentally realized structure. The traces consist of lithographically deﬁned multilayer ribbons of polyimide/Au or Cu/polyimide bonded to a silicone substrate. Scale bar, 1 mm. FEA results for the deformations and distributions of Mises stress in a 3D coil (c) and a 2D serpentine (d) with similar geometries at 0 and 50% uniaxial strain. (e) Distribution of maximum Mises stress for each cross section along the natural coordinate normalized by the arc length, for the 3D helical coil in c, for three different levels of applied strain (0, 25, 50%). (f) Similar results for the case of the 2D serpentine in d. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 a e f x y z x y z d c x y z x y z 200 MPa 0 200 100 0 Maximum mises stress (MPa) 300 = 25% = 50% = 0% 0 0.2 0.4 0.6 1 Normalized position along length 0.8 = 50% = 25% = 0% 0 0.4 0.6 1 Normalized position along length 0.8 200 100 0 Maximum mises stress (MPa) 300 200 MPa 0 b 200 MPa 0 Pre-stretched Strain released 0% uniaxial strain 50% uniaxial strain 0% uniaxial strain 50% uniaxial strain 0.2 Figure 1 \| Assembly of conductive 3D helical coils and their mechanical properties. (a) Process for assembly illustrated by ﬁnite element analysis (FEA). A 2D ﬁlamentary serpentine structure bonded at selected locations to an underling, bi-axially stretched (epre) soft elastomeric substrate (pre-streched). Corresponding 3D helical coils formed by relaxation of the substrate to its initial, unstretched state (strain released). The colour represents the magnitude of Mises stress in the metal layer. (b) Angled and cross-sectional optical images of an experimentally realized structure. The traces consist of lithographically deﬁned multilayer ribbons of polyimide/Au or Cu/polyimide bonded to a silicone substrate. Scale bar, 1 mm. FEA results for the deformations and distributions of Mises stress in a 3D coil (c) and a 2D serpentine (d) with similar geometries at 0 and 50% uniaxial strain. (e) Distribution of maximum Mises stress for each cross section along the natural coordinate normalized by the arc length, for the 3D helical coil in c, for three different levels of applied strain (0, 25, 50%). (f) Similar results for the case of the 2D serpentine in d. Results h In particular, 3D helical interconnects outperform 2D serpentine designs that undergo buckling deformations in the form of local wrinkling, typically by a factor 42.3 for representative prestrains (4100%). As compared to thick serpentine interconnects that are NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 helical interconnects (PI 2.5 mm/Au 1 mm/Cr 5 nm/PI 2.5 mm multilayers with 50 mm in width for each line; PI 2.5 mm/Au 1 mm/Cr 5 nm/PI 2.5 mm/Au 1 mm/Cr 5 nm/PI 2.5 mm multilayers with 50 mm in width for each crossing point) adhered at B500 bonding sites to an elastomeric substrate (E ¼ 20 kPa; Ecoﬂex 00–50, Smooth-on, USA; B4 cm diameter at rest), all encapsulated with an ultra-low-modulus elastomer (E ¼ 3 kPa; Silbione 4717A/B, Bluestar Silicones, France). As described subsequently and demonstrated in Supplementary Movie 1, this platform provides wireless, battery-free capabilities in continuous monitoring of physiological health from mounting locations on the skin. The inset shows the device in a complex state of deformation to illustrate the soft, skin-like physical properties. The overall layout employs a spider-web-like geometry (Supplementary Figs 16–18), selected to avoid failure at any point in the system, to ensure uniform and extreme levels of dominated by planar, scissor-like mechanics, 3D helical inter- connects with a sufﬁcient prestrain (for example, 4100%) exhibit a considerable enhancement (for example, 41.8 times) in the elastic stretchability. Similar improvements, at an even greater factor, apply relative to 2D interconnects with fractal-inspired layouts (Supplementary Fig. 7). Design of 3D interconnect network of helical coils. The versatility in layouts that can be realized by the assembly approach outlined in Fig. 1, the scalability of this process to large areas, and the excellent mechanical behaviours of the 3D helical designs facilitate straightforward implementation even in complex systems (see Supplementary Figs 8–15). The optical image of Fig. 2a presents an example that consists of B50 separate chip-scale electrical components, B250 distinct 3D NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 e Rec. electrode Instru- mentation amplifier High pass filter GND. electrode Ref. electrode Low pass filter Digital accelerometer Bluetooth SoC Internal ADC SPI bus RF Health monitor Log 5200 0 4 2 0 6100 6120 x y z 6140 6160 6180 Time (ms) 6200 6220 6240 6260 –2 –4 1 2 3 5400 –ECG 5600 Time (ms) 5800 6000 6200 Electrophysiology 3-axis acceleration Connect antenna Gain amplifier Wireless power Super- capacitor f FEA SEM 200MPa 0 a d c b Figure 2 \| Three-dimensional network of helical coils as electrical interconnects for soft electronics. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 (a) Optical image of the system at a bi-axially stretched state of 50%, showing B250 3D helices, B500 bonding sites, B50 component chips and elastomers for full encapsulation (Encapsulation material: Silbione 4717A/B, Bluestar Silicones, France; E ¼ 3 kPa; bottom substrate: Ecoﬂex 00-50A/B, Smooth-on, USA; E ¼ 20 kPa) Inset: optical image of the device under a complex state of deformation. Scale bar, 5 mm. Scanning electron micrographs and corresponding FEA results of representative regions of the 3D network, including (b) electrically isolated crossing points and (c,d) interfaces with chip components. Scale bars, 100 mm (b), 1 mm (c) and 1 mm (d). The widths and thicknesses of all coils throughout this system are 50 and 1 mm, respectively. (e) Optical image of a device supported by two ﬁngers. (f) Block diagram of the functional components for a set of electrophysiological (EP) sensors with an analogue signal processing unit that includes ﬁlters and ampliﬁers, a three-axial digital accelerometer, a Bluetooth system on a chip (SoC) for signal acquisition and wireless communication, and a wireless power transfer system for battery-free operation. The signal acquisition involves sampling of the EP sensor output through an internal analogue-to-digital converter (ADC) and data acquisition of the accelerometer output via a serial peripheral interface (SPI). When operated using a custom graphical user interface on a smart phone, this system can capture and transmit a range of information related to physiological health, as shown in Supplementary Movie 1. Figure 2 \| Three-dimensional network of helical coils as electrical interconnects for soft electronics. (a) Optical image of the system at a bi-axially stretched state of 50%, showing B250 3D helices, B500 bonding sites, B50 component chips and elastomers for full encapsulation (Encapsulation material: Silbione 4717A/B, Bluestar Silicones, France; E ¼ 3 kPa; bottom substrate: Ecoﬂex 00-50A/B, Smooth-on, USA; E ¼ 20 kPa) Inset: optical image of the device under a complex state of deformation. Scale bar, 5 mm. Scanning electron micrographs and corresponding FEA results of representative regions of the 3D network, including (b) electrically isolated crossing points and (c,d) interfaces with chip components. Scale bars, 100 mm (b), 1 mm (c) and 1 mm (d). The widths and thicknesses of all coils throughout this system are 50 and 1 mm, respectively. (e) Optical image of a device supported by two ﬁngers. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 (a) Optical image of the system at a bi-axially stretched state of 50%, showing B250 3D helices, B500 bonding sites, B50 component chips and elastomers for full encapsulation (Encapsulation material: Silbione 4717A/B, Bluestar Silicones, France; E ¼ 3 kPa; bottom substrate: Ecoﬂex 00-50A/B, Smooth-on, USA; E ¼ 20 kPa) Inset: optical image of the device under a complex state of deformation. Scale bar, 5 mm. Scanning electron micrographs and corresponding FEA results of representative regions of the 3D network, including (b) electrically isolated crossing points and (c,d) interfaces with chip components. Scale bars, 100 mm (b), 1 mm (c) and 1 mm (d). The widths and thicknesses of all coils throughout this system are 50 and 1 mm, respectively. (e) Optical image of a device supported by two ﬁngers. (f) Block diagram of the functional components for a set of electrophysiological (EP) sensors with an analogue signal processing unit that includes ﬁlters and ampliﬁers, a three-axial digital accelerometer, a Bluetooth system on a chip (SoC) for signal acquisition and wireless communication, and a wireless power transfer system for battery-free operation. The signal acquisition involves sampling of the EP sensor output through an internal analogue-to-digital converter (ADC) and data acquisition of the accelerometer output via a serial peripheral interface (SPI). When operated using a custom graphical user interface on a smart phone, this system can capture and transmit a range of information related to physiological health, as shown in Supplementary Movie 1. a a SEM b d b b FEA 200MPa 0 Rec. electrode Instru- mentation amplifier High pass filter GND. electrode Ref. electrode Low pass filter Digital accelerometer Bluetooth SoC Internal ADC SPI bus RF Health monitor Log 5200 0 4 2 0 6100 6120 x y z 6140 6160 6180 Time (ms) 6200 6220 6240 6260 –2 –4 1 2 3 5400 –ECG 5600 Time (ms) 5800 6000 6200 Electrophysiology 3-axis acceleration Connect antenna Gain amplifier Wireless power Super- capacitor f f e e e Figure 2 \| Three-dimensional network of helical coils as electrical interconnects for soft electronics. (a) Optical im Figure 2 \| Three-dimensional network of helical coils as electrical interconnects for soft electronics. NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 (a) Process for soft encapsulation illustrated with FEA results: ﬁrst, a collection of electronic components joined by an interconnect network in a 2D serpentine design bond at selective sites to a bi-axially prestrained soft elastomeric substrate (Ecoﬂex 00-50A/B, Smooth-on, USA; E ¼ 20 kPa). Partial release of the prestrain mechanically transforms the 2D serpentines into 3D helices. Coating the entire structure with a thin, low-modulus silicone elastomer (Silbione 4717A/B, Bluestar Silicones, France; E ¼ 3 kPa) deﬁnes the encapsulation layer. Finally, releasing the remaining prestrain enhances the 3D geometry of the helices and completes the process. The insets correspond to magniﬁed views of a local region. (b) Elastic stretchability of a 3D circuit system encapsulated at different states of partially released strain in this two-stage encapsulating process (blue for uniaxial stretching along X axis; red for uniaxial stretching along Y axis; black for radial stretching). (c) Bar graph of the elastic stretchability for a 3D circuit system formed by using a one-stage encapsulating process, two-stage encapsulating process and no encapsulation. (d) System-level deformation and distribution of stresses determined by FEA with encapsulation introduced after (d) full and (f) partial release of the prestrain. In all of the FEA images, the colour represents the magnitude of Mises stress in the metal layer. (e) Optical images of a device deformed in similar ways. Scale bars, 1 cm (a–d). The scale bar of the inset of (a) is 1 mm. d Radial 25% X-axis 50% e Radial 70% X-axis 109% Y-axis 111% e Radial 70% X-axis 109% e d Radial 25% X-axis 50% Y-axis 51% f d e f 200 MPa 0 Radial 70% X-axis 109% X-axis 50% X-axis 109% Y-axis 111% Y-axis 51% Y-axis 111% Y-axis 51% Y-axis 111% 200 MPa 0 0 Figure 3 \| Strategy for soft encapsulation and system-level mechanics. (a) Process for soft encapsulation illustrated with FEA results: ﬁrst, a collection of electronic components joined by an interconnect network in a 2D serpentine design bond at selective sites to a bi-axially prestrained soft elastomeric substrate (Ecoﬂex 00-50A/B, Smooth-on, USA; E ¼ 20 kPa). Partial release of the prestrain mechanically transforms the 2D serpentines into 3D helices. Coating the entire structure with a thin, low-modulus silicone elastomer (Silbione 4717A/B, Bluestar Silicones, France; E ¼ 3 kPa) deﬁnes the encapsulation layer. Finally, releasing the remaining prestrain enhances the 3D geometry of the helices and completes the process. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 For example, the length of the line that supplies power must be minimized to reduce resistive dissipation and electromagnetic noise associated with radio frequency power delivery. A large decoupling capacitor (22 mF) at the power source and smaller bypass capacitors at each branch of the supply route, all of which have low equivalent series resistance, help to suppress this noise. The antenna and the electrodes for sensing electrophysiological signals must be spatially separated from the power line to minimize electromagnetic interference, but they must also enable impedance matching (50 O). These and other coupled a x y z Elastic stretchability (%) b c Elastic stretchability (%) 200 MPa 0 d Radial 25% Radial 70% X-axis 50% X-axis 109% Y-axis 51% Y-axis 111% f e 200 MPa 0 0 50 100 150 X-axis Y-axis Radial 30% 0 Partially released strain (%) Radial X-axis Y-axis 0 300 100 One-stage encap. Two-stage encap. No encap. 200 50 100 150 39% egy for soft encapsulation and system-level mechanics. (a) Process for soft encapsulation illustrated with FEA results: ﬁrst, a collection of ponents joined by an interconnect network in a 2D serpentine design bond at selective sites to a bi-axially prestrained soft elastomeric ex 00-50A/B, Smooth-on, USA; E ¼ 20 kPa). Partial release of the prestrain mechanically transforms the 2D serpentines into 3D helices. re structure with a thin, low-modulus silicone elastomer (Silbione 4717A/B, Bluestar Silicones, France; E ¼ 3 kPa) deﬁnes the encapsulation easing the remaining prestrain enhances the 3D geometry of the helices and completes the process. The insets correspond to magniﬁed region. (b) Elastic stretchability of a 3D circuit system encapsulated at different states of partially released strain in this two-stage a x y z 200 MPa 0 Elastic stretchability (%) b 0 50 100 150 X-axis Y-axis Radial 30% 0 Partially released strain (%) 50 100 150 39% b a c Elastic stretchability (%) Radial X-axis Y-axis 0 300 100 One-stage encap. Two-stage encap. No encap. 200 c x y 200 MPa 0 d Radial 25% Radial 70% X-axis 50% X-axis 109% Y-axis 51% Y-axis 111% f e 200 MPa 0 Radial X-axis Y-axis 0 Figure 3 \| Strategy for soft encapsulation and system-level mechanics. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 (f) Block diagram of the functional components for a set of electrophysiological (EP) sensors with an analogue signal processing unit that includes ﬁlters and ampliﬁers, a three-axial digital accelerometer, a Bluetooth system on a chip (SoC) for signal acquisition and wireless communication, and a wireless power transfer system for battery-free operation. The signal acquisition involves sampling of the EP sensor output through an internal analogue-to-digital converter (ADC) and data acquisition of the accelerometer output via a serial peripheral interface (SPI). When operated using a custom graphical user interface on a smart phone, this system can capture and transmit a range of information related to physiological health, as shown in Supplementary Movie 1. electrical performance (details described below), an iterative process guided by FEA allows optimization of all relevant parameters, including the magnitude of the prestrain in the assembly process, the spatial layouts of the components, the conﬁgurations of the bonding sites and the geometries of the 2D serpentine precursors. The use of serpentine unit cells with ﬁxed dimensions across the entire circuit simpliﬁes the design process and yields uniform distributions of Mises stresses. Overlaid on these mechanical considerations is a set of electrical requirements. stretchability and bendability in any direction and to minimize the overall system size. This unusual layout follows from a rigorous, systematic design approach that leverages FEA modelling. Speciﬁcally, for any given design, FEA allows rapid identiﬁcation of locations of high Mises stresses and physical collisions between the different regions of the interconnect network and/or individual components, under various states of deformation within a desired range of strains. Under constraints of interconnect length and connectivity set by considerations in NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 2e,f, the device functions as a soft, skin- mounted technology with important capabilities in monitoring of well-known parameters relevant to physiological health status. Wireless delivery of power to a receiver antenna charges a supercapacitor which then supplies regulated power to the entire system. Sensors allow quantitative measurements of motion, via accelerometry, and electrophysiology, via skin-interfaced electrodes. Raw data pass through a collection of analogue ﬁlters and ampliﬁers prior to wireless transmission using a Bluetooth protocol to a customized app running on a smart phone. Details appear in the Methods and Supplementary Figs 20–23. g pp y g The devices might encounter compression along the thickness direction during practical use, associated with physical contact or normal impact. Quantitative modelling of the encapsulated helical interconnects provides insights into the mechanics associated with such situations. For a variety of prestrains (50–300%) used in the assembly process, compressive forces (for example, B10 kPa) sufﬁciently large to reduce the out-of- plane dimensions of the interconnects to 30% of the original values (that is, a compression ratio of 0.3) lead to maximum principal strains in the metal layer that are below 1.3%, which is much smaller than the fracture strain (45%) of gold or copper. This result indicates that the 3D helical interconnects can survive such compression ratios (0.3) and pressures 410 kPa. Experi- mental demonstrations of robust performance of 3D coil interconnect networks, involve simple test structures a LED to allow visual observation of operation. Each LED interconnect takes the form of a 3D helical coil (width 50 mm, thickness 1 mm), selectively bonded to an elastomeric substrate (Ecoﬂex 00–30, Smooth-on, USA; E ¼ B30 kPa) using interface chemistries described previously and encapsulation with an ultra-soft Soft encapsulation strategy for enhanced mechanics. Practical applications demand encapsulating layers to protect the active and passive components and the interconnects. As compared with liquid encapsulation14, the solid encapsulation approach adopted in this paper avoids the potential for leakage or evaporation. As in previously reported systems that use 2D serpentines, ultra-low- modulus elastomers are attractive due to the minimal mechanical constraints that they impose on the motions of the components and interconnect networks. Typically, introduction of this encapsulation material occurs as a ﬁnal step in the fabrication. An alternative, improved strategy that naturally follows from the 3D assembly process outlined in Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 Near this limit, the elastic stretchability of the encapsulated 3D circuit system approaches that of the unencapsulated counterpart, particularly for uniaxial stretching (for example, 120 versus 144% for X axis and 123% versus 146% for Y axis) (see Supplementary Discussion for more discussions). By comparison, encapsulation at eencap ¼ 0 (that is, encapsulation after complete assembly) retains only limited levels of stretchability (for example, B50% for X axis, 51% for Y axis, and 25% for radial stretching, respectively), as shown in Fig. 3d. The deformation mode in Fig. 3e is, in fact, close to that of an ideal, unencapsulated system (Supplementary Fig. 25). In this construction, a radial strain of 70% induces maximum Mises stress of only B199 MPa (corresponding to its yield strain, B0.3%) in the active materials (Au of the interconnects), consistent with reversible, elastic behaviours. Furthermore, the stress distribution is uniform across most of the circuit (Fig. 3e), as additional evidence of the efﬁcient mechanics. Delamination has the potential to occur at the chip/substrate interface during the process of mechanical-guided assembly, as shown in experimental results (Supplementary Fig. 19). Theoreti- cally, delamination occurs more readily as the prestrain increases or the effective rigidity of the chip increases. Such trends are in qualitative agreement with experimental observations (Suppleme- ntary Fig. 19), in which the delamination area increases as the effective rigidity of the chip increases. In addition, delamination occurs mainly at the periphery regions of the chips. This type of partial delamination does not affect the bonding conditions between electrical interconnects and substrate, and thereby has a minor effect on the integrity of the entire device. The modulus of the top encapsulation has a signiﬁcant effect on the elastic stretchability of the 3D helical interconnects (Supplementary Fig. 26). A low-modulus elastomer (Silbione) reduces mechanical constraints on the deformations of helical interconnects, thereby achieving a much higher stretchability than possible with elastomers with higher modulus values (for example, Ecoﬂex). The circuit can be designed with improved density via the use of increased prestrain during the 3D assembly process. By increasing the prestrain from 150 to 200%, the area of entire circuit can be reduced to B70% of the original area. The elastic stretchability undergoes a relatively small correspond- ing reduction, as in Supplementary Fig. 27. g y As depicted in Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 considerations in mechanics and electronics underpin the optimized design shown in Fig. 2a. 3D helical interconnects via mechanical interactions during ﬁnal release. As illustrated by experimental results (Supplementary Fig. 24), the 3D buckled structures in the system maintain their original forms during the encapsulation process because their equivalent moduli are sufﬁciently high that they are not deformed by shear forces associated with the viscosity of the uncured elastomer or liquid. p g g The scanning electron microscope (SEM) images in Fig. 2b–d highlight some key representative regions of this system, including heterogeneously integrated structures of 3D microcoils, chip-scale components and the supporting elastomeric substrate. In terms of the mechanics, these frames highlight the level of agreement between experiment and FEA predictions across the entire structure. Figure 2b illustrates electrical crossovers enabled by the multilayer construction of the traces. As shown in Fig. 2b–d, the bonding sites retain an undeformed shape due to the low modulus of the underlying elastomer and the relatively large thickness (B6 mm) of the interconnect structure. The result leads to low levels of Mises stresses at these locations. The chip-scale components prevent corresponding near-surface regions of the elastomer from relaxing after release of the prestrain, thereby inducing a certain level of strain concentration. As such, the portions of the helical interconnects that join directly to the chips undergo a slightly enhanced compression, consistent with the increased Mises stresses in Fig. 2d. The FEA results in the lower images show the stress ﬁeld associated with the 3D microcoil. The maximum computed stress in the metal layer across the entire 3D interconnect structure is B130 MPa. This value corresponds to B0.19% strain, which is below the yielding point (B0.3%). q Quantitative mechanics modelling reveals signiﬁcant associated improvements in the elastic stretchability (Fig. 3b,c), compared to the usual case of encapsulation at the ﬁnal stage of fabrication and assembly. Speciﬁcally, the enhancement corresponds to a factor of B2.2 and 2.8 times for uniaxial and radial stretching, respectively, when eencap ¼ 30% and epre ¼ 150% for the device in Fig. 2a (Esubstrate ¼ 20 kPa and Eencapsulation ¼ 3 kPa). Modelling is critical for the practical implementation of this concept, simply because the use of eencap above a certain threshold (439% for the case examined here) can lead to plastic yielding of the interconnects before full release (Fig. 3b). NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 The insets correspond to magniﬁed views of a local region. (b) Elastic stretchability of a 3D circuit system encapsulated at different states of partially released strain in this two-stage encapsulating process (blue for uniaxial stretching along X axis; red for uniaxial stretching along Y axis; black for radial stretching). (c) Bar graph of the elastic stretchability for a 3D circuit system formed by using a one-stage encapsulating process, two-stage encapsulating process and no encapsulation. (d) System-level deformation and distribution of stresses determined by FEA with encapsulation introduced after (d) full and (f) partial release of the prestrain. In all of the FEA images, the colour represents the magnitude of Mises stress in the metal layer. (e) Optical images of a device deformed in similar ways. Scale bars, 1 cm (a–d). The scale bar of the inset of (a) is 1 mm. 5 NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications Methods Fi it l Finite element analysis. Three-dimensional FEA techniques allowed prediction of the mechanical deformations and stress distributions of helical interconnects and entire circuit systems, during processes of compressive buckling and re-stretching. Four-node shell elements with a three-layer (PI/metal/PI) composite modelled the interconnects, and eight-node solid elements modelled the substrate and encapsulation. Reﬁned meshes ensured computational accuracy using commercial software (Abaqus). The critical buckling strains and corresponding buckling modes determined from linear buckling analyses served as initial imperfections in the postbuckling analyses to determine the deformed conﬁgura- tions and strain distributions. To evaluate stretchability in the encapsulated condition, 3D helical interconnects determined by postbuckling analyses were embedded in an encapsulation solid that covered the entire area of substrate. The elastic stretchability corresponds to point at which the Mises stresses in the metal layer exceed the yield strength (B199 MPa for Au and 357 MPa for Cu, both corresponding to B0.3% yield strain) across at least one quarter of the width of any section of the interconnect. Previous experimental studies32,55 support the use of this type of criterion. A typical hyper-elastic constitutive relation, that is, the Mooney–Rivlin law, captured the properties of the elastomeric substrate (elastic modulus Esubstrate ¼ 20 kPa and Poisson’s ratio nsubstrate ¼ 0.49) and encapsulation material (Eencapsulation ¼ 3 kPa and nencapsulation ¼ 0.49). The relevant material parameters are (C10 ¼ 2.68 kPa, C01 ¼ 0.67 kPa, D1 ¼ 0.006 kPa 1) for the substrate and (C10 ¼ 0.40 kPa, C01 ¼ 0.10 kPa, D1 ¼ 0.04 kPa 1) for the encapsulation. The other material parameters are EAu ¼ 70 GPa and nAu ¼ 0.44 for gold; ENi ¼ 200 GPa and nNi ¼ 0.31 for nickel; ECu ¼ 119 GPa and nNi ¼ 0.34 for copper; and EPI ¼ 2.5 GPa and nPI ¼ 0.27 for PI. Demonstration of soft electronics built with 3D helical coil. Figure 4a and Supplementary Figs 33–35 present optical images of the system in various states of deformation, with an FEA result for the rightmost frame. Functionally, devices with these designs offer reliable operation in all such circumstances (Supplementary Figs 34 and 35). Figure 4b,c shows capabilities in three-axis accelerometry for tracking of 3D motion and respiration. As in Fig. Methods Fi it l 4d, simultaneous monitoring of electrophysiological signals is also possible, including capture of electrocardiogram (ECG), electromyogram (EMG), electrooculogram (EOG) and electro- encephalogram (EEG) data for quantitative evaluation of cardiac, muscle, eye and brain activity, respectively. Multimodal operation depicted in Fig. 4e, Supplementary Fig. 36 and Supplementary Movie 1 involves recording of three-axis acceleration and EP signals simultaneously, where the former provides important contextual information on the latter. As shown by Supplementary Fig. 37, ECG data collected using a device in an undeformed state are identical to those collected using a device stretched radially to a strain of 50%. This invariance in operation is consistent with electrical resistances of the coils that remain constant under mechanical deformation. The 3D design approaches, the coupled mechanical/electrical considerations in layout and the two-stage encapsulation method, are each critically important in the properties and the operation of such systems. Fabrication of networks of 3D helical coils for fundamental study. Spin-casting poly(methyl methacrylate) (PMMA; B100 nm in thickness, Microchem, USA) formed a thin sacriﬁcial layer on a glass substrate. Spin-casting polyimide (PI; 1B3 mm in thickness, Sigma-Aldrich, USA), depositing thin layers of metal by electron beam evaporation (Cr/Au or Cu, thickness 0.1–1 mm), performing photolithography, wet-etching, spin-casting another layer of polyimide followed by oxygen reactive ion etching deﬁned a network of 2D serpentine structures, referred to here as the 2D precursor. Dissolving the PMMA by immersion in acetone for 10 min allowed retrieval of the 2D precursor onto the surface of a piece of water- soluble tape (Water-Soluble Wave Solder 5414, 3M, USA). Selective deposition of Ti (5 nm)/SiO2(50 nm) by electron beam evaporation through a shadow mask (free-standing patterned sheet of a photodeﬁnable epoxy with thickness of 0.1 mm; SU-8, Microchem, USA) deﬁned sites for strong bonding to a silicone elastomer substrate formed by spin-casting and curing a prepolymer (thickness 1 mm; Ecoﬂex 00-50A/B, Smooth-on, USA) on a glass plate. A custom mechanical stage allowed application of precisely controlled levels of biaxial strain to this elastomer, selected using guidance from computation to achieve the required geometrical transfor- mation from 2D to 3D. Laminating the 2D precursor onto a prestrained substrate and heating (10 min, 70 C in an oven) the system activated formation of strong siloxane bonds between the patterned SiO2 layer on the 2D precursor and the surface of the silicone substrate. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 elastomer (Silbione 4717A/B, Bluestar Silicones, USA; E ¼ B3 kPa) as outlined above. The computational results and experimental results are in Supplementary Figs 28 and 29. The experiments show that an LED system constructed with networks of helical interconnects can survive different types of compressive loadings that might occur in practical use, consistent with modeling results. microsystems technologies and unusual classes of materials and devices. A combination of established and developing elements in overall architectures that leverage both 2D and 3D layout features affords powerful opportunities not only in bio-integrated electronics but also in many other areas of emerging interest, from soft robotics to systems for virtual reality to hardware for autonomous navigation. g Experimental measurements of the mechanical responses also show good agreement with modelling, even at the full system level of B250 helical interconnects and B50 chips (Fig. 3d–f). Overlays of optical images with FEA results (Supplementary Fig. 30) facilitate comparisons. Local regions can be examined quantitatively by using the index of structural similarity (SSIM)54. The results reveal SSIM indices of B0.81, 0.75 and 0.77, respectively, for the cases of radial, X axis and Y axis stretching shown here (see Supplementary Fig. 31 for details). For reference, comparisons of images of local areas to themselves after translation or rotation (Supplementary Fig. 32) indicate that an SSIM value of 0.75 corresponds to a 2.3% relative X axis offset, a 2.0% relative Y axis offset or a 3.3 rotation relative to the image centre; an SSIM of 0.81 corresponds to a 1.8% X axis offset, a 1.5% Y axis offset or a 2.5 rotation. Methods Fi it l Dissolving the tape by immersion in DI water and releasing the prestrain transformed the 2D precursors into an extended network of 3D helical coils via compressive buckling. NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 1 involves applying this material as a liquid precursor at a state of partial release of the substrate prestrain, eencap, crosslinking it into a solid form and then completing the release, as shown in Fig. 3a. This last step deforms the cured material in a manner that softly embeds the NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 6 ARTICLE 8 NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunication b Amplitude (mV) Time 1 –1 0 0 2 Acceleration c Normalized amplitude Time 1 –1 0 Acceleration Respiration rate (bpm) 40 0 20 At rest X Y X Y Z a Reconfigured 3D motion Analysed exercise effect 0.5 1.5 1 0 2 0.5 1.5 1 Z After exercise a a b Amplitude (mV) Time 1 –1 0 0 2 Acceleration X Y 0.5 1.5 1 Z b Amplitude (mV) Time 1 –1 0 0 2 Acceleration X Y X Y Z Reconfigured 3D motion 0.5 1.5 1 Z c Normalized amplitude Time 1 –1 0 Acceleration Respiration rate (bpm) 40 0 20 At rest Analysed exercise effect 0 2 0.5 1.5 1 After exercise X Y Z Reconfigured 3D motion b c Normalized amplitude 0 Z X d Amplitude (mV) Time 1 –1 0 Frequency (Hz) Time 14 10 12 0 Amplitude (mV) Time 1 –1 0 0 1 2 3 4 5 6 7 Amplitude (mV) Time 1 –1 0 0 0.5 1 1.5 2 P Q R S T P Q R S T Upside Downside Left Centre Right EOG EMG ECG Eyes closed Eyes opened EEG 1 2 3 4 0 1 2 3 4 5 6 7 d Amplitude (mV) Amplitude (mV) Time 1 –1 0 0 1 2 3 4 5 6 7 Amplitude (mV) Time 1 –1 0 0 0.5 1 1.5 2 P Q R S T P Q R S T Left Centre Right EMG ECG d Amplitude (mV) Time 1 –1 0 Frequency (Hz) Time 14 10 12 0 Upside Downside EOG Eyes closed Eyes opened EEG 1 2 3 4 0 1 2 3 4 5 6 7 1 Amplitude (mV) Frequency (Hz) Amplitude (mV) e e Running Standing Walking Amplitude (n.d.) Acceleration (n.d.) Time 10 70 130 0 ECG Chest movement 20 30 40 50 60 80 90 100 110 120 140 150 160 170 180 Running Figure 4 \| Mechanical and operational characteristics of the complete and encapsulated system. (a) Optical images of a device deformed in different ways, with corresponding FEA results highlighted in red coloured boxes. (b) Representative recordings of three-axis acceleration from a device on the left forearm and inferred 3D patterns of motion. ARTICLE d Amplitude (mV) Time 1 –1 0 Frequency (Hz) Time 14 10 12 0 Amplitude (mV) Time 1 –1 0 0 1 2 3 4 5 6 7 Amplitude (mV) Time 1 –1 0 0 0.5 1 1.5 2 P Q R S T P Q R S T Upside Downside Left Centre Right EOG EMG ECG Eyes closed Eyes opened EEG e Running Standing Walking Amplitude (n.d.) Acceleration (n.d.) Time 10 70 130 0 ECG Chest movement b Amplitude (mV) Time 1 –1 0 0 2 Acceleration c Normalized amplitude Time 1 –1 0 Acceleration Respiration rate (bpm) 40 0 20 At rest X Y X Y Z a Reconfigured 3D motion Analysed exercise effect 0.5 1.5 1 0 2 0.5 1.5 1 Z After exercise 20 30 40 50 60 80 90 100 110 120 140 150 160 170 180 1 2 3 4 0 1 2 3 4 5 6 7 Figure 4 \| Mechanical and operational characteristics of the complete and encapsulated system. (a) Optical images of a device deformed in differen ways, with corresponding FEA results highlighted in red coloured boxes. (b) Representative recordings of three-axis acceleration from a device on the lef forearm and inferred 3D patterns of motion. (c) Results for respiration rate extracted from frequency analysis of accelerometer data from a device placed on the chest, for cases at rest and after physical exercise. (d) Electrophysiological recordings with inset images of the device on the skin: electrocardiogram (ECG), electromyogram (EMG), electrooculogram (EOG) and electroencephalogram (EEG). (e) Wireless, multimodal monitoring of body activity, through simultaneous measurements of ECG (plotted in green colour) and movements of the chest by accelerometry (plotted in blue colour) during a time interva that includes standing (0–60 s), walking (60–120 s) and running (120–180 s). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 noise ratio. A high-pass ﬁlter (4.8 Hz) and a low-pass ﬁlter (54.1 Hz) in a Sallen- Key topology, deﬁned the passband of the circuit. A non-inverting ampliﬁer magniﬁed the ﬁltered signal to provide an overall gain of 40 dB. The circuit used single-ended power at 3.3 V. A DC offset of 1.65 V allows capture of signal in the range between 0 and 3.3 V. An 8-bit analogue-to-digital converter integrated into the wireless chip (nRF51822, Nordic Semiconductor) enabled digital acquisition at a sampling rate of 250 Hz. The same circuit measured electrocardiogram (ECG) and electroencephalogram (EEG) data. The circuit also can perform electro- oculography (EOG) and electromyography (EMG) with a gain of 80 dB and a passband of 10–500 Hz and 0.5–20 Hz, respectively. the wireless chip (nRF51822, Nordic Semiconductor) enabled digital acquisition at a sampling rate of 250 Hz. The same circuit measured electrocardiogram (ECG) and electroencephalogram (EEG) data. The circuit also can perform electro- oculography (EOG) and electromyography (EMG) with a gain of 80 dB and a passband of 10–500 Hz and 0.5–20 Hz, respectively. Key topology, deﬁned the passband of the circuit. A non-inverting ampliﬁer magniﬁed the ﬁltered signal to provide an overall gain of 40 dB. The circuit used single-ended power at 3.3 V. A DC offset of 1.65 V allows capture of signal in the range between 0 and 3.3 V. An 8-bit analogue-to-digital converter integrated into range between 0 and 3.3 V. An 8 bit analogue to digital converter integrated into passband of 10 500 Hz and 0.5 20 Hz, respectively. Discussion Th l The results presented here establish concepts, as well as routes for practical implementation, for 3D microstructure designs in soft electronics. Speciﬁc ﬁndings include quantitative advantages of ideal, spring-like mechanics in 3D helical coils compared to traditional 2D layouts; scalable approaches for forming 3D helical frameworks as interconnect networks between advanced micro- system components; combined electrical/mechanical techniques for optimizing system design; encapsulation materials and methods for ideal, 3D mechanics; and multimodal, wireless, skin-mounted demonstration devices for health monitoring. These ideas in 3D design have relevance not only to interconnect networks but also to other sub-systems, such as the antennas, the sensor structures and certain of the active and passive device components as well. Compatibility of the 3D assembly approaches with the most advanced methods in 2D micro/ nanofabrication provides alignment both with state-of-the-art Fabrication process for soft wireless electronics. Processes similar to those described in the previous section, but with additional steps in spin-casting, metal deposition, photolithography and reactive ion etching yielded networks with electrically isolated crossing points and contact pads as interfaces to electronic components. A conductive alloy (In97Ag3, Indalloy 290, Indium Corporation, USA) enabled bonding of the contacts associated with the components to the pads in the interconnect network, while still in a 2D geometry on a bi-axially prestrained elastomer substrate. Partially releasing this prestrain followed by casting a uniform layer of an ultra-low-modulus elastomer (Silbione 4717A/B, Bluestar Silicones, France; E ¼ B3 kPa) encapsulated the entire system while pre- serving freedom of motion of the helical coils upon application of strain. This layer physically protected the system from the surroundings during handling and use. Circuit design for electrophysiological sensing module. The electro- cardiography (ECG) circuit used an instrumentation ampliﬁer (INA333, Texas Instruments, common-mode rejection ratio ¼ 100 dB) as a pre-ampliﬁer for differential signal inputs to suppress common-mode noise. A driven ground with negative feedback allowed further noise reduction, thereby improving the signal-to- NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 7 References 1. Rogers, J. A., Someya, T. & Huang, Y. G. Materials and mechanics for stretchable electronics. Science 327, 1603 (2010). 1. 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With this system, 60 mW can be transmitted at 2 W and a 1 cm distance, capable of operating the system, which had a peak power consumption of 30–40 mW (Supplementary Fig. 38). 26. Lacour, S. P., Jones, J., Wagner, S., Li, T. & Suo, Z. G. Stretchable interconnects for elastic electronic surfaces. Proc. IEEE 93, 1459 (2005). 27. Li, T., Suo, Z. G., Lacour, S. P. & Wagner, S. Compliant thin ﬁlm patterns of 27. Li, T., Suo, Z. G., Lacour, S. ARTICLE (c) Results for respiration rate extracted from frequency analysis of accelerometer data from a device placed on the chest, for cases at rest and after physical exercise. (d) Electrophysiological recordings with inset images of the device on the skin: electrocardiogram (ECG), electromyogram (EMG), electrooculogram (EOG) and electroencephalogram (EEG). (e) Wireless, multimodal monitoring of body activity, through simultaneous measurements of ECG (plotted in green colour) and movements of the chest by accelerometry (plotted in blue colour) during a time interval that includes standing (0–60 s), walking (60–120 s) and running (120–180 s). NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms15894 Acknowledgements Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ This work was supported by the Center for Bio-Integrated Electronics. K.-I.J. acknowledges the support from the Ministry of Science (#2017R1C1B2007795). Y.Z. acknowledges support from the National Natural Science Foundation of China (#11672152) and the National Basic Research Programme of China (#2015CB351900). Y.H. acknowledges the support from the NSF (#CMMI1300846, #CMMI1400169 and #CMMI1534120). How to cite this article: Jang, K.-I. et al. Self-assembled three dimensional network designs for soft electronics. Nat. Commun. 8, 15894 doi: 10.1038/ncomms15894 (2017). 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 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/ Author contributions K.-I.J., K.L., H.U.C., Y.H., Y.Z., J.A.R. designed idea and wrote manuscript. S.X., H.N.J., J.W.K., H.H.J., J.S., H.U.C., B.H.K., J.-H.K., J.L., K.J.Y., J.K., J.W.L., J.-W.J., Y.M.S. performed experiments and analysed the experimental data. K.L., Y.H. and Y.Z. led the structural designs and mechanics modelling, with assistance from C.Y., A.W. and Z.L. Others contributed to the analysis of experimental results. References Nat. Commun. 5, 5032 (2014). 55. Zhang, Y. H. et al. Experimental and theoretical studies of serpentine microstructures bonded to prestrained elastomers for stretchable electronics. Adv. Funct. Mater. 24, 2028 (2014). 21. Wang, C. et al. User-interactive electronic skin for instantaneous pressure visualization. Nat. Mater. 12, 899 (2013). 9 NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications r The Author(s) 2017 NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications Additional information Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing interests: The authors declare no competing ﬁnancial interests. 10 NATURE COMMUNICATIONS \| 8:15894 \| DOI: 10.1038/ncomms15894 \| www.nature.com/naturecommunications
https://openalex.org/W2778275005	https://www.frontiersin.org/articles/10.3389/fonc.2017.00319/pdf	English	null	The Central Role of Amino Acids in Cancer Redox Homeostasis: Vulnerability Points of the Cancer Redox Code	Frontiers in oncology	2,017	cc-by	14,496	Abbreviations: AA(s), amino acid(s); ALDH1L2, 10-formyl-THF dehydrogenase; AOD, antioxidant defense; ARE, antioxidant response element; ASCT2, alanine-serine-cysteine transporter 2; ATM, ataxia telangiectasia mutated gene; eIF2α, eukaryotic initiation factor 2α; ER, endoplasmic reticulum; ETC, electron transport chain; FOXO, forkhead box O; GCL, glutamate– cysteine ligase; GCN2, general control non-derepressable 2; GLS1/2, cytoplasmic/mitochondrial glutaminase; GLUD1, glutamate dehydrogenase; GOT1/2, aspartate transaminase 1/2; GR, glutathione reductase; GS, glutathione synthetase; GSH, glutathione; GSH-Px, glutathione peroxidase; GSTs, glutathione S-transferases; GSSG, glutathione oxidized; [H2O2], hydrogen peroxide; [HO⋅], peroxyl radical; [HO−], hydroxyl anion; KEAP1, Kelch-like ECH-associated protein 1; LAT1, L-type amino acid transporter 1; ME1/2, malic enzyme 1/2; MTHFD, methylene tetrahydrofolate dehydrogenase; mTORC1, mechanistic target of rapamycin complex 1; NAD(P)H, nicotinamide adenine dinucleotide (phosphate); NNT, energy-linked transhydro- genase; [NO⋅], nitric oxide; NOX, NADPH oxidase; NRF2, nuclear factor (erythroid-derived-2)-like 2; [1O2], singlet oxygen; [ ] O2⋅−, superoxide anion radical; PDAC, pancreatic ductal adenocarcinoma; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PHGDH, phosphoglycerate dehydrogenase; PPP, pentose phosphate pathway; PRXs, peroxiredoxins; Rb, retinoblastoma; [ROO⋅], peroxyl radicals; [ROOH], organic hydroperoxides; ROS, reactive oxygen species; SHMT2, serine hydroxymethyl transferase 2; SNAT1-2, system A amino acid transporter 1–2; SOD, superoxide dismutase; SSP, serine synthesis pathway; TCA, tricarboxylic acid; THF, tetrahydrofolate; TRXs, thioredoxins; TSC, tuberous sclerosis complex; TSP, trans- sulfuration pathways; CySSCy, cystine (oxidized cysteine); xCT, system xc-light chain. The Central Role of Amino Acids in Cancer Redox Homeostasis: vulnerability Points of the Cancer Redox Code Milica Vucˇetic′1, Yann Cormerais1, Scott K. Parks1 and Jacques Pouysse´ gur1,2 1 Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco, Monaco, 2 Institute for Research on Cancer and Aging (IRCAN), CNRS, INSERM, Centre A. Lacassagne, Université Côte d’Azur, Nice, France Milica Vucˇetic′1, Yann Cormerais1, Scott K. Parks1 and Jacques Pouysse´ gur1,2 1 Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco, Monaco, 2 Institute for Research on Cancer and Aging (IRCAN), CNRS, INSERM, Centre A. Lacassagne, Université Côte d’Azur, Nice, France A fine balance in reactive oxygen species (ROS) production and removal is of utmost importance for homeostasis of all cells and especially in highly proliferating cells that encounter increased ROS production due to enhanced metabolism. Consequently, increased production of these highly reactive molecules requires coupling with increased antioxidant defense production within cells. This coupling is observed in cancer cells that allocate significant energy reserves to maintain their intracellular redox balance. Glutathione (GSH), as a first line of defense, represents the most important, non-enzymatic antioxidant component together with the NADPH/NADP+ couple, which ensures the maintenance of the pool of reduced GSH. In this review, the central role of amino acids (AAs) in the maintenance of redox homeostasis in cancer, through GSH synthesis (cysteine, gluta- mate, and glycine), and nicotinamide adenine dinucleotide (phosphate) production (serine, and glutamine/glutamate) are illustrated. Special emphasis is placed on the importance of AA transporters known to be upregulated in cancers (such as system xc-light chain and alanine-serine-cysteine transporter 2) in the maintenance of AA homeostasis, and thus indirectly, the redox homeostasis of cancer cells. The role of the ROS varies (often described as a “two-edged sword”) during the processes of carcinogenesis, metasta- sis, and cancer treatment. Therefore, the context-dependent role of specific AAs in the initiation, progression, and dissemination of cancer, as well as in the redox-dependent sensitivity/resistance of the neoplastic cells to chemotherapy are highlighted. Keywords: cancer, amino acids, redox homeostasis, glutathione, NADPH/NADP+ Edited by: Edited by: Sergio Giannattasio, Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari (IBIOM), Italy Reviewed by: Cesare Indiveri, University of Calabria, Italy Paula Ludovico, University of Minho, Portugal Correspondence: Milica Vucˇetic′ milica@centrescientifique.mc; Jacques Pouysségur jacques.pouyssegur@unice.fr Reviewed by: Cesare Indiveri, University of Calabria, Italy Paula Ludovico, University of Minho, Portugal Reviewed by: Cesare Indiveri, University of Calabria, Italy Paula Ludovico, University of Minho, Portugal Correspondence: Milica Vucˇetic′ milica@centrescientifique.mc; Jacques Pouysségur jacques.pouyssegur@unice.fr Keywords: cancer, amino acids, redox homeostasis, glutathione, NADPH/NADP+ Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology Received: 02 October 2017 Accepted: 08 December 2017 Published: 21 December 2017 Received: 02 October 2017 Accepted: 08 December 2017 Published: 21 December 2017 Review Review published: 21 December 2017 doi: 10.3389/fonc.2017.00319 published: 21 December 2017 doi: 10.3389/fonc.2017.00319 Edited by: Sergio Giannattasio, Istituto di Biomembrane, Bioenergetica e Biotecnologie Molecolari (IBIOM), Italy Reviewed by: Cesare Indiveri, University of Calabria, Italy Paula Ludovico, University of Minho, Portugal *Correspondence: Milica Vucˇetic′ milica@centrescientifique.mc; Jacques Pouysségur jacques.pouyssegur@unice.fr PARTIALLY REDUCED OXYGEN— “ACTIVATED” OXYGEN According to the previous paragraph, it seems that a pro-oxidative state could facilitate initiation and progression of tumorigenesis. However, when reactive and very short living species such as ROS are considered, the situation is not so clear. Accordingly, studies on the effects of antioxidant supplements for cancer prevention and treatment showed opposed and mainly unpromising results, giving rise to confusion and the “antioxidant paradox” (1). Another redox consideration for cancer treatment includes increased ROS levels in cancer cells that already “walk on the edge of oxidative abyss” (23–25). This stand point arises from the very well know concept of hormesis that has been recognized since the XVI century by Paracelsus’s—“Die Dosis macht das Gift” or “the dose makes the poison” (26). The concept of hormesis, which revolutionized mod- ern toxicology, claims that the dose–response curve is U-shaped, generally meaning that a drug/stimulus can have opposite effects in small and large doses [for more details refer to Calabrese and Baldwin (27) and papers stemming from it]. Indeed, it has been shown that a mild oxidative state promotes all hallmarks of cancer cells; however, if the threshold is exceeded (reaching the top of the arm of the U-shaped curve), influence of the oxidative environ- ment can easily become anti-carcinogenous, promoting cell-cycle arrest, senescence, programmed cell death, or necrosis (Figure 1). The first steps in understanding oxygen toxicity occurred in the mid-twentieth century when Gerschman et al. (7–9) proposed that the damaging effects of oxygen could be attributed to the formation of oxygen radicals. At approximately the same time, research with [18O2] and mass spectrometry showed that oxygen atoms from molecular oxygen [O2] could be introduced into bio- molecules (10, 11). The susceptibility of biomolecules to oxidation gave a biological frame to oxygen toxicity, and together with the discovery of superoxide dismutase [SOD; (12)] fueled research in the field of oxidative damage in biological systems. The term “oxidative stress” was introduced into scientific literature for the first time in 1985 (13). i Now it is clear that the oxidative capacity of molecular oxygen in vivo is minimal, but that is not the case for its partially reduced counterparts known as “reactive oxygen species—ROS.” ROS is a term widely used to describe a number of reactive molecules and free radicals derived from molecular oxygen. However, we feel obliged to emphasize the generic nature of this term. ROS IN CANCER The terms “ROS” and “cancer” cover a wide range of molecules and diseases, which makes broad generalizations almost impos- sible. Is it possible, however, to conceptualize some common denominators of the cancer redox state? Widespread opinion is that virtually all malignant cells are in a pro-oxidative state, mostly due to oncogene-driven altered and/or intensified cell metabolism [reviewed in Ref. (17–21)]. However, Halliwell (20) raised important questions regarding ROS measurement in malignant (and other) cells in classical culture conditions that include 21% oxygen and media that is usually deficient in antioxidants/antioxidant precursors and contains free iron ions. These conditions, per se, favor ROS generation, and thus special attention should be paid in extrapolating results obtained in vitro to the in vivo state. Considering this point in combination with current advances in the cancer redox field, a major conclusion that can be drawn is that cancer cells indeed experience mild oxi- dative pressure in comparison to normal cells (Figure 1) that can help them to exhibit characteristic cancer hallmarks [for detailed review refer to Hornsveld and Dansen (22)]. p p p y g Although the idea of AA dependency of cancer antioxidant defense (AOD) has received more attention recently, a unified review on this subject is lacking. In 2015, Jones and Sies (6) labeled the nicotinamide adenine dinucleotide (NAD, NADP) and thiol/ dysulfide [glutathione (GSH)/glutathione oxidized (GSSG) in the first place] systems together with thiol redox proteome as carriers of the cellular “Redox Code.” According to this principle, spatiotemporal organization of these systems is fundamental for physiology, while its disruption inevitably leads to pathology. Interestingly, accumulating literature indicates that AA availabil- ity and metabolism are upstream and superior to these systems, especially in cancer cells. Our review will address this particular aspect of redox regulation in tumors. However, before consider- ing the involvement of AA homeostasis in cancer redox balance, it is necessary to point out some important findings, as well as delusions, that exist in the complex cancer redox field. Citation: Vucˇetic′ M, Cormerais Y, Parks SK and Pouysségur J (2017) The Central Role of Amino Acids in Cancer Redox Homeostasis: Vulnerability Points of the Cancer Redox Code. Front. Oncol. 7:319. doi: 10.3389/fonc.2017.00319 December 2017 \| Volume 7 \| Article 319 1 Frontiers in Oncology \| www.frontiersin.org AAs in Cancer Redox Homeostasis Vucˇetic′ et al. Frontiers in Oncology \| www.frontiersin.org INTRODUCTION radical, [HO⋅]; peroxyl radicals, [ROO⋅]; nitric oxide, [NO⋅]) and non-radical (hydrogen peroxide, [H2O2]; hydroxyl anion, [HO−]; singlet oxygen, [1O2]; organic hydroperoxides, [ROOH]) species, which differ significantly in terms of half-life, water/lipid solubil- ity and reactivity. For example, the cellular half-life of lipophobic [HO⋅] is only ~10−9 s because of its reactivity, compared to ~1 ms for [H2O2], which also can diffuse through lipid cellular compart- ments (14). However, use of the common term ROS is sometimes unavoidable (15) due to the complex nature of biological systems, an inability to exactly measure the species generated in a spati- otemporal manner in addition to the so-called theory of “kindling radicals” by which a few primary ROS “inflame” a cascade of ROS amplification by stimulating the sources of secondary ROS (16). The potential of targeting redox homeostasis for both cancer prevention and development of novel anticancer treatments has been recognized during past decades. However, despite intensive efforts, development of an effective redox-based therapy remains challenging. A main reason for this is cancer cell plasticity but also our inability to adequately perceive the complexity of redox homeostasis. Namely, antioxidant prophylaxis led to the “antioxi- dant paradox” (1, 2), while use of chemotherapeutics that com- promise the oxidative status of cancer cells encountered resistance (3) and the ability of some cancer cells to upregulate antioxidant protective mechanisms (4). Currently, most attention on targeting redox homeostasis focuses on the attack and downregulation of endogenous antioxidant tumor cell defense mechanisms (5). In this review, we approach cancer redox balance from a different perspective with the main players involving amino acids (AAs). PARTIALLY REDUCED OXYGEN— “ACTIVATED” OXYGEN However, if ROS burst induces irreversible damage and/or there is not enough components required for repair systems (e.g., glutathione), cancer cells experience programmed cell death or necrosis (cytotoxic effects of ROS). Figure 1 \| Reactive oxygen species (ROS) can (i) promote cancer, (ii) cause growth arrest, and (iii) be cytotoxic. In normal cells, increased (endogenous or exogenous) oxidative pressure leads to adequate upregulation of cellular antioxidant defense (AOD), which prevent mutagenic events and initiation of cancer formation. However, AOD is not 100% efficient, and thus, these “challenging states” also represent well-known risk factors for cancer development. Once formed, cancer progression seems to be further stimulated by a mild pro-oxidative state due to intensified metabolism, ROS-producing foci, etc. Importantly, this state is still maintained within “redox homeostatic range” thanks to strongly upregulated AOD of cancer cells. However, due to maximized AOD, cancer cells do not support further increase in ROS levels and thus cross the threshold into the state of “oxidative stress.” If ROS level increase further (e.g., due to chemotherapy), the only way for cancer cells to prevent further damage is by decreasing ROS production via cell-cycle arrest to repair damage and prevent cell death (cytostatic effects of ROS). However, if ROS burst induces irreversible damage and/or there is not enough components required for repair systems (e.g., glutathione), cancer cells experience programmed cell death or necrosis (cytotoxic effects of ROS). become detrimental (Figure 1). Indeed, antioxidant pathways known to respond to increased oxidative pressure in normal cells are constitutively activated in some cancers. The best example is the nuclear factor (erythroid-derived-2)-like 2 (NRF2)-signaling pathway [reviewed elsewhere in great detail (34, 35)]. NRF2 is the main transcription factor regulating expression of AOD enzymes. Under normal conditions, NRF2 is constantly ubiquitinated by Kelch-like ECH-associated protein 1 (KEAP1) and degraded by the proteasome. Oxidants/electrophiles inactivate Keap1 and stabilize NRF2, which then translocates into the nucleus, binds to the antioxidant response element, and activates the transcription of many cytoprotective genes that encode detoxifying enzymes and antioxidant proteins. Constitutive activation of NRF2, due to gain-of-function mutations in NRF2 (36), or loss-of-function mutation in its negative regulator KEAP1, was observed in different types of cancers (37–41). In addition, several tumor- suppressor genes act to repress tumor cell proliferation or cause cells to enter permanent cell-cycle arrest in response to ROS overproduction. These include retinoblastoma, p16INK4A, JNK, p38, p53, and forkhead box O. PARTIALLY REDUCED OXYGEN— “ACTIVATED” OXYGEN Most of these tumor-suppressor proteins sense changes in the cellular oxidative status and respond accordingly by inhibiting the cell cycle, and thus allowing cells time to recover after oxidative stress, and/or to induce expression of AOD enzymes (22). Thus, it has been shown that increased oxidative pressure in the blood, if not adequately balanced by internal AOD, may limit the efficiency of melanoma cells to form distant tumors (28). These results are a textbook example of the antioxidant paradox suggest- ing how dietary supplementation of antioxidants may promote the metastatic potential of the cancer cells.hf Thus, it has been shown that increased oxidative pressure in the blood, if not adequately balanced by internal AOD, may limit the efficiency of melanoma cells to form distant tumors (28). These results are a textbook example of the antioxidant paradox suggest- ing how dietary supplementation of antioxidants may promote the metastatic potential of the cancer cells.hf p The anticancer effects of many conventional therapies, includ- ing irradiation and DNA-damaging chemotherapeutics (cispl- atin, doxorubicin, gemcitabine, and 5-fluorouracil), rely mostly or partially on increased ROS production, due to mitochondria damage and dysfunction, as well as activation of NADPH oxidase (NOX) enzymes (29–33). However, these treatments encounter resistance with initial response being followed by the develop- ment of protective mechanisms against these oxidative/genotoxic insults. The mechanisms of resistance are complex involving drug modification, inhibition, degradation, and/or efflux [for further readings refer to Housman et al. (3)]. In spite of this complexity, the central role that AOD plays in these processes provided the rational for developing anticancer therapies targeting this aspect of cancer redox balance. PARTIALLY REDUCED OXYGEN— “ACTIVATED” OXYGEN ROS includes both radical (superoxide anion radical, [ ] O2 ⋅−; hydroxyl December 2017 \| Volume 7 \| Article 319 2 Vucˇetic′ et al. AAs in Cancer Redox Homeostasis Figure 1 \| Reactive oxygen species (ROS) can (i) promote cancer, (ii) cause growth arrest, and (iii) be cytotoxic. In normal cells, increased (endogenous or exogenous) oxidative pressure leads to adequate upregulation of cellular antioxidant defense (AOD), which prevent mutagenic events and initiation of cancer formation. However, AOD is not 100% efficient, and thus, these “challenging states” also represent well-known risk factors for cancer development. Once formed, cancer progression seems to be further stimulated by a mild pro-oxidative state due to intensified metabolism, ROS-producing foci, etc. Importantly, this state is still maintained within “redox homeostatic range” thanks to strongly upregulated AOD of cancer cells. However, due to maximized AOD, cancer cells do not support further increase in ROS levels and thus cross the threshold into the state of “oxidative stress.” If ROS level increase further (e.g., due to chemotherapy), the only way for cancer cells to prevent further damage is by decreasing ROS production via cell-cycle arrest to repair damage and prevent cell death (cytostatic effects of ROS). However, if ROS burst induces irreversible damage and/or there is not enough components required for repair systems (e.g., glutathione), cancer cells experience programmed cell death or necrosis (cytotoxic effects of ROS). Figure 1 \| Reactive oxygen species (ROS) can (i) promote cancer, (ii) cause growth arrest, and (iii) be cytotoxic. In normal cells, increased (endogenous or exogenous) oxidative pressure leads to adequate upregulation of cellular antioxidant defense (AOD), which prevent mutagenic events and initiation of cancer formation. However, AOD is not 100% efficient, and thus, these “challenging states” also represent well-known risk factors for cancer development. Once formed, cancer progression seems to be further stimulated by a mild pro-oxidative state due to intensified metabolism, ROS-producing foci, etc. Importantly, this state is still maintained within “redox homeostatic range” thanks to strongly upregulated AOD of cancer cells. However, due to maximized AOD, cancer cells do not support further increase in ROS levels and thus cross the threshold into the state of “oxidative stress.” If ROS level increase further (e.g., due to chemotherapy), the only way for cancer cells to prevent further damage is by decreasing ROS production via cell-cycle arrest to repair damage and prevent cell death (cytostatic effects of ROS). AOD IN CANCER As mentioned previously, oncogenic mutations lead to a pro- oxidative state of cancer cells. However, these cells are still required to maintain ROS levels below the threshold that would Antioxidant defense is divided into enzymatic and non- enzymatic parts. Enzymatic AOD includes enzymes such as SODs, December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 3 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. However, these two nicotinamide nucleotide systems have some- what different roles in metabolism. Namely, while NADH/NAD+ is involved in catabolism and energy supply, NADPH/NADP+ is central for anabolism, defense, and redox homeostasis [reviewed in Ref. (6)]. The redox potential of these two systems also dif- fers significantly in cells. Namely, the cytosolic redox potential of NADH/NAD+ is more oxidized (−241 mV) (52, 53) while in mitochondria, it operates at a more negative redox potential (−318 mV) (54), providing reductive force for ATP synthesis. Meanwhile, NADPH/NADP+ operates at more negative redox potential than the NAD system both in cytosol (−393 mV) and mitochondria (−415 mV) (53).h catalases, gluthatione peroxidases (GSH-Px), and glutathione S-transferases, as well as redox proteins such as thioredoxins (TRXs), peroxiredoxins, and glutaredoxins. Non-enzymatic AOD com- ponents are low-molecular weight compounds such as the key AOD tripeptide glutathione (GSH), vitamins (vitamins C and E), β-carotene, and uric acid. Complementary to these AOD components is the reducing equivalent NADPH that maintains catalases in active forms, serves as a cofactor for TRX and glu- tathione reductase [which converts oxidized glutathione (GSSG) into its reduced state (GSH)], and acts as a reducing agent for regeneration of glutaredoxins.h The concept of the Redox Code proposed recently by Jones and Sies (6) secludes GSH and NADH/NADPH as main determinants of the dynamic nature of redox signaling and control in multi- dimensional biological systems. This is even more pronounced in cancer cells due to increased and imbalanced metabolism, mutation accumulation during tumor progression and activated ROS-producing foci (such as defected mitochondria or NOX enzymes). The main reason why GSH and nicotinamide adenine dinucleotide (phosphate) are in the spotlight is the fact that these are the ultimate reducing factors of the cell. Glutathione NADPH production occurs via the pentose phosphate path- way (PPP), folate metabolism, and malic enzymes (MEs). The importance of AAs for NADPH-producing pathways, especially in cancer cells, is discussed below. Glutathione, a tripeptide γ-glutamyl-cysteinyl-serine, appears in two forms: the predominant reduced form (GSH), which reaches millimolar concentrations in the cell, and the minor oxidized form (GSSG), which is estimated to be less than 1% of the total GSH (42). The bulk of GSH is found in the cytosol (~90%), while the rest is localized mainly in mitochondria and the endoplasmic reticulum (ER) (43). GSH functions to detoxify electrophilic compounds including xenobiotics, which makes it central to cellular anticancer drug resistance (44). Owing to the sulfhydryl (−SH) group of cysteine, GSH can serve as an electron donor for reduction of peroxides (reactions catalyzed by GSH-Px) or disulfides. GSH can also directly react with various oxidants in a non-enzymatic manner, although these reaction kinetics are generally very slow (45). In addition, GSH is important in its cysteine-storage function (γ-glutamyl cycle).f AAs SENSING FROM A REDOX PERSPECTIVE Glucose, AAs, and fatty acids are the crucial building blocks of cellular biomolecules. Tight regulatory mechanisms have evolved to maintain the level of each within homeostatic range. The two main protein kinases involved in sensing and regulation of AA homeostasis are the mechanistic target of rapamycin complex 1 (mTORC1) and general control non-derepressable 2 (GCN2) [for an extensive reviews refer to Bar-Peled and Sabatini (59), Efeyan et al. (60), and Broer and Broer (61)]. Briefly, mTORC1 is a major sensor of specific AAs (Leu, Arg, and Lys), which also receives integrated, growth factors, hormonal, environmental and stress signals regulating growth, and proliferation. Although mecha- nisms of mTORC1 activation have progressed considerably in the past 20 years, the precise effects of individual AAs on mTORC1 activation have remained elusive. Sabatini’s group has illuminated AA sensing by demonstrating that mTORC1 translocation to lysosomes, is critical for its activation (59). Interestingly, recent studies revealed that this lysosomal localization allows mTORC1 sensing of AA levels (Arg and Gln), not only in cytoplasm but also in lysosomal compartement via the lysosomal membrane- resident transport protein SLC38A9 that constitutes a physical and functional part of the AA-sensing machinery (62, 63). Conversely, GCN2-kinase senses AA-uncharged tRNA, result- ing in a general suppression of protein translation, paralleled by induction of the mechanisms to increase the cellular AA pool. Data regarding redox dependency of these pathways are still scarce and mechanically unclear. Similar to ROS, GSH effects can be pro- or antitumorigenic (46). Although it is important in carcinogen detoxification, increased GSH levels and GSH-dependent biotransformation in many tumors may increase resistance to chemotherapy and radiotherapy (47–50). In addition, high GSH levels are associated with cancer hallmarks such as genomic instability, suppression of apoptosis, invasion, and metastatic activity [for further reading refer to Balendiran et al. (46)]. AOD IN CANCER The energy-linked mitochondrial enzyme NNT that transfers electrons from NADH to NADPH thus connecting the two systems is of utmost importance in cancers containing mutations in the tricarboxylic acid (TCA) cycle (fumarate hydratase or succinate dehydrogenase) or the electron transport chain (ETC, complex I or III), which have been shown to promote utiliza- tion of glutamine by reductive carboxylation (55, 56). Namely, adequate citrate production in these conditions requires high NADPH/NADP+ ratios (57), which are achieved by the activity of the NNT (58). CYSTEINE LEVELS DETERMINES GSH LEVELS Two cytosolic ATP-dependent enzymes are involved in GSH synthesis: glutamate–cysteine ligase (GCL), which catalyzes formation of a particular gamma-peptidic bond between Glu and Cys, and glutathione synthetase. The rate-limiting step in GSH synthesis is the reaction catalyzed by GCL (85). Genetic deletion of the GCL catalytic subunit was lethal in the mouse embryo, while knockout mice for the modifier subunit of the enzyme, although viable and fertile, show a significant decrease of tissue GSH levels (9–16% of wt) (86). The Km of mouse GCL for cysteine is estimated at ~0.2 mM (87), which is near the upper limit of typical cellular cysteine concentrations, while the Km for glutamate is at or below the cellular glutamate concentration for Drosophila, mouse, or human GCLholo enzymes (88–90). Hence, it is not surprising that cysteine is the main regulator of GCL activity, and thus GSH synthesis (Figure 2). The main downstream target of activated GCN2 is the eukaryotic initiation factor 2α (eIF2α), whose phosphorylation results in a general reduction of translation initiation, while specific mRNAs containing upstream open-reading frames (e.g., ATF4) are actively translated. However, it has been recognized that GCN2 can be activated by a number of different stresses [osmotic, UV, oxidative (such as [H2O2]), and ER] independently of AA depletion/imbalance (75–77). Interestingly, although the mechanisms are not yet known, it is recognized that the response of GCN2 to stressors such as [H2O2] or UV radiation are very fast in comparison to the gradual accumulation of uncharged tRNAs.l y y g In physiological conditions, cysteine is not an essential AA as it can be synthetized through trans-sulfuration pathways (TSP) from methionine, mainly in the liver. Approximately 50% of the cysteine in hepatic GSH is derived from methionine via TSP (91). However, high demand for cysteine in cancer cells, make TSP insufficient (Figure 2). Furthermore, some tumors have shown significantly lower expression of TSP enzymes mostly due to transcriptional silencing (92, 93). Consequently, Cramer and coworkers (94) showed that depletion of cyst(e)ine with pharmacologically optimized cyst(e)inase enzymes induced cell- cycle arrest and cancer cell death due to GSH depletion and ROS accumulation, both in vitro and in vivo. In turn, the AA-sensing pathways also influence cellular redox balance. Namely, ATF4, an effector molecule of the GCN2-pathway, also serves as a dimerization partner of the cap “n” collar transcription factor NRF2 (78, 79) promoting resist- ance to oxidative stress (79, 80). NADPH/NADP+ Couple Antioxidant defense is completely ineffective without the NADPH/NADP+ cofactor, which serves as a main electron donor for both antioxidant enzymes and catabolic reactions. NADPH supplies reducing equivalents to maintain vital AOD components including the maintenance of active catalase and the regenera- tion of glutathione, TRX, and glutaredoxin. The NADH/NAD+ system is also involved in reversible 2-electron transfer catalysis and is connected with the NADPH/NADP+ system by activity of mitochondrial energy-linked transhydrogenase (NNT) (51). December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 4 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. Earlier studies showed that UV radiation activates mTORC1 signaling through MAP kinase activation by promoting phospho- rylation of its downstream target p70S6k in an [H2O2] concentra- tion and time-dependent manner (64, 65). mTORC1 activation was also observed when cells were treated with oxidizing agents, and surprisingly, even in AA-depleted conditions (66, 67). By contrast, subcellular localization of the mTORC1-interacting protein complex tuberous sclerosis complex at the peroxisome is responsible for mTORC1 repression and autophagy induction in response to ROS (68). Also, the tumor-suppressor ataxia tel- angiectasia mutated gene, appears to regulate autophagy through repression of mTORC1 in response to oxidative stress (69, 70). Thus, it seems that net effects of ROS on mTORC1 activity are context, time, and dose dependent. However, it should be emphasized that although the AAs leucine, arginine, and lysine are identified as key stimuli for mTORC1 activation, recent work on hepatoma HepG2 cells revealed significant sensitivity of both mTORC1 and GCN2 kinases to cysteine depletion (71). Prompt (within 60 min) inhibition of mTORC1 upon cysteine removal was observed. Considering that the Cys proteome coevolved with advanced [O2] sensing and [H2O2] signaling systems (72–74), this effect of cysteine on mTORC1 from a redox perspective may be of higher importance than the effects of ROS, per se. The results listed earlier favor the hypothesis that redox and AA balance are tightly intertwined. How AAs specifically influ- ence the cellular “Redox Code” (GSH and NADPH levels) will be discussed below with special attention placed on the pathways that might represent “vulnerability points” for design of novel anticancer therapeutics. CYSTEINE LEVELS DETERMINES GSH LEVELS Consistently, it has been shown that mouse fibroblasts lacking Atf4 depend on supplemental reducing substances, such as glutathione, N-acetyl cysteine, or β-mercaptoethanol in their growth media (81). Recent work on HT1080 and A549 tumor cells showed the phosphorylation of eIF2 by protein kinase RNA-like endoplasmic reticulum kinase increases the ability of these cells to cope with increased oxida- tive pressure in an ATF4-independent manner by activating Akt (82). The importance of the GCN2 kinase in maintaining redox balance was also proved in vivo. Mice lacking GCN2 exhibited an increase in protein carbonylation in response to a leucine- imbalanced diet (83).f Frontiers in Oncology \| www.frontiersin.org xc-Transport Systemi Different transporters are involved in the import of the reduced, cysteine (CySH), and oxidized, cystine (CySSCy) form of this semi-essential AA. The heavy-chain transporter subunit of system xc-light chain (xCT) seems to play a pivotal role in the import of CySSCy, the predominant form of cysteine in circulation. After import, CySSCy is reduced by cystine reductase and used for different purposes including GSH biosynthesis. Import of cysteine can occur via ASCT (alanine/serine/cysteine transporter) and other transporters (x). The importance of the cystine/glutamate antiporter in redox regulation was further implicated in the newly described type of cell death—ferroptosis (128, 129). Ferroptosis is described as an iron-dependent, programmed form of cell death driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4 and subsequent accumulation of membrane lipid peroxides (130). The first described inducer of ferroptosis in Ras-mutated human foreskin fibroblasts was the xCT inhibitor erastin (131). Depletion of intracellular GSH levels due to inhibition of xCT and subsequent increase of ROS levels seems to be sufficient to trigger erastin-dependent cell death. The same results were observed with sulfasalazine, which is another inhibitor of xCT (109, 132). Interestingly, it has been shown that a loss of cysteinyl- tRNA synthetase might prevent erastin-induced cell death by inducing the TSP (133), suggesting that trans-sulfuration can contribute to resistance to inhibition of xCT and ferroptosis induction. (106), this induction of the system xc- in culture conditions is caused by the high partial pressure of oxygen. Consistent with this hypothesis, prolonged cultivation of fibroblasts in reduced oxygen partial pressure caused a significant decrease in the system xc-activity (106). Considering that AA transporters are necessary for tumor cell proliferation, it is not surprising that xCT is upregulated in many patient samples and tumor cell lines including hepatoma, lymphoma, glioma, colon, breast, prostate, and pancreatic (95, 101, 107–113). Expression of the xCT subunit seems to be under direct control of oncogenes including NRF2 and Ets-1 (114–116). In addition, the promoter region of the SLC7A11 gene contains an AA response element, which allows the transcription factor ATF4 to enhance expression of xCT in response to AA depletion and/or oxidative stress (115, 117). p System xc-light chain mediates import of cystine into cells thus regulating GSH levels (118, 119). Since GSH is the most abundant non-enzymatic antioxidant within the cell, upregulation of xCT satisfies the highly proliferative phenotype of cancer cells. xc-Transport Systemi This is supported by complete growth inhibition of lymphoma cells and certain glioma, breast, prostate, lung, and pancreatic cancer cells upon pharmacological inhibition of xCT by sulfasalazine or by the cyclic glutamate analog (109, 111). Besides its role in tumor growth, knockdown or pharmacological inhibition of xCT increased adhesion and inhibited tumor cell invasion in vitro and decreased metastases in vivo (120). In addition, xCT was shown to associate with CD44v, a major adhesion molecule for the extracel- lular matrix, which is involved in tumor invasion and metastasis in lethal gastrointestinal tumors (121) along with the metabolic interplay between tumors and host tissue (122). Furthermore, xCT plays a pivotal role in the chemoresistance of tumor cells (123–125), particularly to anticancer drugs that produce high amounts of ROS, such as geldanamycin and celastrol (126, 127). xc-Transport Systemi Multiple tissue-specific transporters are responsible for the import of cystine (CySSCy), the oxidized and predominant form of the AA in circulation (40–50 µM), and/or cysteine, which is present at substantially lower concentrations (8–10 µM) (95–97). However, increasing data in the literature points toward the xc-system as being crucial for CySSCy import in can- cer cells (Figure 2). The system xc- acts as a Na+-independent and Cl−-dependent antiporter of the anionic forms of cystine and glutamate and is composed of the transporter light-chain (xCT, encoded by SLC7A11 gene) and a chaperone heavy-chain (CD98hc aka 4F2hc, encoded by SLC3A2 gene) subunit [for a comprehensive review, see Lewerenz et al. (98)]. Interestingly, although the system xc- seems to be a ubiquitous marker of almost all cells cultured in vitro, its in vivo distribution in humans appears restricted mainly to the CNS, pancreas, fibro- blasts, and immune cells (99–105). According to Bannai et al. As for the effect of mTOR on redox homeostasis, a recent study showed that mTORC1 controls ATF4 activity by regulating the translation and stability of its mRNA (84). These results indicate that mTORC1, besides promoting anabolism and consequently increased ROS production, may also contribute to maintenance of the cellular redox equilibrium through “antioxidant proper- ties” of ATF4. December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 5 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. Figure 2 \| Cystine import is the rate-limiting step in glutathione biosynthesis. Cysteine can be synthesized within the cell through the trans-sulfuration pathway. However, this pathway is often insufficient in cancer cells and therefore cysteine must be imported. Different transporters are involved in the import of the reduced, cysteine (CySH), and oxidized, cystine (CySSCy) form of this semi-essential AA. The heavy-chain transporter subunit of system xc-light chain (xCT) seems to play a pivotal role in the import of CySSCy, the predominant form of cysteine in circulation. After import, CySSCy is reduced by cystine reductase and used for different purposes including GSH biosynthesis. Import of cysteine can occur via ASCT (alanine/serine/cysteine transporter) and other transporters (x). Figure 2 \| Cystine import is the rate-limiting step in glutathione biosynthesis. Cysteine can be synthesized within the cell through the trans-sulfuration pathway. However, this pathway is often insufficient in cancer cells and therefore cysteine must be imported. Frontiers in Oncology \| www.frontiersin.org SERINE/FOLATE PATHWAY AND NADPH PRODUCTION Textbooks have stated for years that the main cellular NADPH- producing system is the PPP. Surprisingly, a recent comprehensive study (134) showed that serine-driven one-carbon metabolism (folate cycle) gives almost the same contribution in the NADPH production as the PPP and MEs in proliferating cells. It is also interesting to note that enzymes of both PPP and the serine synthesis pathway (SSP, from which the folate cycle streams out) are induced by NRF2 (135, 136). The function of the folate cycle is ascribed to the collection of one-carbon units from AAs, and subsequent incorporation of these moieties into biomolecules in biosynthetic or methylation reactions. One of the major December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 6 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. branching points of the folate cycle is 10-formyl-tetrahydrofolate (10-formyl-THF), which in mitochondria may be used for ATP regeneration [methylene tetrahydrofolate dehydrogenase (MTHFD) reaction], formylation of the mitochondrial initiator N-formylmethionine-tRNA or metabolized to [CO2], generat- ing NADPH (10-formyl-THF dehydrogenase reaction). On the other side, in cytosol, 10-formyl-THF can be used for purine or NADPH synthesis, while its counterpart 5,10-methylene-THF is used for thymidylate synthesis and homocysteine remethylation in the methionine cycle. In cancer, mitochondrial 10-formyl-THF is mainly used for NADPH production due to overexpression of corresponding enzyme, while in citosol, this reaction is prevented so one-carbon unit, required for purine synthesis, would not be wasted (137, 138). Default mitochondria-to-cytosol directional- ity of the folate cycle is achived by different expression of enzymes branching points of the folate cycle is 10-formyl-tetrahydrofolate (10-formyl-THF), which in mitochondria may be used for ATP regeneration [methylene tetrahydrofolate dehydrogenase (MTHFD) reaction], formylation of the mitochondrial initiator N-formylmethionine-tRNA or metabolized to [CO2], generat- ing NADPH (10-formyl-THF dehydrogenase reaction). On the other side, in cytosol, 10-formyl-THF can be used for purine or NADPH synthesis, while its counterpart 5,10-methylene-THF is used for thymidylate synthesis and homocysteine remethylation in the methionine cycle. In cancer, mitochondrial 10-formyl-THF is mainly used for NADPH production due to overexpression of corresponding enzyme, while in citosol, this reaction is prevented so one-carbon unit, required for purine synthesis, would not be wasted (137, 138). Default mitochondria-to-cytosol directional- ity of the folate cycle is achived by different expression of enzymes in these compartments, as well as more reductive, i.e., oxidative environment in cytosol and mitochondria respectively (139). SERINE/FOLATE PATHWAY AND NADPH PRODUCTION Two mitochondrial reactions of the folate cycle contribute to NADPH production; one is catalyzed by MTHFD, and the other is catalyzed by 10-formyl-THF dehydrogenase (ALDH1L2) (Figure 3). Fan et al. showed that depletion of either of these enzymes decreased NADPH/NADP+ and consequently GSH/ GSSG ratios and impaired cellular resistance to imposed oxida- tive stress (134). Similarly, Piskounova et al. showed that redox balancing effects of these enzymes is fundamental for metastatic potential of melanoma cells in vivo (28). Namely, this study showed that knockdown of either MTHFD or ALDH1L2 pre- vents distant metastasis of melanoma cells that encounter high- oxidative pressure in the blood and visceral organs. Besides, it was reported that the first mitochondrial enzyme of the folate cycle, Figure 3 \| The folate cycle is fueled by the serine synthesis pathway (SSP) and extracellular serine. SSP diverges from glycolysis at the level of 3-phosphoglycerate, which is converted into 3-phospho-hydroxipyruvate by the action of the enzyme phosphoglycerate dehydrogenase (PHDGH) and ultimately to serine following further enzymatic steps. This pathway is of great importance in cancers with mutated or overexpressed PHDGH, while serine import plays a pivotal role in maintenance of the serine cellular balance in cells with unaltered PHDGH activity. The folate cycle in the vast majority of the cells starts in mitochondria by the action of serine hydroxymethyl transferase 2 (SHMT2) which generates glycine and 5,10-methylene-tetrahydrofolate (5,10-methylene-THF). The next reaction can produce NADH or NADPH depending if methenyltetrahydrofolate dehydrogenase 2 (MTHD2) or MTHD2-like (MTHD2L) is used to convert 5,10-methylene-THF into 5,10-methenyl-THF. The same enzyme than generate one-carbon unit—10-formyl-THF, which can be used for ATP production by the enzyme (MTHD1L) or NADPH generation in the reaction catalyzed by 10-formyTHF dehydrogenase (ALDH1L2). If ATP is generated, 10-formylTHF is converted into a format that is transported into the cytosol and used by trifunctional MTHFD1 enzyme to regenerate 10-formylTHF for purine synthesis, 5,10-methylene-THF for thymidylate synthesis and homocysteine remethylation in the methionine cycle. The unidirectionality of the folate cycle seems to be provided by more oxidative mitochondrial redox state that favors use of NAD(P)+ by mitochondrial MTHD2(L). Figure 3 \| The folate cycle is fueled by the serine synthesis pathway (SSP) and extracellular serine. SERINE/FOLATE PATHWAY AND NADPH PRODUCTION Consequently, serine depletion both in vitro and in vivo decreases proliferation and induces metabolic remodeling, commencing with SSP induction, to replenish cellular serine pool (143). SERINE/FOLATE PATHWAY AND NADPH PRODUCTION SSP diverges from glycolysis at the level of 3 phosphoglycerate which is converted into 3 phospho hydroxipyruvate by the action of the enzyme phosphoglycerate dehydrogenase (PHDGH) and ultimately to Figure 3 \| The folate cycle is fueled by the serine synthesis pathway (SSP) and extracellular serine. SSP diverges from glycolysis at the level of 3-phosphoglycerate, which is converted into 3-phospho-hydroxipyruvate by the action of the enzyme phosphoglycerate dehydrogenase (PHDGH) and ultimately to serine following further enzymatic steps. This pathway is of great importance in cancers with mutated or overexpressed PHDGH, while serine import plays a pivotal role in maintenance of the serine cellular balance in cells with unaltered PHDGH activity. The folate cycle in the vast majority of the cells starts in mitochondria by the action of serine hydroxymethyl transferase 2 (SHMT2) which generates glycine and 5,10-methylene-tetrahydrofolate (5,10-methylene-THF). The next reaction can produce NADH or NADPH depending if methenyltetrahydrofolate dehydrogenase 2 (MTHD2) or MTHD2-like (MTHD2L) is used to convert 5,10-methylene-THF into 5,10-methenyl-THF. The same enzyme than generate one-carbon unit—10-formyl-THF, which can be used for ATP production by the enzyme (MTHD1L) or NADPH generation in the reaction catalyzed by 10-formyTHF dehydrogenase (ALDH1L2). If ATP is generated, 10-formylTHF is converted into a format that is transported into the cytosol and used by trifunctional MTHFD1 enzyme to regenerate 10-formylTHF for purine synthesis, 5,10-methylene-THF for thymidylate synthesis and homocysteine remethylation in the methionine cycle. The unidirectionality of the folate cycle seems to be provided by more oxidative mitochondrial redox state that favors use of NAD(P)+ by mitochondrial MTHD2(L). December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org Frontiers in Oncology \| www.frontiersin.org AAs in Cancer Redox Homeostasis Vucˇetic′ et al. termed serine hydroxymethyl transferase 2 (SHMT2) is essential for maintaining mitochondrial NADPH and GSH level during hypoxia in neuroblastoma cell lines. This study detected a cor- relation between high expression of SHMT2 and poor prognosis in neuroblastoma patients (140). Expression of SHMT2 in neuro- blastoma cells seems to be controlled by the collaborative action of c-Myc and HIF1α. However, numerous oncogenes are reported to affect enzymes of the folate cycle. For example, it is shown that common KRAS mutation associates with increased expression of MTHFD2 in non-small cell lung cancer cell lines (141), while mTORC1-dependent induction of MTHFD2 is reported in both normal and cancer cells (142). Serine Synthesis Pathwayh y y The importance of serine for cancer physiology came from earlier studies that showed increased flux through the SSP in cancer cells (148). However, this was somewhere neglected until the recent discovery that the first enzyme of SSP, phos- phoglycerate dehydrogenase (PHGDH), is genetically amplified in breast cancer and melanoma (149, 150), and overexpression of the SSP components are correlated with poorer prognosis in breast cancer patients (151). Consistently, suppression of PHGDH in cell lines characterized with elevated expression of this enzyme decreases cell proliferation and serine synthesis. What is even more interesting is that in non-tumorigenic breast cancer cells, overexpression of PHGDH alone lead to disrup- tion of the acinar cellular morphology and predisposed them to neoplastic transformation (149, 152), making the PHGDH a bona fide oncogene (153).i Besides production of NADPH, the folate cycle contributes to production of GSH by intersecting with the methionine cycle (Figure 4). Considering the role of methionine and homocyst- eine in the TSP (cysteine synthesis), as well as that glycine is product of serine metabolism (folate cycle), it is not surprising that serine depletion results in reduced level of glutathione (143), while activation of serine synthesis is now well identified as a bypass of glycolysis flux contributing to GSH synthesis (136, 144). Serine, just like cysteine, can be transported into the cell by different transporters [such as the sodium-dependent transport system ASC that will be mentioned later in the text, and transporter system A, as well as sodium-independent system asc (145, 146)], or synthesized de novo from glycolytic intermediate 3-phosphoglycerate through the SSP. Highly proliferating cancer cells both in culture conditions and in vivo consume significant amount of exogenous serine (143, 147). Amplification of PHGDH de-sensitizes tumors to exogenous serine levels but also represents a vulnerability point for poten- tial cancer treatment. Namely, PHGDH knockdown strongly decreased proliferation and some of the SSP outputs [such as α-ketoglutarate (α-KG)] only in cells with amplified PHGDH expression (150). Interestingly, PHGDH also prevents conver- sion of glycine to serine suggesting that the folate cycle relies exclusively on serine synthesis in PHGDH overexpressing Figure 4 \| Crossroads of NADPH-producing pathways (marked dark blue) and the pathways from which they diverge or to which they converge (marked light blue). Amino acids involved in these pathways are marked in red. GLUTAMATE AND NADPH PRODUCTION tumors (154). This was demonstrated by PHGDH knockdown decreasing cell proliferation even when exogenous serine was present (154). In addition to the PPP and folate cycle, MEs are known to regu- late NADPH/NADP+ balance, which is seemingly dependent of glutamine metabolism in cancer. One of the main metabolic characteristics of many cancers, besides the Warburg effect (158, 159), is increased consumption of glutamine to the extent where exogenous level of this AA limit tumor cell survival. This “glutamine addiction” has been recognized for more than 50 years (160, 161); however, diverse contributions of glutamine to intermediary metabolism, cell signaling, and gene expression are still not fully understood (162).h Several other oncogenes also induce expression of the SSP enzymes, such as c-Myc and HER2 (155, 156). Also, in line with its involvement in maintaining redox balance, the SSP enzyme expression is induced by NRF2 in an ATF4-dependent manner in NSCLC cells (136). Interestingly, Maddocks and coworkers (143) showed that serine can be a vulnerable point of cancer metabolism even in tumors that do not have multiplication of the PHGDH gene, but lack p53. Namely, they showed that the p53–p21 axis is fundamental for metabolic adaptation upon serine deprivation, while loss of p53 in the conditions of serine depletion leads to impaired glycolysis and elevated ROS levels. The vast majority of glutamine in the cell is converted into glutamate either by cytoplasmic glutaminase (GLS1) or by the mitochondrial isoform of this enzyme (GLS2). Glutamate is then converted to α-KG by the enzyme glutamate dehydrogenase. α- KG can then have one of two fates (Figure 5). (1) Canonically, produced α-KG enters the TCA and replenishes it, or (2) it is carboxylated to isocitrate, pushing the TCA in the opposite direc- tion (163). Interestingly, pharmacological inhibition of the SSP could also influence flux through the PPP. Namely, inhibition of the SSP would increase intracellular levels of 3-phosphoglycer- ate, which has been shown to inhibit 6-phosphogluconate dehydrogenase that catalyzes the second step in the oxidative PPP (157). Figure 5 \| Glutamine/glutamate fates in cancer cells. Different transporters are proposed to fuel the “Glutamine addiction” of cancer cells including alanine-serine- cysteine transporter 2 (ASCT2), SNAT1/2, and L-type amino acid transporter 1 (LAT1). Once inside the cell, Gln can be use for uptake of essential AAs by LAT1. Serine Synthesis Pathwayh Figure 4 \| Crossroads of NADPH-producing pathways (marked dark blue) and the pathways from which they diverge or to which they converge (marked light blue). Amino acids involved in these pathways are marked in red. PH-producing pathways (marked dark blue) and the pathways from which they diverge or to which they converge (marked light ese pathways are marked in red. December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org Frontiers in Oncology \| www.frontiersin.org AAs in Cancer Redox Homeostasis Vucˇetic′ et al. GLUTAMATE AND NADPH PRODUCTION However, the vast majority of Gln is promtly deaminated to glutamate by the action of cytoplasmic or mitochondrial glutaminase (GLS1 and GLS2, respectively). If deaminated in cytosol, Glu is transferred into mitochondria, and there it is further converted into α-ketoglutarate (α-KG) to replenish the tricarboxylic acid (TCA). However, the fate of α-KG can be dual. It can follow normal TCA flow until oxaloacetate (OAA), which is then converted into asparate by aspartate dehydrogenase (GOT2) and translocated into cytoplasm or used for synthesis of asparagine and arginine (protein synthesis). However, if the α-KG is carboxylated to isocitrate and then converted into citrate, citrate is exported into the cytosol where it is used for lipid synthesis in the form of acetyl-CoA. Glutamate-derived aspartate can also be converted into OAA by cytoplasmic GOT1, commonly induced in KRAS-mutated tumors. OAA is then converted first into malate by malate dehydrogenase 1 (MDH1) and then into pyruvate by malic enzyme (ME), generating reducing power in the form of NADPH. Besides involvement in anaplerosis and NADPH production, Glu has an important role as a component of GSH, as well as a substrate for system xc-light chain (xCT) in allowing entrance of cystine into the cell. Figure 5 \| Glutamine/glutamate fates in cancer cells. Different transporters are proposed to fuel the “Glutamine addiction” of cancer cells including alanine-serine- cysteine transporter 2 (ASCT2), SNAT1/2, and L-type amino acid transporter 1 (LAT1). Once inside the cell, Gln can be use for uptake of essential AAs by LAT1. However, the vast majority of Gln is promtly deaminated to glutamate by the action of cytoplasmic or mitochondrial glutaminase (GLS1 and GLS2, respectively). If deaminated in cytosol, Glu is transferred into mitochondria, and there it is further converted into α-ketoglutarate (α-KG) to replenish the tricarboxylic acid (TCA). However, the fate of α-KG can be dual. It can follow normal TCA flow until oxaloacetate (OAA), which is then converted into asparate by aspartate dehydrogenase (GOT2) and translocated into cytoplasm or used for synthesis of asparagine and arginine (protein synthesis). However, if the α-KG is carboxylated to isocitrate and then converted into citrate, citrate is exported into the cytosol where it is used for lipid synthesis in the form of acetyl-CoA. Glutamate-derived aspartate can also be converted into OAA by cytoplasmic GOT1, commonly induced in KRAS-mutated tumors. GLUTAMATE AND NADPH PRODUCTION OAA is then converted first into malate by malate dehydrogenase 1 (MDH1) and then into pyruvate by malic enzyme (ME), generating reducing power in the form of NADPH. Besides involvement in anaplerosis and NADPH production, Glu has an important role as a component of GSH, as well as a substrate for system xc-light chain (xCT) in allowing entrance of cystine into the cell. December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 9 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. When glutamine-derived α-KG follows the canonical path- way, the TCA works normally (clockwise) until oxaloacetate (OAA), which is usually converted into aspartate by aspartate transaminase (GOT2) and exported into the cytosol, or alterna- tively, it can be converted into asparagine and arginine and fuel protein synthesis. Interestingly, a recent study on KRAS-mutated pancreatic ductal adenocarcinoma (PDAC) showed that GOT2 regulates glutamine flux by producing α-KG and aspartate from glutamate and OAA (164). Aspartate is then shuttled into the cytosol where it is converted back into OAA by cytoplasmic GOT1. The OAA produced is converted first to malate and then to pyruvate and NADPH by the action of cytoplasmic malic enzyme 1 (ME1). Considering that KRAS-mutated PDACs have decreased flux through the PPP (165), glutamine-fueled ME1 in these cells may be seen as a major contributor to the NADPH homeostasis. Indeed, ME1 suppression increased ROS accumulation and decreased tumor cell growth both in vitro and in vivo, while suppressing glutamine utilization and sensitizing cells to oxidative damage (164). Conversely, it remains to be determined if inhibitors of glutamine import or its conversion to glutamate would have the same effects on oxidative status and cell growth. cancer cell glutamine addiction might be a direct consequence of xCT activity, which consumes large amounts of glutamate derived from extracellular glutamine thereby restricting nutrient flexibility of the cell (175, 176).ht l The importance of glutamine in cancer cells often dominates ASCT2 experimental interpretations. However, it is important to remember ASCT2’s ability to transport other AAs such as serine. As mentioned, some cancer cells remain highly dependent on the uptake of exogenous serine (143). Since ASCT2 display a strong affinity for serine, it would be interesting to investigate the role of this transporter in serine metabolism and redox homeostasis in general. Furthermore, the name of ASCT2: alanine–serine– cysteine transporter may be misleading. Alanine-Serine-Cysteine Transporter 2 (ASCT2) ( ) Alanine-serine-cysteine transporter 2 (SLC1A5) is a Na+-depen dent transporter carrying small neutral AAs such as alanine, serine, cysteine, glutamine, and asparagine (Km ~20 μM) in addition to long-chain AAs such as threonine, valine, and methionine with lower affinity (Km ~300–500 μM). ASCT2 is proposed to play a central role in sustaining cancer cell glutamine homeostasis based on work from Myc-driven cancers, which are particularly addicted to glutamine, and fuel their “glutamine addiction” by promoting high ASCT2 expression (167–169). Also, ASCT2 together with xCT and L-type amino acid transporter 1 (LAT1), comprise the “minimal set” of transporters required for cancer AA homeostasis and the group known to be highly upregulated in cancer (170, 171). Consequently the glutamine import activity of ASCT2 has been proposed to be fundamental for the activity of other AA transporters upregulated in cancer, such as xCT and LAT1 (leucine-for-glutamine exchanger) (171–173). However, recent findings demonstrated that ASCT2 inhibition can be overcame in certain cancer cell types partly by expressing the Na+-dependent glutamine transporters system A amino acid transporter 1–2, questioning the functional redundancy for certain AA transport- ers in tumor growth (174). Regardless, glutamine import (via ASCT2 or other transporters) is indeed of great importance for normal functioning of LAT1 and xCT. Recent studies showed that GLUTAMATE AND NADPH PRODUCTION Namely, ASCT2 is structurally related to the glutamate transporter and neutral AA transporter ASCT1 and when expressed in Xenopus laevis oocyte ASCT2 indeed exhibits Na+-dependent uptake of AA similar to ASCT1 (177). However, the same study of Utsunomiya-Tate and collaborators revealed that ASCT2 exhibits different tissue distri- bution, as well as substrate selectivity and functional properties when compared to ASCT1. Thus, for example, glutamate uptake by ASCT1 is electrogenic, while in the case of ASCT2 lowering pH enhances uptake, which suggests electroneutral uptake. Also, it seems that cysteine is not a substrate for ASCT2, but an allosteric inhibitor of its activity. 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YC, SP, and JP were funded by the Centre Scientifique de Monaco (CSM) and a Grant from GEMLUC. MV and JP made substantial contributions to conception and design, revised manuscript critically, and gave final approval CONCLUDING REMARKS For a long time, the mild pro-oxidative redox state of cancer cells has been recognized as a vulnerable point of these highly metabolically active cells. However, in the context of chemo- therapy, we are still struggling to find the adequate approach to the vast majority of ROS-producing therapeutics that encounter cellular resistance and frequent disease relapse. During the past decade, an approach involving suppression of the internal AOD of cancer has attracted more attention. Within highly complex and intertwined AOD system, GSH and NADPH play the most universal and important role in determining the characteristic redox cellular profile. Considering that AA import and metabo- lism seems to be upstream of these AOD systems, we have emphasized here the specific molecules and pathways that show great, but still insufficiently examined, potential for anticancer therapy from a redox standpoint. In conclusion, the transport and internal synthesis pathways for cysteine, serine, glutamine, and to some extent glycine appear to be the most interesting targets for the development of novel redox-based therapeutics. Targeting AA transport systems (xCT, ASCT2, and SNAT) is promising considering that import of these semi-essential December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 10 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. of the version to be submitted. YC and SP revised manuscript critically and gave final approval of the version to be submitted. AAs are not required in normal cells, while they are absolutely required for cancer cell survival. 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Proc Natl Acad Sci U S A (2008) 105(48):18782–7. doi:10.1073/pnas.0810199105 December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 14 AAs in Cancer Redox Homeostasis Vucˇetic′ et al. 168. Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, et al. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature (2009) 458(7239):762–5. doi:10.1038/ nature07823 175. Muir A, Danai LV, Gui DY, Waingarten CY, Lewis CA, Vander Heiden MG. Environmental cystine drives glutamine anaplerosis and sensitizes can- cer cells to glutaminase inhibition. Elife (2017) 6:e27713. doi:10.7554/ eLife.27713 169. Ren P, Yue M, Xiao D, Xiu R, Gan L, Liu H, et al. ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 activation. J Pathol (2015) 235(1):90–100. doi:10.1002/path.4429 176. Shin CS, Mishra P, Watrous JD, Carelli V, D’Aurelio M, Jain M, et al. 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Amino acid transporters in cancer and their relevance to “glutamine addiction”: novel targets for the design of a new class of anticancer drugs. Cancer Res (2015) 75(9):1782–8. doi:10.1158/0008-5472.CAN-14-3745 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 172. Banjac A, Perisic T, Sato H, Seiler A, Bannai S, Weiss N, et al. REFERENCES The cystine/ cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death. Oncogene (2008) 27(11):1618–28. doi:10.1038/sj.onc.1210796 173. Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell (2009) 136(3):521–34. doi:10.1016/j.cell.2008.11.044 Copyright © 2017 Vučetić, Cormerais, Parks and Pouysségur. 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. 174. Broer A, Rahimi F, Broer S. Deletion of amino acid transporter ASCT2 (SLC1A5) reveals an essential role for transporters SNAT1 (SLC38A1) and SNAT2 (SLC38A2) to sustain glutaminolysis in cancer cells. J Biol Chem (2016) 291(25):13194–205. doi:10.1074/jbc.M115.700534 December 2017 \| Volume 7 \| Article 319 Frontiers in Oncology \| www.frontiersin.org 15
https://openalex.org/W3120192807	https://zenodo.org/record/1567515/files/article.pdf	English	null	Automatic Ore Unloader	Scientific American	1,901	public-domain	4,009	AUTOMATIC ORE UN LOADER. BY W. FRANK M'CLURE. Three great automatic iron ore unloaders, the first of their kind in the world, will be in operation upon the docks of the Carnegie Company at Conneaut, Ohio, The total weight of the first machine was found to be 400 tons and its height 55 feet. The all-important part of the machine is the bucket, which grasps ten tons at a single lift, or ten times that lifted by the largest ore bucket previously used. This great bucket is at tached to a revolving pending leg, which in turn swings from a long and gigantic arm. This arm is carried forward and back upon a track, to a point above the vessel when the bucket is to be filled and to a point above the car when the bucket is to be dumped. THE SCOOP GATHERING UP ITS LOAD. The bucket is first lowered part way into the vessel's hold. Next the scoop is opened and then lowered until it strikes the ore cargo and sinks deep into it. When open, the bucket has a spread of nineteen feet. The scoop is closed upon the ore by hydraulic power. It is then ready to be raised and conveyed to a point over the car into which the ore is to be dropped. Where the ore is to be placed on the stock piles, it is dropped into a trolley car which will convey it. The second coal region, the Upper Silesian, is even larger. Here the carboniferous mountains reach a depth of 7,000 meters, on an area of 2,162 square miles, and the 114 workable layers have an averag.?, thickness of 170 meters. When calculating the quan tity of coal workable to a depth of 1,000 meters, it must be taken into account that the carboniferous mountains generally are covered by younger layers about 200 meters in thickness, and that, when carrying on operations to a depth of 1,000 meters, about 33 1-3 per cent has to be deducted for safety constructions, loss, etc. There would thus remain, according to Prof. Schulz's estimates, a quantity of 62,800,000,000 tons of workable coal down to 1,000 meters. During the period from 1748 to 1900 there have been extracted 500,000,000 tons, thus leaving 62,300,000,000 tons to be mined. According to the Upper Silesian mining re turns, the increase in the output from decade to decade has been 43.5 per cent. AUTOMATIC ORE UN LOADER. BY W. FRANK M'CLURE. In 1899, when the deepest shaft was but 594 meters, the output amounted to 23,500,000 tons. Supposing that it reaches within 50 years three times the present output, the deposits down to 1,000 meters would last for 890 years, and would probably not be exhausted until the year 2790. At a depth of 1,000 to 1,500 meters there are further . available 101,550,000,000 tons, and from 1,500 to 2,000 meters another _ 140,800,000,000 tons, the mining of which would require 1,450 and 2,000 years respectively. But, even at that time, there would yet be immense quantities of coal available, as huge deposits extend over the mountains deeper than 2,000 meters. ---.<---- The automatic ore un loader is expected to take out from 90 to 95 per cent of the ore in a vessel. The bucket when below the hatch of a vessel can be swung around lengthwise, in which case it reaches about nine feet from the edge of the hatch in either direction. The small amount of ore which cannot be reached by the scoop is shoveled by hand to a point within its grasp, In the accompanying photograph showing three ma chines, the big scoop or bucket of the machine in the distance is below the hatch of the vessel, reaching into the ore. The scoop of the next machine is raised to a point above the vessel, and the view nearest the reader shows the scoop after it has been conveyed and the contents dumped into a railroad car. Four machines, side by side, can be used in the a ver age vessel at one time. Each machine is expected to remove 300 tons of ore per hour when fully perfected. Four machines, thus unloading 1,200 tons per hour, would empty the largest vessel on the lakes in a com paratively short time. The largest cargo of ore hauled last year on the Great Lakes aggregated 7,446 gross tons. If such rapid handling of iron ore can be se cured, the work of many men will be saved. One of the ore unloaders can be operated by six men. Three of this number remain in the vessel to shovel ore within the reach of the scoop. Three operate the machine. With four machines working in a vessel twenty-four men would thus do the work which usually requires 100 men. THE SCOOP GATHERING UP ITS LOAD. ---.<---- How the Welsbach Mantle is Made. ---.<---- How the Welsbach Mantle is Made. The "mantle" of the Welsbach light is an ash con sisting mainly of the oxides of certain rare metals- lanthanum, yttrium, zirconium, etc., which are rendered incandes cent by heating to a high temperature. A six·cord cotton thread is woven on a knitting machine into a tube of k n i t t e d fabric of a rather open mesh. This web has the grease and dirt thoroughly washed out of it, is dried and is cut into l e n g t h s double that required for a single mantle. It is then saturated in a so lution containing the requisite oxides, wrung out, s t r e t c h e d over spools and dried. Next, the double·length pieces are cut into two, the top of each piece is doubled back and sewed with a platinum wire, which draws the top in and provides a means of supporting the man tle, when finished, from the wire holder. After stretching the mantle over a form, smooth ing it down and fasten ing the platinum wire to the wire mantle holder, the mantle is burned out by touching a Bunsen burner to the top. The cotton burns l'OWllUUL AUrOMAr ---.<- How the Welsbach Ma The "mantle" of the Welsbach sisting mainly of the oxides of lanthanum, yttrium, zirconium, etc., which are rendered incandes cent by heating to a high temperature. A six·cord cotton thread is woven on a knitting machine into a tube of k n i t t e d fabric of a rather open mesh. This web has the grease and dirt thoroughly washed out of it, is dried and is cut into l e n g t h s double that required for a single mantle. It is then saturated in a so lution containing the requisite oxides, wrung out, s t r e t c h e d over spools and dried. Next, the double·length pieces are cut into two, the top of each piece is doubled back and sewed with a platinum wire, which draws the top in and provides a means of supporting the man tle, when finished, from the wire holder. ---.<---- How the Welsbach Mantle is Made. After stretching the mantle over a form, smooth ing it down and fasten ing the platinum wire to the wire mantle holder, the mantle is burned out by touching a Bunsen burner to the top. The cotton burns How the Welsbach Mantle is Made. The "mantle" of the Welsbach light is an ash con sisting mainly of the oxides of certain rare metals- lanthanum, yttrium, zirconium, etc., which are rendered incandes cent by heating to a high temperature. A six·cord cotton thread is woven on a knitting machine into a tube of k n i t t e d fabric of a rather open mesh. This web has the grease and dirt thoroughly washed As yet no attempts have been made to establish speed records. Otherwise the tests are reportEd to be very e n c o u r a g i n g. George H. Hulett, a me chanical engineer 0 f Akron, Ohio, is the in ventor. On completing the first machine some time ago it l'OWllUUL AUrOMArIC ORE UNLOADER IN USE, SHOWHiG DIFFERENt l'OSIrIONS OF THE MAIN ARltt AND BUCUr. AUTOMATIC ORE UN LOADER. BY W. FRANK M'CLURE. Harbor the coming season. The complete success of these machines will mean their general adoption along the Great Lakes, and, incidentally, the realization of the fondest hopes of many of the big dock companies. Their use at all the ports will revolutionize the ore handling industry. For years pessimists have prophesied that a success ful automatic iron ore unloader was an impossibility. Futile attempts to build such a machine have been made from time to time in the pasȰ decade. The an nouncement, therefore, that Andrew Carnegie was to build an automatic ore unloader at a cost of $100,000 occasioned no little interest. MARCH 9, 1901. 153 was found necessary to rebuild it. Additional bear ings in particular were found to be needed. Each test of the machine has been more satisfactory than the former one, and when last year the Carnegie Company ordered two more machines of the same pattern com pleted for this season's business, at a cost of $100,- 000 each, it was apparent that the steel king felt sure of their success. The three machines have now been completed. The accompanying photograph shows them, side by side, each in operation but in different positions. off slowly, leaving a skeleton mantle of metallic oxides, which preserves the exact shape and detail of every cotton fiber. The soft oxides are then hardened in a Bunsen flame. A stronger mantle is made upon lace making machinery.-The Keystone. . Germany's Two Great Coal Regions. BY H. L G EISSrL. Prof. Schulz, German Privy Councilor of Mines, and a recognized authority on European mining matters, has just given out some interesting figures on the coal deposits of the two great German mining dis tricts. Prof. Schulz says that the Rhenish West phalian mining region extends over an area of 60 square miles. To a depth of 700 meters there are yet available OOld exploitable 11,000,000,000 tons of bitu minous coal; from that depth to 1,000 meters there remain 18,300,000,000 tons; and in the depth between 1,{\00 and 1,500 meters, well accessible under present mining conditions, there are another 25,000,000,000 tons. or, altogether to the last-named depth of 1,500 meters, 54,300,000,000 tons. Prof. Schulz expresses the opinion that science wUI improve our present means to such a degree that in time it will be possible to safely carry on operations at a greater depth than 1,500 meters, whereby another 75,000,000,000 tons of coal would be obtainable. Thus, the total quantity of coal still buried in the Rhenish Westphalian dis trict amounts to 129,300,000,000 tons. Supposing the future annual output of the district should average 100,000,000 tons-that is to say, about twice the pres ent output--the coal deposits available down to a depth of 1,000 meters would still last for 293 years, and to 1,500 meters for 543 years. A VALVE MOTION F)R SMALL HIGH-PRESSURE PUMPS. The use of high pressures with pumps of small di mensions presents difficulties, as the valves are liable to be kept open by the pressure. To overcome this drawback, Adolph Richter, 1138 First Avenue, Manhat tan, New York city, employs a special device for press ing the valves firmly against their seats during the time they should remain stationary, the valves being released shortly before they are to be shifted. A NOVEL LIVE·BAIT FAlL. feeding sufficient oxygen to enable the. fish to live not only for hours, but for clays, has been very ingeniously overcome in an invention for which Mr. Cassius M. Fisk, of Napoleon, Ohio. has taken out a patent. The valves are turned by means of the levers shown at the end of the elevation, each lever being connected with one suction valve and one delivery valve by links and crank arms. These levers are struck periodically by an arm on the front end of a shaft which is jour naled in a slide moving together with the pump piston and provided with a pinion rolling on a stationary rack. This same shaft carries at its rear end another THE MONTGOMERY COPY-HOLDER. Mr. Fisk's invention is a pail which is provided with an air-chamber in its bottom and with a hand pump secured to the side. The lower end of the pump cylinder communicates with the air-chamber by means of a pipe; and the air-chamber communicates with the body of the pail by means of a flexible pipe. The pail having been filled with water and the minnows placed therein, the hand-pump is operated to fill the air·chamber with compressed air. Such is the pres sure that the air is spontaneously supplied to the fish in the water through the flexible pipe. It is ne cessary to pump fresh air into the chamber only at very long intervals; for the construction of the flexible pipe is such that the air is very gradually discharged. ries parallel supporting-rods. On these rods the book holder is slidably mounted. In order to hold the book open (particularly a thick book), two spreaders are employed, one for the bottom, one for the top, of the page. One spreader is mounted on the book-holder, and the other on the parallel supporting-rods abov€ the holder. A NEW BOOK OR COPY HOLDER FOR TYPEWRITERS. inal properties with farm animals; that the oil of the sunflower seed is not produced commercially in the United States; and that in the cultivation of the sun flower the methods pursued for growing Indian corn are to be followed, and the plant is capable of cultiva tion over almost as wide an area as Indian corn. A NEW BOOK OR COPY HOLDER FOR TYPEWRITERS. The dithculty of hOlding books, loose sheets, or docu ments used by public speakers, copyists, and typewrit ers is overcome by means of a holder recently patented by Burgess T. Montgomery, of 752 Ninth Street, Wash ington, S. E., D. C. vary from zero to 8.6 per cent. It is not the propor tion of ammoniacal salts in the solution, but th·ȱ amount of free ammonia which determines the pro portion. The superior limit is reached when the liquor contains 350 per 1,000 of ammonia (20 per cent strength) or 69 parts by which of NH". The salt ob tained under these conditions is a tri-ammoniac ar· seniate of cobalt, having the formula (As 0,), Co, + 3 NH3 + 5 H, 0, which approaches the zinc salts ob- tained bv M. Bette. On the other hand , the action of this salt upon the salts of cobalt in presence of ammonium salts (without free 8.mmonia) gives a pale rose salt, crystallized in fine needles, in cotton-like clusters. This salt contains no ammonium, and its compositioa (As 0,), Co" + "H, 0, is that of natural erythrine; the crystalline form and grouping are the same. M. Du cru has isolated two of these salts which lie between erythrine and the first mentioned compound; the mono-ammoniac salt (As 0,), Co" + NH" + 7H,O iil formed when the solution contains 15 per 1,000 of free ammonia, while the di-ammoniac salt (As 0,), Co" +- 2NH" + 6H,o is obtained at a concentration of 60 pel' 1,000. The four salts thus obtained appear to be dis tinct compounds and not mixtures. The experimentȲr intends to describe a similar series of nickel salts anrl also a new method of analysis for arsenic which is based upon these experiments . The device comprises a rotary pedestal which car- .. ĺ .... ---+ . ... .. - - The Sunflower. Dr. Harvey W. Wiley, Chief of the Division of Chemistry, U n i t e d States Department of Agriculture, in a spe cial report shows that the sunflower can be grown successfully over large areas in t h e United States; that it is a crop which makes a considerable drain on the elements of soil fer tilizers; that one of the most valuable cOIC.stitu ents of the plant is the oil, which exists in large quantities in the seeds; that the eco nomic production of the sunflowers is now COll fined almost exclusively to Russia, where it is a n agricultural indus try of considerable im portance; that in the United States i t is grown as an ornament and for the production of seeds, which are used chiefly for poultry and bird feeding and for condimental and medic- l'OWllUUL AUrOMArIC ORE UNLOADER IN USE, SHOWHiG DIFFERENt l'OSIrIONS OF THE MAIN ARltt AND BUCUr. © 1901 SCIENTIFIC AMERICAN, INC. © 1901 SCIENTIFIC AMERICAN, INC. The Current Supplement. The current SrpPLE1\1E:'iiT, No. 1314, is commenced by a most'interesting article on the maple sugar industry, accompanied by engravings showing the tapping of trees and the boiling of sirup. "Dock Equipment for the Rapid Handling of Coal and Ore on the Great Amer icr.n Lakes" is the continuation of an important article. "Some Links Between Natural History and Medicine" is by J. Arthur Thomson. "Progress of Agriculture in the United States" is by George K. Holmes. "A Model System of Water Works" is by F. 0. Jones, and is ac companied by working drawings. The usual trade sug gestions from the United States Consuls and Trade Notes and Receipts are published. PERSPECTIVE VIEW OF A ROUGH MODEL OF THE VALVE GEAR. been turned they are pressed against their seats tight ly, thus allowing high pressures to be obtained without danger of leakage. been turned they are pressed against their seats tight ly, thus allowing high pressures to be obtained without danger of leakage. A VALVE MOTION F)R SMALL HIGH-PRESSURE PUMPS. The essential feature of each spreader is an axial rod passing through a bearing in a line at right angles to the plane of the book-holder, the rod having two bearing-arms offset to the same side of the axial center, so that when rotated about the center the arms will both pass off the book to allow the page to be turned. The axial rod of each spreader is longitu dinally adjustable to accommodate books of different thicknesses. SIDE ELEVATION OF THE VALVE-GEAR. The inventor assures us that he has subjected his device to most severe tests. Forty fish, among them minnows so delicate that they cannot ordinarily be kept alive for more than ten hours, were placed in the pail and supplied with air in the manner described. So efficient was lhe apparatus that after twelve days the fish were all alive, although the water had not been changed during the interval and had b<lcome very foul. "Vhen the supply of air was at that time cut off, the fish came gasping for oxygen to the surface. The same minnows could not be kept alive in the same amount of fresh water for mone) than fourteen or fifteen hours. SIDE ELEVATION OF THE VALVE-GEAR. Pivoted leaves or wings at the bottom of the holder form extensionG for books of various sizes and thick nesses. arm which at the end of each stroke operates a set of toggle levers connected with spring arms exerting an axial pressure on the valves when the toggle levers are in line with each other. The parts are so timed in operation that the axial pressure on the valves is relieved before they are turned, and after they have Another feature of the invention which deserves to be mentioned is a line-spacer or indicator pivotally mounted on a side-rod between two friction-clamps. From the two parallel supporting-rods extending up wardly from the base an arm extends outwardly, which carries an electric incandescent lamp, so that the copy can be illuminated, if it be so desired. PERSPECTIVE VIEW OF A ROUGH MODEL OF THE VALVE GEAR. The holder has every motion that can be demanded by the copyist or reader, and is provided with means for receiving all kinds of copy, thick or thin, long or short. • • • Ne'v ('ollli)UlIlld!lli oC Coba1t. M. Ducru has recently presented to the Academie des Sciences the results :.>f a 3ericls of experiments in which he has formed s<'veral new compounds of cobalt. If til a solution of cobalt containing ammoniacal salts and a sufficient proportion of free ammonia is added arsenic acid or a soluble arseniate, a gelatinous precipitate, very voluminous, is seen to form, its color being mOi'e or less violet. At the ordinary temperature these prf' cipitates are not modified, but if the flask containing the liquid and precipitate is kept near the boiling poim in a water bath, the precipitate is slowly modified; it contracts and is transformed into another of a rath8r dark red which microscopic examination shows to be entirely crystallized. The duration of the experiment is variable; with concentrated solutions in the proper proportion it may take but a few minutes, but with weak solutions as long as 100 to 150 hours are re- quired. The crystalline compounds thus ob- tained are cobaltous salts; under the Illl- croscope they are seen as needles groupel in clusters, and sometimes in rhomboidal plates. They ha.ve a marked action upon polarizerl light, and belong to the clinorhombic system. They are insoluble in water and weak ammoniacal solu tions, but easily soluble in the mineral acids. At the ordinary temperature they lose ammonia, but very slowly. These compounds, which M. Ducru has thus obtained for the first time, are ammoniacal arseniates of cobalt, but their composition varies. While the prQ portion of cobalt and arsenic is practicallv the same for all these products, that of the ammonium may A PAIL FOR LIVE BAIT, Every fisherman knows how difficult it is to keep minnows alive, If the fish are kept in a pail, the water must be constantly changed to furnish a new supply of oxygen, The difficulty thus presented of THE MONTGOMERY COPY-HOLDER. A NOVEL LIVE BAIT FAlL 'rhe Bre,,6a Prize. The Academie Royale des Sciences de Turin an nounces that a Prix Bressa of 9,600 francs ($1,920) is open to competition among investigators and inventors of all nationalities. The prize will be awarded to the person who, in the opinion of the Academy, made the most brilliant or useful discovery in the four years 1897-1900, or who produced the most celebrated work in pure or applied science. Works intended for con sideration in connection with the prize must be sent to the president of the Academy before the end of next year. The contest will close December 31, 1902. The right is reserved to award the prize to an investigator whose work is considered to be the most worthy of the honor, even though he does not submit an account of it. Content!!'. (Illustrated anjclp.Ļ are marked with an R..st.erisk.l Automohile. ga solille* .... ...... 148 Automobile news ............... , 1[J) Bait. pHil for live.... . 1[)4 Book hold er. ....... ... .. 1;)4 Bre]^a prize.... . .. lfi4 California treei"i and pJanti"i . Hi} Coal regiolls, Germany's... . If}) Cobalt. enmpnullGs of .......... . 1.14 Notes and queries.......... . 155 Ore, unloader* .. ... .. . _ . . ... . . ... .. ]53 Patent decii"iion ..... ...... . ... .... . ]46 Patent w ith a bistory. . ...... ]47 Phonograph records, permanent 1<11 Pollok prize... .. 141 San Gimig-IlflJlo.. .. ............ 14R _cience notes. . .... 1.11 IIJlectrical Hotes... . .. 1fl} Rhl'imps. elIlLII`e] of collJl' in ..... ]49 }1Nllgilleerillg Ilotes .. . IfJl Stal'. IlPW 111 Perseus ...... . ....... Hfj FiKhillg with steam }Jump ... . ... lr.O nJlJllement. ('urrent . ...... . . ... ]54 Ingenllity. trium ph of.... In Telegraphy. Slaby, i"iystem ....... . ]46 InventIOns, index of 156 TeleOraphy, Slahy. wire]es ... 152 Malaria, transmission of.. .. .. 14R 'l'ypesetting machine. Paige.145, 150 Mantle, Welsbach... . 15;-; Valve motion for pnmps . ....... 165 Content!!'. (Illustrated anjclp.Ļ are marked with an R..st.erisk.l Content!!'. © 1901 SCIENTIFIC AMERICAN, INC.
https://openalex.org/W1862440859	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0141909&type=printable	English	null	Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics	PloS one	2,015	cc-by	6,411	OPEN ACCESS Citation: Li L-b, Leung DYM, Goleva E (2015) Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics. PLoS ONE 10(10): e0141909. doi:10.1371/journal.pone.0141909 Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES Copyright: © 2015 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by National Institutes of Health grants AI070140 (DL), HL036577 (DL) and National Institutes of Health/National Center for Advancing Translational Sciences Colorado Clinical and Translational Institute grant UL1 TR001082. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics Ling-bo Li1, Donald Y. M. Leung1,2, Elena Goleva1* g , g , 1 Department of Pediatrics, National Jewish Health, Denver, Colorado, United States of America, 2 Department of Pediatrics, University of Colorado Denver, Aurora, Colorado, United States of America * GolevaE@njhealth.org Abstract OPEN ACCESS Citation: Li L-b, Leung DYM, Goleva E (2015) Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics. PLoS ONE 10(10): e0141909. doi:10.1371/journal.pone.0141909 Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES Received: August 28, 2015 Accepted: October 14, 2015 Published: October 30, 2015 Copyright: © 2015 Li 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 Steroid resistance is a significant problem in management of chronic inflammatory dis- eases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% pre- dicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was col- lected from patients prior to oral prednisone administration. Phosphorylated mitogen acti- vated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexametha- sone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14+ mono- cytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no differ- ence was found in phosphorylation of ERK or JNK in CD14+ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid resistant asthmatics, suggesting that p38 and MSK1 phosphorylation can serve as blood biomarkers of steroid resistance. OPEN ACCESS Citation: Li L-b, Leung DYM, Goleva E (2015) Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics. PLoS ONE 10(10): e0141909. doi:10.1371/journal.pone.0141909 Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES Received: August 28, 2015 Accepted: October 14, 2015 Published: October 30, 2015 Copyright: © 2015 Li 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. RESEARCH ARTICLE Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics Ling-bo Li1, Donald Y. M. Leung1,2, Elena Goleva1* 1 Department of Pediatrics, National Jewish Health, Denver, Colorado, United States of America, 2 Department of Pediatrics, University of Colorado Denver, Aurora, Colorado, United States of America Patients We enrolled 24 adult asthma patients with airflow limitation (baseline FEV180% predicted) and either airway hyperresponsiveness (PC20 methacholine < 8mg/ml) or bronchodilator responsiveness (>12% improvement in FEV1% predicted after 180 mcg metered-dose inhaler albuterol). Corticosteroid response of asthmatics was classified based on their prebronchodila- tor morning FEV1% predicted response to a one week course of 40mg/day oral prednisone. Asthmatics were defined as SR if they had less than 10% improvement in FEV1 and steroid sensitive (SS) if they showed significant improvement (12%). Informed written consent was obtained from all patients before enrollment in this study. The Institutional Review Board at National Jewish Health, Denver, Colorado approved this study. Research has been conducted according to the principles expressed in the Declaration of Helsinki. Subject characteristics are presented in Table 1. P-p38 MAPK in Monocytes of SR Asthmatics [1, 2]. It is estimated that up to 20% of asthmatics do not respond to GCs, these patients are referred to as steroid resistant (SR) asthmatics[3]. SR asthmatics are characterized by increased airway inflammation that cannot be suppressed by GC treatment. The role of race, smoking, obesity, vitamin D level, allergens, and infection in steroid resistance is under active investiga- tion[4–6]. Endotoxin exposure has recently been identified as an important factor that alters cellular response to GCs[7–9]. Our research group recently demonstrated alterations in airway micro- biome of SR asthma patients, with the expansion of Gram-negative LPS producing bacteria [10]. We also reported significant levels of endotoxin in the bronchoalveolar lavage (BAL) fluid of SR asthmatics[8, 10]. Along with high endotoxin levels in BAL fluid, BAL macrophages of these patients demonstrated classical macrophage activation and induction of LPS signaling pathways[8]. Stimulation with LPS has been shown to result in the phosphorylation and activa- tion of p38, ERK and JNK in monocytes and macrophages[11, 12]. Several studies have dem- onstrated that mitogen activated protein kinase (MAPK) pathways are involved in activation of transcription factors, such as NF-κB and AP-1[13, 14]; these transcription factors play a crit- ical role in LPS-induced expression of proinflammatory genes, such as TNF-α, IL-1β, IL-6, IL- 8, MCP-1, E-selectin, VCAM-1 and ICAM-1. Cytoplasmic glucocorticoid receptor (GCR) mediates cellular response to GCs. Activated GCR translocates to the cell nuclei and acts as a transcriptional factor. GCR can inhibit pro- inflammatory MAPK signaling by inducing nuclear mitogen activated kinase phosphatase (MKP1) expression[15, 16]. At the same time, GCR activity is subject to kinase modulation, activated MAPKs can inhibit GCR function via phosphorylation that will inhibit GCR nuclear translocation in response to GC treatment, cause the GCR to return to the cytoplasm or modify GCR transcriptional activity[17, 18]. In this manuscript, we evaluated evidence for MAPK acti- vation in peripheral blood of SR and SS asthmatics and asked whether MAPK activation in peripheral blood can serve as a biomarker of SR asthma. Introduction Glucocorticoids (GCs) are potent anti-inflammatory drugs used for treatment of asthma and other inflammatory diseases. However, a number of patients are refractory to GC therapy Competing Interests: The authors have declared that no competing interests exist. 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 Reagents and antibodies Phosflow Lyse/Fix buffer, Phosflow Perm/Wash Buffer Ι and Stain Buffer were purchased from BD Pharmingen (San Diego, CA). Primary fluorophore-conjugated antibodies specific to phos- phorylated proteins: mouse anti-p38 MAPK (pT180/pY182) Alexa 647 (cat. #612595) and mouse anti-p44/42 MAPK(pT202/pY204) Alexa 647 (cat. #612593), mouse IgG1 Alexa 647 (cat. #557783) were purchased from BD Pharmingen (San Diego, CA); mouse antibody against phosphorylated JNK (T183/Y185) Alexa 647 (cat. #9257) was from Cell Signaling Technology 2 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 P-p38 MAPK in Monocytes of SR Asthmatics Table 1. Patient characteristics. SR asthma SS asthma n = 13 n = 11 Age, yrs 38.5±3.3 41.4±4.0 Gender (Male/Female) 6/7 3/8 Race (C/AA/Other) 8/5/0 9/0/2 BMI, kg/m2 30.8±2.3 32.4±2.6 IgE, U/ml 195±84 115±28 Baseline FEV1% predicted 73.7±2.4 62.1±5.5 FEV1% reversal with Albuterol 16.6±3.3 37.0±9.8 FEV1% change after Prednisone burst 1.5±1.6 39.8±10.6 Medications*: ICS/LABA 4 4 Medications: ICS 3 2 Medications: None 6 5 The values shown are Mean±SE. p<0.001 as compared to SS asthmatics. For the SR and SS asthmatics that received ICS/LABA or ICS the Mean±SE of the ICS dose in budesonide equivalents was 1163±402 μg and 1100±336 μg, respectively. d i 10 1371/j l 0141909 001 Table 1. Patient characteristics. (Beverly, MA). Fluorophore-conjugated antibodies against cell-specific surface proteins: CD3 FITC, CD4 PE, CD8 PerCp-Cy5.5, CD14 FITC, CD16 PE, CD20 PerCP-Cy5.5 were purchased from BD Pharmingen. Anti-total and phosphorylated rabbit monoclonal antibodies to p38 MAPK (cat. #8690 and #4511), p44/42 MAPK (cat. #4695 and #4370), JNK (#4672 (rabbit polyclonal antibody) and #4668) and MSK1 (#3489 and #9595 (rabbit polyclonal antibody)) antibodies for Western blot analysis, as well as anti-Hsp90 (#4877) antibody were purchased from Cell Signaling Technology. Peripheral Blood Mononuclear Cell (PBMC) preparation Heparinized peripheral blood was collected from all study subjects. Whole blood samples were used for flow cytometry analysis. PBMC were isolated by Ficoll-Hypaque1 density gradient centrifugation. Isolated cells were resuspended at 1x106 cells/ml in RPMI 1640 medium con- taining 10% charcoal-filtered fetal calf serum (Gemini Bio-Products, Calabasas, CA). Western blot Whole cell extracts were prepared from freshly isolated PBMC using the ice-cold complete lysis buffer (1x Cell Signaling Lysis Buffer, #9803, Cell Signaling Technology, Inc., Danvers, MA)) with 10μL each PMSF, protease inhibitor cocktail, and sodium orthovanadate (#sc- 24948, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) per 1ml of lysis buffer). Cells were lysed for 5 min, scraped of the plates, sonicated for 10s and spun for 10 min at 10,000g at 4°C. 50μg of protein per condition were run on a 4–25% gradient gel (Bio-Rad Laboratories, Hercu- les, CA) and transferred to nitrocellulose membranes. The membranes were blotted with 1:1000 primary antibodies overnight at 4°C. After serial washes with TBS containing 0.1% Tween 20, membranes were incubated with appropriate peroxidase-conjugated secondary anti- bodies for 1 h at room temperature. Immunoreactive bands were visualized using a chemilumi- nescent kit (GE Healthcare) and images were captured on film (GE Healthcare). Developed X- ray films were scanned and densitometry of the bands were quantified with NIH Image soft- ware (version 1.63) (this software is available on the Internet at http://rsb.info.nih.gov/nih- image). Hsp90 detection was used to control the quality of protein preparation and to ensure that equal amounts of cellular proteins were loaded per lane. LPS-induced cytokine production by PBMC in the presence of MAPK inhibitors and corticosteroids PBMC were resuspended in RPMI 1640 (BioWhittaker) containing 10% charcoal filtered heat inactivated steroid-free FCS (Gemini Bio Products, Calabasas, CA), 40 μmol/l L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 20 mmol/l HEPES (GIBCO BRL Life Tech- nologies) at a concentration of 1×106/ml, pretreated with 1μM p38 MAPK inhibitor (SB203580) or with 10μM ERK inhibitor (PD98059) for 1h, followed by incubation with 10 ng/ ml LPS in the absence or presence of 10-7M dexamethasone (DEX). Cells were collected after 24h of incubation and IL-8 mRNA expression was examined by real time PCR. P-p38 MAPK in Monocytes of SR Asthmatics Real time PCR (RT-PCR) Total RNA was prepared using RNeasy Mini kit (Qiagen, Valencia, CA). After reverse tran- scription, 500 ng cDNA from each sample were analyzed by RT-PCR using the dual-labeled fluorogenic probe method on an ABI Prism 7300 RT-PCR system (Applied Biosystems). The expression of human IL-8 mRNA and 18s RNA was determined. All primers were purchased from Applied Biosystems (Foster City, CA). Statistical analysis Results were expressed as the Mean±SE. Statistical analysis was conducted using GraphPad Prism, version 5 (GraphPad Software, La Jolla, CA). Unpaired Student’s t test and nonparamet- ric tests were used. P<0.05 were considered significant. Cell surface and intracellular phospho-MAPK flow cytometry analysis 300μl of blood was fixed in 6 ml of BD Phosflow Lyse/Fix buffer at 37°C for 10 min and then washed once with PBS. After centrifugation, the cell pellet was permeabilized in BD Phosflow Perm/Wash Buffer Ι for 10 min at room temperature. Cell pellet was resuspended in 200μl of staining buffer, and cell suspension was equally divided between four FACS tubes containing 50μl of monoclonal antibodies (CD surface antibodies plus anti-phospho-MAPK or isotype control). Cells were incubated at room temperature for 45 min in the dark. After the final wash, cells were resuspended in 1% paraformaldehyde (200μl) and stored at 4°C. Cells were examined using FACScaliber (San Jose, CA) and data were analyzed using CellQuest software. We tested the levels of total MAPK expression in several cell types from SR and SS asthma patients by flow cytometry. We did not observe the difference in total MAPK expression between the cells from two study groups (data not shown). Therefore we chose only to assess MAPK phosphorylation status by flow cytometry. 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 P-p38 MAPK in Monocytes of SR Asthmatics Fig 1. MAPKs phosphorylation in freshly isolated PBMC of SS asthmatics and SR asthmatics as assessed by Western blot. (A) Cell lysates were prepared from the PBMC of nine subjects with SS asthma and nine subjects with SR asthma. 50 μg of protein from each patient PBMC cell extract were run on a 4–20% gradient gel and blots were probed with antibodies against phosphorylated forms of p38, ERK and JNK. Membranes were stripped and re- probed with antibodies against total p38, ERK and JNK, respectively. Hsp90 detection was used to monitor protein loading. (B) Densitometry values of phosphorylated form of p38 to total p38 in PBMC of SR and SS asthmatics. Fig 1. MAPKs phosphorylation in freshly isolated PBMC of SS asthmatics and SR asthmatics as assessed by Western blot. (A) Cell lysates were prepared from the PBMC of nine subjects with SS asthma and nine subjects with SR asthma. 50 μg of protein from each patient PBMC cell extract were run on a 4–20% gradient gel and blots were probed with antibodies against phosphorylated forms of p38, ERK and JNK. Membranes were stripped and re- probed with antibodies against total p38, ERK and JNK, respectively. Hsp90 detection was used to monitor protein loading. (B) Densitometry values of phosphorylated form of p38 to total p38 in PBMC of SR and SS asthmatics. doi:10.1371/journal.pone.0141909.g001 defined as SR or SS based on changes in lung function after one week of oral prednisone burst. Asthma patients recruited had airway obstruction (Table 1). Despite bronchodilator respon- siveness, SR asthma patients did not demonstrate improvement in lung function after oral prednisone burst, while SS patients were bronchodilator responsive and significantly improved their lung function after one week of oral prednisone treatment (Table 1). Western blot analy- ses were performed using whole protein extracts prepared from PBMC of SR and SS asthmatics. We observed presence of the activated form of p38, phosphorylated p38 MAPK (p-p38) in PBMC of all subjects with asthma (Fig 1). Moreover, significantly increased phosphorylation of p38 MAPK was detected in PBMC extracts from SR asthmatics compared with SS asth- matics (p<0.0116). The expression of total p38, however, in cells of the two groups of asth- matics was similar. In contrast, phosphorylated ERK was found in the cell lysates of only one SR asthma subject out of 18 asthmatics tested. No JNK phosphorylation was detected in PBMC extracts from 18 asthmatics examined. The activity of anti-phospho-JNK antibody was validated with the lysates from LPS stimulated PBMC (data not shown). p38 phosphorylation in PBMC of asthmatics Previous studies have documented p38 MAPK activation in BAL macrophages of severe asthma patients[19, 20]. Given our recently reported data on classical macrophage activation in BAL macrophages of SR asthmatics[8] and evidence for the expansion of LPS producing bacteria in the airways of SR asthmatics,[10] we evaluated whether MAPK activation can be observed in the peripheral blood of SR asthmatics as compared to SS asthmatics. Patients were 4 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 P-p38 MAPK in Monocytes of SR Asthmatics Fig 2. Flow cytometry assessment of MAPK phosphorylation in whole blood samples of SR and SS asthma patients. Whole blood aliquots were stained with either anti-CD3, CD4, CD8 antibody cocktail or CD14, CD16, CD20 antibody cocktail plus one of the phospho-MAPK antibodies (i.e. p-p38, p- ERK, p-JNK) or corresponding IgG isotype control antibodies (to detect nonspecific antibody binding). (A) Phosphorylation of p38 in CD14+ monocytes of subjects with SR asthma vs. SS asthma (MFI). (B) Phosphorylation of ERK in CD14+ cells of subjects with SR asthma vs. SS asthma (MFI). Fig 2. Flow cytometry assessment of MAPK phosphorylation in whole blood samples of SR and SS asthma patients. Whole blood aliquots were stained with either anti-CD3, CD4, CD8 antibody cocktail or CD14, CD16, CD20 antibody cocktail plus one of the phospho-MAPK antibodies (i.e. p-p38, p- ERK, p-JNK) or corresponding IgG isotype control antibodies (to detect nonspecific antibody binding). (A) Phosphorylation of p38 in CD14+ monocytes of subjects with SR asthma vs. SS asthma (MFI). (B) Phosphorylation of ERK in CD14+ cells of subjects with SR asthma vs. SS asthma (MFI). doi:10.1371/journal.pone.0141909.g002 subsets in PBMC and therefore could not be detected by Western blot analysis using the PBMC extracts. To do this, we analyzed p38, ERK and JNK phosphorylation by flow cytometry using hepa- rinized whole blood samples from asthmatics. We discovered that there was a significantly stronger signal for activated p38 in CD14+ monocytes from SR than SS asthmatics (p = 0.014) (Fig 2A), whereas no difference in phosphorylation of ERK and JNK in CD14+ cells was observed in the two groups of asthmatics (Fig 2B). In addition, no significant difference in phosphorylated p38, ERK, or JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from SR vs. SS asthmatics (Table 2). Evidence for p38 pathway activation in cells from SR asthmatics To support the importance of p38 activation in the cells of SR asthmatics, we evaluated phos- phorylation of mitogen- and stress-activated kinase 1 (MSK1), a kinase directly downstream of p38. MSK1 phosphorylation was examined by Western blot using the same cell lysates pre- pared to assess p38 phosphorylation in the study subjects. The levels of MSK1 phosphorylation were found to be significantly higher in PBMC lysates from SR asthmatics as compared to SS asthmatics (Fig 3A and 3B), p< 0.001. Increased p38 phosphorylation in peripheral blood CD14+ monocytes of SR asthmatics We further asked what specific subsets of PBMC contribute to the increased p38 phosphoryla- tion in PBMC of SR asthmatics. At the same time, ERK and JNK activation in PBMC was eval- uated, as it was possible that activation of these kinases was only present in specific rare cell 5 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 P-p38 MAPK in Monocytes of SR Asthmatics Fig 3. Activation of the p38 pathway in the cells of SR asthmatics. (A) Significantly elevated p-MSK1 levels in lysates from PBMC of SR as compared to SS asthma patients. Representative Western blot results of p-MSK1 and MSK1 in PBMC are shown. PBMC protein lysates prepared to assess p38 phosphorylation (Fig 1) were examined for p-MSK1 expression. (B) Densitometric values of phosphorylated form of MSK1 to total MSK1 in PBMC of SR and SS asthmatics. Fig 3. Activation of the p38 pathway in the cells of SR asthmatics. (A) Significantly elevated p-MSK1 levels in lysates from PBMC of SR as compared to SS asthma patients. Representative Western blot results of p-MSK1 and MSK1 in PBMC are shown. PBMC protein lysates prepared to assess p38 phosphorylation (Fig 1) were examined for p-MSK1 expression. (B) Densitometric values of phosphorylated form of MSK1 to total MSK1 in PBMC of SR and SS asthmatics. doi:10.1371/journal.pone.0141909.g003 MAPK remains phosphorylated after treatment with SB203580, but its catalytic activity is inhibited. Phosphorylation of the downstream kinase MSK1 was chosen as a read out for p38 suppression by SB203580. MSK1 phosphorylation was significantly decreased in the cells of SR asthmatics pretreated with p38 MAPK inhibitor, but not with the ERK inhibitor, PD98059 (Fig 4A and 4B). Importantly, cells of SR asthmatics that were pretreated with the p38 inhibi- tor became significantly more responsive to the suppressive effects of GCs. As shown by MAPK remains phosphorylated after treatment with SB203580, but its catalytic activity is inhibited. Phosphorylation of the downstream kinase MSK1 was chosen as a read out for p38 suppression by SB203580. MSK1 phosphorylation was significantly decreased in the cells of SR asthmatics pretreated with p38 MAPK inhibitor, but not with the ERK inhibitor, PD98059 (Fig 4A and 4B). Importantly, cells of SR asthmatics that were pretreated with the p38 inhibi- tor became significantly more responsive to the suppressive effects of GCs. As shown by Fig 4. Inhibition of p38 MAPK activation using p38 inhibitor SB203580 results in increased DEX suppression LPS-induced IL-8 mRNA production. P38 MAPK inhibition by p38 inhibitor SB203580 (SB), but not ERK inhibitor, PD98059 (PD) results in selective inhibition of downstream kinase activation (MSK1 phosphorylation) in the cells of SR asthmatics as shown by Western blot (A, B). After preincubation of PBMC from SR asthmatics with a selective p38 inhibitor the cells became steroid sensitive, i.e. doi:10.1371/journal.pone.0141909.g004 Effects of p38 MAPK inhibitor on responses of cells from SR asthmatics to GCs Effects of p38 MAPK inhibitor on responses of cells from SR asthmatics to GCs To evaluate the role of p38 activation in steroid resistance, PBMC from SR asthmatics were preincubated with a selective p38 MAPK inhibitor, SB203580. As previously reported[21], p38 Table 2. MFI of phosphorylated MAPK staining in various cell types in the blood samples of asthmatics. Cell type MFI p-ERK MFI p-p38 MFI p-JNK SS asthma SR asthma SS asthma SR asthma SS asthma SR asthma CD3+CD4+ (T helper cells) 1.26±0.31 1.29±0.40 1.18±0.31 0.81±0.23 7.34±1.51 8.07±1.41 CD3+CD8+ (T cytotoxic cells) 1.83±0.85 1.95±0.86 0.95±0.34 1.83±0.85 4.24±1.30 3.75±1.04 CD14-CD16+ (NK cells) 1.75±0.48 1.84±0.39 2.63±0.73 1.74±0.48 4.06±0.86 3.96±0.59 CD14-CD20+ (B cells) 0.38±0.09 0.68±0.18 1.30±0.37 0.79±0.27 1.56±0.39 0.86±0.25 doi:10.1371/journal.pone.0141909.t002 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 6 / 11 rylated MAPK staining in various cell types in the blood samples of asthmatics. Table 2. MFI of phosphorylated MAPK staining in various cell types in the blood samples of asthmatics. 6 / 11 Discussion Steroid resistance is a common problem in many chronic inflammatory diseases, including asthma. Our current study demonstrates that there is a selective increase in p38 activation in peripheral blood monocytes of SR asthma patients compared to the cells of SS asthmatics. This differential p38 activation was not observed in CD4+, CD8+ T cells, B cells or NK cells. The functional significance of p38 pathway activation in SR asthmatics was supported by our obser- vation of a significant increase in MSK1 phosphorylation and lack of GC suppression of IL-8 mRNA production by the cells from SR asthmatics. Importantly, ERK and JNK phosphoryla- tion in PBMC of SR asthmatics was not remarkable or negative. This data suggests that periph- eral blood p38 MAPK pathway activation can serve as a selective biomarker for SR asthmatics. In a previous study, we demonstrated the expression of multiple inflammatory genes related to classical macrophage activation in BAL cells from SR asthmatics as compared with SS asth- matics[8]. Activated ERK, JNK and p38 MAPK had been found in airway epithelial cells and alveolar macrophages of patients with asthma[20, 22, 23] and studies have demonstrated asso- ciation between the clinical severity of asthma and expression of p-p38 in asthmatic airways [24]. P38 MAPK was also demonstrated to regulate the production of many pro-inflammatory cytokines such as IL-8, IL-6, TNF-α; increased levels of IL-8 and TNF-α have been found in BAL macrophages from SR asthmatics[24]. In a recent study, Bhavsar et al. stimulated alveolar macrophages from asthma patients of different severities with LPS and concluded that steroid responsiveness was correlated with the degree of p38 MAPK activation[19]. Our current study found enhanced p38 phosphorylation also in circulating CD14+ cells of SR asthmatics. To the best of our knowledge, this is the first report to compare the levels of phosphorylated MAPK in components in PBMC and the first study that demonstrates increased p38 phosphorylation in circulating blood CD14+ from patients with SR asthma as compared to SS asthma. P38 consists of four subtypes (p38 alpha, beta, gamma and delta), which regulate cellular activities by serving as phosphorylation substrates of its upstream kinases, and also phosphory- lates specific serines and threonines of its downstream substrates. Studies suggest that different p38 isoforms have overlapping, but also distinct physiological roles[25, 26]. The anti-p-p38 MAPK antibody used in this study recognizes all p38 MAPK isoforms. greater suppression of LPS induced IL-8 mRNA by DEX, as shown by RT-PCR (C). d i 10 1371/j l 0141909 004 Fig 4. Inhibition of p38 MAPK activation using p38 inhibitor SB203580 results in increased DEX suppression LPS-induced IL-8 mRNA production. P38 MAPK inhibition by p38 inhibitor SB203580 (SB), but not ERK inhibitor, PD98059 (PD) results in selective inhibition of downstream kinase activation (MSK1 phosphorylation) in the cells of SR asthmatics as shown by Western blot (A, B). After preincubation of PBMC from SR asthmatics with a selective p38 inhibitor the cells became steroid sensitive, i.e. greater suppression of LPS induced IL-8 mRNA by DEX, as shown by RT-PCR (C). Fig 4. Inhibition of p38 MAPK activation using p38 inhibitor SB203580 results in increased DEX suppression LPS-induced IL-8 mRNA production. P38 MAPK inhibition by p38 inhibitor SB203580 (SB), but not ERK inhibitor, PD98059 (PD) results in selective inhibition of downstream kinase activation (MSK1 phosphorylation) in the cells of SR asthmatics as shown by Western blot (A, B). After preincubation of PBMC from SR asthmatics with a selective p38 inhibitor the cells became steroid sensitive, i.e. greater suppression of LPS induced IL-8 mRNA by DEX, as shown by RT-PCR (C). doi:10.1371/journal.pone.0141909.g004 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 7 / 11 P-p38 MAPK in Monocytes of SR Asthmatics RT-PCR, preincubation of PBMC from SR asthmatics with SB203580 resulted in significantly greater DEX-mediated suppression of LPS induced IL-8 mRNA (Fig 4C). RT-PCR, preincubation of PBMC from SR asthmatics with SB203580 resulted in significantly greater DEX-mediated suppression of LPS induced IL-8 mRNA (Fig 4C). PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 Discussion On the other hand, p38 MAPK inhibitor, SB203580, that was used in this study, is an inhibitor for p38 MAPK alpha and beta isoforms; p38 MAPK gamma and delta isoforms are insensitive to this class of pyridi- nyl imidazole p38 MAPK inhibitors[27]. Pretreatment of PBMC with SB203580 inhibited p38 MAPK activity in the cells and enhanced ability of DEX to suppress IL-8 production by PBMC. This suggests that phosphorylated p38 MAPK in the cells of SR asthma patients is likely an alpha/beta isoform rather then gamma/delta. A recent study demonstrated inhibition of p38 MAPK gamma isoform by formeterol in PBMC pretreated by IL-2/IL-4[28]. In our study we did not observe the difference in p38 MAPK activation between patients treated with ICS/ LABA as compared to patients treated with ICS only (data not shown). Although steroids have been reported to inhibit p38 MAPK phosphorylation in airway epithelial cells,[29] we did not observe the difference in p38 MAPK activation between steroid users and non-steroid users in both SR and SS asthma groups (data not shown). Downstream of p38, there is MSK1, which can mediate phosphorylation of histone H3 on serine at different inflammatory gene promoters to stimulate their transcription[30]. However, GCs counteract the recruitment of activated MSK1 at inflammatory gene promoters resulting in the inhibition of NF-κβ, p65 transaction and concurrent histone H3 phosphorylation[31]. 8 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 P-p38 MAPK in Monocytes of SR Asthmatics Another example of interaction between the GCR and MSK1 is that MSK1 predominantly localizes in the nucleus, however, activated GCR can translocate p-MSK1 from the nucleus to the cytoplasm, therefore inhibiting MSK1 effects on its downstream targets[32]. In our current study, we detected an increase in activated MSK1 that paralleled the increase of phosphorylated p38 in PBMC from SR asthma, indicating the potential involvement of MSK1 in regulation of cellular steroid responses. Our data is consistent with previous observations by several research groups that p38 MAPK inhibition in severe asthma may enhance cellular responsiveness to GCs[9, 19, 33, 34]. Several studies have also demonstrated effectiveness of p38 inhibition in the reversal of GC insensitivity of cells from COPD patients[35, 36]. However previous studies have not identified the cell type in peripheral blood with selective p38 MAPK activation in severe and/or steroid resistant asthma. Discussion Our study demonstrates for the first time p38 and MSK1 activation in periph- eral blood monocytes of SR asthmatics. Peripheral blood from patients can be readily analyzed. Thus, p-p38 and p-MSK1 should be further evaluated in PBMC as potential biomarkers of asthmatic responsiveness to GC treatment. Author Contributions Conceived and designed the experiments: LL EG DL. Performed the experiments: LL. Analyzed the data: LL EG. Wrote the paper: LL EG DL. PLOS ONE \| DOI:10.1371/journal.pone.0141909 October 30, 2015 References Lim W, Gee K, Mishra S, Kumar A. 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https://openalex.org/W4200216503	https://pub.uni-bielefeld.de/download/2959873/2960349/ChemistryOpen%20-%202021%20-%20Nonnhoff%20-%20Process%20Development%20of%20the%20Copper%20II%20%25u2010Catalyzed%20Dehydration%20of%20a%20Chiral%20Aldoxime%20and.pdf	English	null	Process Development of the Copper(II)‐Catalyzed Dehydration of a Chiral Aldoxime and Rational Selection of the Co‐Substrate	ChemistryOpen	2,021	cc-by	4,851	Full Papers doi.org/10.1002/open.202100230 ChemistryOpen www.chemistryopen.org Process Development of the Copper(II)-Catalyzed Dehydration of a Chiral Aldoxime and Rational Selection of the Co-Substrate Jannis Nonnhoff and Harald Gröger*[a] Dedicated to Professor Dr. Albrecht Berkessel on the occasion of his 65th birthday Dedicated to Professor Dr. Albrecht Berkessel on the occasion of his 65th birthday The access towards chiral nitriles remains crucial in the synthesis of several pharmaceuticals. One approach is based on metal-catalyzed dehydration of chiral aldoximes, which are generated from chiral pool-derived aldehydes as substrates, and the use of a cheap and readily available nitrile as co- substrate and water acceptor. Dehydration of N-acyl α-amino aldoximes such as N-Boc-l-prolinal oxime catalyzed by copper (II) acetate provides access to the corresponding N-acyl α-amino nitriles, which are substructures of the pharmaceuticals Vilda- gliptin and Saxagliptin. In this work, a detailed investigation of the formation of the amide as a by-product at higher substrate loadings is performed. The amide formation depends on the electronic properties of the nitrile co-substrate. We could identify an acceptor nitrile which completely suppressed amide formation at high substrate loadings of 0.5 m even when being used with only 2 equivalents. In detail, utilization of trichlor- oacetonitrile as such an acceptor nitrile enabled the synthesis of N-Boc-cyanopyrrolidine in a high yield of 92% and with full retention of the absolute configuration. Figure 1. Chiral 2-cyanopyyrolidine based DPP-4 inhibitors. ChemistryOpen 2022, 11, e202100230 (1 of 6) © 2021 The Authors. Published by Wiley-VCH GmbH [a] J. Nonnhoff, Prof. Dr. H. Gröger Chair of Industrial Organic Chemistry and Biotechnology Faculty of Chemistry Bielefeld University, Universitätsstrasse 25 33615 Bielefeld (Germany) E-mail: harald.groeger@uni-bielefeld.de Supporting information for this article is available on the WWW under https://doi.org/10.1002/open.202100230 Part of the “Industrial Organic Synthesis” Special Collection. © 2021 The Authors. Published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1. Introduction Nitriles represent an important class of compounds for bulk and fine chemicals with a particular focus on their occurrence as chiral building blocks in pharmaceuticals.[1] An example are chiral acylated α-amino nitriles (Figure 1) such as Saxagliptin 1,[2] distributed by AstraZeneca/BMS, and Vildagliptin 2,[3,4] distributed by Novartis, with sales of over $1 billion per year. Both pharmaceuticals belong to the class of dipeptidyl peptidase-4 (DPP-4) inhibitors and are used in the treatment of type II diabetes. In both molecules, the (S)-2-cyanopyrrolidine structure plays a crucial role in the mechanism of action and, thus, represents a key structure. Figure 1. Chiral 2-cyanopyyrolidine based DPP-4 inhibitors. catalysts for this transformation, the use of toxic cyanides is a drawback of this approach. The utilization of toxic cyanides in the synthesis of nitriles can be avoided by dehydration of amides and aldoximes. Such a process is used, for instance, in the synthesis of Vildagliptin 2, and proceeds through the dehydration of N-acyl protected l- proline amide, which is easily accessible from the chiral pool reagent l-proline amide by using a suitable Vilsmeier reagent.[3,4] The preparation of the Vilsmeier reagent is based on the utilization of phosphoryl chloride as a substrate. As a result, various efforts have been made to discover new ways of synthesizing chiral nitriles and especially α-amino nitriles. One example is given by the asymmetric Strecker synthesis. Starting from prochiral aldehydes, chiral α-amino nitriles can be obtained using amines, cyanides and chiral catalysts.[5] Despite great progress in the development of Dehydration of aldoximes represents a promising alternative for synthesizing nitriles. For the dehydration of aldoximes, several stochiometric reagents have been described, such as oxalyl chloride,[6] iodine and triphenylphosphane,[7] trifluoro- acetic anhydride,[8] Swern-like conditions[9] or chlorotropylium,[10] while the aldoximes are simply obtained from their aldehydes by condensation with hydroxylamine. Some one-pot syntheses based on the dehydration of aldoximes, obtained in situ from their aldehydes, have also been described. In particular, a dehydration with HMDS and DBU,[11] the use of ethyl chlorophosphates[12] or acid-catalyzed dehydration in DMSO[13] were reported. [a] J. Nonnhoff, Prof. Dr. H. Gröger Chair of Industrial Organic Chemistry and Biotechnology Faculty of Chemistry Bielefeld University, Universitätsstrasse 25 33615 Bielefeld (Germany) E-mail: harald.groeger@uni-bielefeld.de Supporting information for this article is available on the WWW under https://doi.org/10.1002/open.202100230 Part of the “Industrial Organic Synthesis” Special Collection. © 2021 The Authors. Published by Wiley-VCH GmbH. 2.1. Variation of Catalyst Loading and Temperature First, the influence of the amount of copper acetate on the progress of the reaction and side product formation was studied by varying the catalyst loading between 0.5 and 5 mol% (see Supporting Information). While no difference was observed between 1 and 5 mol% with respect to the total conversion as well as to the amide formation, the total conversion at a catalyst loading of 0.5 mol% decreased to 38% (28% nitrile 5 and 10% amide 6). Thus, a catalyst loading of 2 mol% was chosen for the subsequent study of the impact of the reaction temperature on conversion and amide formation. The temperature was varied in the range of 70°C to 20°C using the standard reaction conditions (Table 1). The dehydra- tion of aldoxime 4 takes place at all selected reaction temper- atures, although with different rates. While the conversions at 70°C and 50°C are quantitative after four hours, the reaction time must be extended at lower temperatures in order to achieve full substrate consumption. It is also noteworthy that a reaction at 20°C, without stirring, also leads to the formation of both nitrile and amide. To evaluate the effect of the temperature on amide formation, the reactions were carried out until complete conversion of the oxime was observed. When comparing the results, only a small effect of temperature can be noted, but there seems to be a tendency for a higher degree of amide formation at lower temperature. After complete con- During our research on scaling up this process and improving the space-time yield of the dehydration step by raising the substrate loading of Boc-prolinal oxime 4 to 0.5 m in the presence of acetonitrile as reagent (“acceptor nitrile”), the formation of N-Boc-proline amide 6 in significant amounts was observed, which also occurred in a developed one-pot process.[19] The reason for the formation of Boc-proline amide 6 as a by-product can be found when taking into account the reaction mechanism of the desired dehydration step. 1. Introduction This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. A recent focus has been on the development of environ- mentally friendly and cost-effective versions of this type of ChemistryOpen 2022, 11, e202100230 (1 of 6) © 2021 The Authors. Published by Wiley-VCH GmbH Full Papers doi.org/10.1002/open.202100230 ChemistryOpen Scheme 1. De novo synthesis of enantiomerically highly enriched N-acyl amino nitrile 5 by Rommelmann et al.[18] Scheme 1. De novo synthesis of enantiomerically highly enriched N-acyl amino nitrile 5 by Rommelmann et al.[18] 2. Results and Discussion reaction, leading to the use of various metal catalysts such as copper,[14] palladium,[15] iron,[16] cobalt and others.[17] In contrast to the use of classical dehydration reagents, these catalytic versions often use other (cheap) nitriles such as acetonitrile as water acceptors, thus forming the corresponding amides as by- products. This work is focusing on an investigation of the influence of the reaction parameters on the suppression of the undesired amide formation as a side reaction. As a standard reaction, dehydra- tion of Boc-prolinal oxime 4 at a substrate concentration of 0.5 m, a catalyst loading of 2 mol% and the use of 10 equivalents of acetonitrile in ethyl acetate for 4 h at 70°C was selected (Scheme 2 ). In previous studies of our group,[3,4,18] an alternative syn- thesis of enantiomerically highly enriched N-acyl amino nitriles, based on a copper(II) acetate-catalyzed dehydration of aldox- imes, was described, using acetonitrile as water acceptor (Scheme 1). Starting from substrates derived from the chiral pool such as N-Boc-l-prolinal 3, condensation with hydroxyl- amine, thus forming Boc-prolinal oxime 4, and subsequent dehydration enabled a de novo-access to N-Boc-(S)-2-cyano- pyrroldine (5).[3,4,18] This process was transferred to other N- acylated pyrrolidines, which could serve as intermediates in the synthesis of Vildagliptin.[3,4,18] Despite the advantages of such a cyanide-free synthesis of acylated N-amino nitriles, this access however showed disadvantages in terms of applicability on technical scale. The dehydration suffered from a low substrate concentration (80 mm), a low space-time yield and from being a two-step process. © 2021 The Authors. Published by Wiley-VCH GmbH 2.1. Variation of Catalyst Loading and Temperature While almost no conversion of prolinal oxime 4 after 18 h was observed at room temperature, 7% of nitrile 5 and 13% of amide 6 were detected at 70°C. This supports the assumption that the formation of the amide side product proceeds, at least in part, through the reaction mechanism of the aldoxime to nitrile conversion (in which the formed nitrile 5 then acts as a nitrile acceptor). At the same time, the presence of acetonitrile leads to an increased formation of amide 6 at both studied temperatures. This can be explained by the formation of the desired cyanopyrrolidine 5 in higher amount, which then acts as an intermediate in the undesired formation of the proline amide side product 6. version at 70°C, 29% of amide 6 are formed, in contrast to 37% at 30°C and 32% at room temperature. To gain insight into the amide formation as a side reaction, a reaction without acetonitrile at room temperature and at 70°C was also studied (Table 1, entries 9, 10). While almost no conversion of prolinal oxime 4 after 18 h was observed at room temperature, 7% of nitrile 5 and 13% of amide 6 were detected at 70°C. This supports the assumption that the formation of the amide side product proceeds, at least in part, through the reaction mechanism of the aldoxime to nitrile conversion (in which the formed nitrile 5 then acts as a nitrile acceptor). At the same time, the presence of acetonitrile leads to an increased formation of amide 6 at both studied temperatures. This can be explained by the formation of the desired cyanopyrrolidine 5 in higher amount, which then acts as an intermediate in the undesired formation of the proline amide side product 6. In contrast, a reduction of the substrate concentration to 100 mm using 10 equivalents of acetonitrile in ethyl acetate as a solvent only leads to a reduced reaction rate, but not to a relevant reduction of the formed undesired proline amide 6 in the reaction mixture (entry 11). These results also support the described mechanism of an amide formation via a nitrile intermediate. Accordingly, an increased amount of acetonitrile as acceptor nitrile and, thus, co-substrate decreases the probability of a further reaction of the desired Boc-protected nitrile 5 as such a co-substrate in the further course of the reaction. 2.1. Variation of Catalyst Loading and Temperature With these findings on the impact of various reaction conditions on the dehydration of aldoxime 4 in hand, we next focused on the preparation of Boc-2 cyanopyrrolidine 5 within a one-pot process starting from aldehyde 3 (Scheme 3). The reaction was carried out at a substrate concentration of 0.1 m using an aqueous hydroxylamine solution in acetonitrile. 2.1. Variation of Catalyst Loading and Temperature In particular at high substrate loading, in situ formed Boc- cyanopyrrolidine 5 can also take over the function of the acceptor nitrile in the dehydration of unreacted aldoximes.[20,21] Likewise, a transformation of several aldoximes and nitriles into their primary amides using copper(II) acetate in aqueous and organic media has been described in the presence of aldoximes or hydroxylamine.[20,22] Mechanistic studies with isotopically labelled substrates show that amide formation does not occur by simple water addition. Instead, an in situ formed nitrile acts as a (formal) water acceptor in the dehydration of further aldoximes.[23] Scheme 2. Standard synthesis of N-Boc-(S)-2-cyanopyrrolidine (5). Scheme 2. Standard synthesis of N-Boc-(S)-2-cyanopyrrolidine (5). ChemistryOpen 2022, 11, e202100230 (2 of 6) Full Papers doi.org/10.1002/open.202100230 ChemistryOpen Table 1. Screening of temperature in copper(II)-catalysed dehydration of prolinal oxime 4. Table 2. Influence of acetonitrile equivalents and substrate loading on copper(II)-catalyzed dehydration of prolinal oxime 4. Table 2. Influence of acetonitrile equivalents and substrate loading on copper(II)-catalyzed dehydration of prolinal oxime 4. Table 1. Screening of temperature in copper(II)-catalysed dehydration of prolinal oxime 4. Entry T [°C] Time [h] Aldoxime 4 [%] Nitrile 5 [%] Amide 6 [%] 1 70 4 – 71 29 2 50 4 – 67 33 3 30 4 7 64 30 4 30 18 – 63 37 5 rt 4 21 63 16 6 rt 18 – 68 32 7 rt[a] 18 2 65 32 8 20[a] 18 81 14 5 9 rt[b] 18 97 2 1 10 70[b] 18 80 7 13 [a] Without stirring; [b] without acetonitrile. pp ( ) y y p Entry Equiv. MeCN Substrate concentration [mm] Aldoxime 4 [%] Nitrile 5 [%] Amide 6 [%] 1 235 80 – 99 1 2 150 127 – 96 4 3 125 152 – 96 4 4 100 190 – 95 5 5 75 253 – 94 6 6 64 300 – 93 7 7 48 400 – 91 9 8 38 500 – 89 11 9 10[a] 500 – 74 26 10 10[a] 300 – 73 27 11 10[a] 100 9 68 23 [a] Ethyl acetate was used as solvent. version at 70°C, 29% of amide 6 are formed, in contrast to 37% at 30°C and 32% at room temperature. To gain insight into the amide formation as a side reaction, a reaction without acetonitrile at room temperature and at 70°C was also studied (Table 1, entries 9, 10). ChemistryOpen 2022, 11, e202100230 (3 of 6) © 2021 The Authors. Published by Wiley-VCH GmbH 2.2. Variation of the Excess of the Acceptor Nitrile In addition, the electronic properties of the aldoxime substrate should then play a role on both conversion and side product formation. While a general influence of electron donating substituents in this type of reaction is mentioned, an influence on amide formation under the chosen conditions has not yet been described.[24] 7a to 0.83 for p-(N,N-dimethyl)aminobenzaldoxime 7e, defin- ing the electronic characteristics of the p-substituents. Further- more, n-octanal oxime (8) was investigated as an aliphatic substrate. We were pleased to find that the dehydration reaction under standard conditions (Scheme 1) indeed pro- ceeded for all selected substrates. However, there is a considerable difference in the selectivity of the reaction. For the aliphatic substrate n-octanal oxime (8), only a small amount of the side product n-octanamide (12) (3%) is observed at complete conversion of the aldoxime (Figure 2). When using the 4-substituted benzaldoximes 7a–e as substrates, there is a clear dependency of the selectivity on the Hammett parameter and, thus, on the electron density at the carbon atom of the aldoxime group (Figure 2). When using benzaldoxime 7e containing the dimethylamino group as the most electron-donating substituent in this study as a substrate, formation of the resulting benzamide 11e in only 9% yield was observed. However, when reducing the degree of electron donation, associated with an increasing σp value, the selectivity decreases sharply. While in the dehydration of unsubstituted benzaldoxime 7c to benzonitrile 9c already 17% amide is formed, the selectivity decreases further for electron-withdraw- ing substituents. An amide formation of 31% and 35% in the dehydrations towards p-chlorobenzonitrile 9b (σp=0.23) and p- nitrobenzonitrile 9a (σp=0.78), respectively, was observed, although only a conversion of 95% was noted in the latter case. These observations confirm that the reactivity of the aldoxime and the resulting nitrile significantly influence the progress and selectivity of the reaction. The electron-withdrawing substituent in para-position lowers the electron density at the cyanocarbon of the in situ formed p-nitrobenzonitrile 9a, favoring a nucleophilic attack in the dehydration of unreacted aldoxime 7a compared to the higher electron density of acetonitrile, which served as the acceptor nitrile in the initial dehydration step. In the conversion of octanal oxime 8, this effect is only slightly observed, since the resulting octanenitrile 10 has a similar electron density at the reactive center as acetonitrile. 2.2. Variation of the Excess of the Acceptor Nitrile Compared to the one-step dehydration of prolinal oxime 4, a longer reaction time was needed to achieve full conversion of the starting material. After 24 h, N-Boc-(S)-2-cyanopyrrolidine (5) was isolated in a yield of 85%, whereas only 5% conversion to amide 6 was observed (Scheme 3). Note that an optical rotation analysis revealed a full retention of the chiral information. Since an influence of the acetonitrile on conversion and side- product formation could be observed, the used equivalents were subsequently varied in a standard reaction at 70°C. In detail, the number of equivalents was varied between 235 and 10, which in most cases has also been associated with a change in substrate concentration (Table 2). By increasing the amount of acetonitrile, the formation of side product 6 in the dehydration of prolinal oxime 4 can be reduced or almost suppressed. For example, an increase from 10 to 38 equivalents of acetonitrile in a reaction at 0.5 m substrate concentration leads to a significant decrease of the formation of side product 6 (26% to 11%, entries 8, 9). By successively increasing the number of equivalents of acetonitrile up to 235, which corresponds to a substrate concentration of 80 mm of 4 in pure acetonitrile, led to a further decrease of formation of the undesired amide 6 to only 1% at a complete conversion of the aldoxime after 4 h (entry 1). Although using a large excess of acetonitrile enables suppression of the side product and high conversion, the resulting substrate concentrations of 80 or 100 mm are not Scheme 3. One-pot synthesis of N-Boc-(S)-2-cyanopyrrolidine (5) in pure acetonitrile. Scheme 3. One-pot synthesis of N-Boc-(S)-2-cyanopyrrolidine (5) in pure acetonitrile. ChemistryOpen 2022, 11, e202100230 (3 of 6) © 2021 The Authors. Published by Wiley-VCH GmbH Full Papers doi.org/10.1002/open.202100230 ChemistryOpen attractive for an industrial application. Thus, other options to inhibit amide formation have been considered and, in partic- ular, we became interested in a fine-tuning of the electronic properties of the acceptor nitrile with respect to the ability to add water within the dehydration step with aldoxime 4 as a substrate. We envisioned that reducing the electron density at the carbon atom of the nitrile moiety of the “acceptor nitrile” by electron-withdrawing substituents could lead to a faster (formal) addition of water during the dehydration process of the aldoxime substrate. 2.2. Variation of the Excess of the Acceptor Nitrile Here, the 10-fold excess of acetonitrile plays a more significant role, which leads to the suppression of the undesired amide formation in this case. In order to verify this hypothesis, at first the influence of the electron density at the aldoxime on amide formation in the standard reaction was investigated (Figure 2). For this purpose, besides n-octanal oxime, various p-substituted benzaldoximes were chosen based on their Hammett values and used as substrates in a dehydration reaction. Initially, the benzaldoximes 7a–e with different substitution pattern in 4-position were prepared from the respective aldehydes in good to excellent yields. Similarly, amides 11a-e were obtained from the corresponding aldoximes as reference compounds in low to moderate yields by a copper(II)-catalyzed rearrangement in toluene. The Hammett values σp of the aromatic aldoximes ranged from 0.78 for p-nitrobenzaldoxime Figure 2. Influence of substrate structure on dehydration of aldoximes using acetonitrile as an acceptor nitrile. Figure 2. Influence of substrate structure on dehydration of aldoximes using acetonitrile as an acceptor nitrile. Figure 2. Influence of substrate structure on dehydration of aldoximes using acetonitrile as an acceptor nitrile. © 2021 The Authors. Published by Wiley-VCH GmbH 2.3. Variation of Acceptor Nitrile Based on these findings, a modification of the acceptor nitrile with regard to suppression of amide formation was investigated next. At first, different monosubstituted, aliphatic acceptor nitriles such as butyronitrile or octanenitrile were used, but these acceptor nitriles showed no positive influence on the selectivity in the dehydration of prolinal oxime 4 (see Support- ing Information). However, when using more electrophilic acceptor nitriles we found a significant increase of the reactivity with respect to water addition, as expected. In detail, we tested two different acceptor nitriles with such an expected higher reactivity, namely trichloroacetonitrile and succinonitrile (Fig- ure 3). Trichloroacetonitrile exhibits a strong electrophilicity due to the trifold chlorine substitution, whereas succinonitrile has Figure 2. Influence of substrate structure on dehydration of aldoximes using acetonitrile as an acceptor nitrile. ChemistryOpen 2022, 11, e202100230 (4 of 6) Full Papers doi.org/10.1002/open.202100230 ChemistryOpen proportion of the undesired p-nitrobenzamide product was observed. With this improved nitrile acceptor in hand were applied to the dehydration of Boc-prolin next step. When utilizing either ten equiva shown) or even just two equivalents of trichloro were pleased to find a complete conversion o without formation of the undesired amid (Scheme 4). For the experiment with two trichloroacetonitrile, work-up and product isola ducted, which led to the isolation of the desi cyanopyrrolidine 5 in 92% yield. Furthermore, a full retention of the absolute configuration was o Figure 3. Influence of different acceptor nitriles on dehydration of p-nitro benzaldoxime 7a. Scheme 4. Dehydration of Boc-prolinal oxime 4 with trich the optimized nitrile acceptor. Figure 3. Influence of different acceptor nitriles on dehydration of p-nitro benzaldoxime 7a. Scheme 4. Dehydration of Boc-prolinal oxime 4 with trichloroacetonitrile as the optimized nitrile acceptor. Figure 3. Influence of different acceptor nitriles on dehydration of p-nitro benzaldoxime 7a. Scheme 4. Dehydration of Boc-prolinal oxime 4 with trichloroacetonitrile as the optimized nitrile acceptor. proportion of the undesired p-nitrobenzamide 11a as a side product was observed. With this improved nitrile acceptor in hand, these findings were applied to the dehydration of Boc-prolinal oxime 4 as a next step. When utilizing either ten equivalents (data not shown) or even just two equivalents of trichloroacetonitrile, we were pleased to find a complete conversion of the aldoxime without formation of the undesired amide side-product (Scheme 4). Acknowledgements We gratefully acknowledge generous support from the European Regional Development Fund (ERDF) within the project “Sustain- able production of nitrile industrial chemicals” (Grant no. EFRE- 0400138). 3. Conclusion just a slightly lower electron density at the cyano-carbon atom compared to acetonitrile, but has the advantage of a bifunc- tionality. This bifunctionality theoretically enables to halve the number of equivalents compared to those needed for acetonitrile. In this work, the relationship between the ratio of the reactivity of the acceptor and product nitrile and its influence on the formation of the undesired amide side product in a dehydration of aldoximes was investigated. Toward this end, the dehydra- tion of para-substituted benzaldoximes with different electron densities at the carbon atoms of the aldoxime and cyano groups and using acetonitrile as the co-substrate was analyzed. Depending on the chosen substrate, amide formation can be reduced or completely suppressed by a high excess of acetonitrile or by appropriate choice of a more electrophilic acceptor nitrile such as trichloroacetonitrile. By suppressing the undesired amide formation, the yields were significantly improved for p-nitrobenzonitrile 9a and N-Boc-cyanopyrrolidine 5, leading to a high yield of 92% in the latter case. The reaction was first carried out for both acceptor nitriles under standard conditions with p-nitrobenzaldoxime 7a as aldoxime substrate, which, in previous experiments, had shown the highest tendency to amide formation. Ten equivalents of the nitrile functionalities were used in each case in the first place, resulting in the conversion of aldoxime 7a to nitrile 9a in both cases. When five molar equivalents of succinonitrile are used, a significant improvement of the selectivity (12% amide) was observed in comparison with acetonitrile (35% amide). On the other hand, reduction to two molar equivalents of succinonitrile leads to a non-complete conversion (96%) of 7a after 4 h with a similarly high amide 11a content (33%) compared to the experiment with ten equivalents of acetonitrile. When using trichloroacetonitrile as a co-substrate, we were pleased to find that this compound showed even better properties as a nitrile acceptor (Figure 3). Here, a complete conversion of the para-substituted benzaldoxime 7a to nitrile 9a was observed in the presence of 10, 5 and 2 equivalents. Furthermore, extractive and column chromato- graphic work-up gave the resulting product 4-nitrobenzonitrile 9a in 89% yield, which is a considerable increase compared to the synthesis using acetonitrile (58%). Only with just an equimolar use of this acceptor nitrile, analogous to succinoni- trile, an incomplete conversion and, with 35%, a high 2.3. Variation of Acceptor Nitrile For the experiment with two equivalents of trichloroacetonitrile, work-up and product isolation were con- ducted, which led to the isolation of the desired N-Boc-(S)-2- cyanopyrrolidine 5 in 92% yield. Furthermore, also in this case full retention of the absolute configuration was observed. Figure 3. Influence of different acceptor nitriles on dehydration of p-nitro benzaldoxime 7a. Data Availability Statement [12] J. Zhu, F. Lee, J. Wu, C. Kuo, K. Shia, Synlett 2007, 1317. [13] a) W.-Y. Fang, H.-L. Qin, J. Org. Chem. 2019, 84, 5803; b) S. T. Chill, R. C. Mebane, Synth. Commun. 2009, 39, 3601; c) J. K. Augustine, A. Bombrun, R. N. Atta, Synlett 2011, 2223. The data that support the findings of this study are available from the corresponding author upon reasonable request. [14] O. Attanasi, P. Palma, F. Serra-Zanetti, Synthesis 1983, 741. [15] K. Tambara, G. D. Pantoş, Org. Biomol. Chem. 2013, 11, 2466. [16] K. Hyodo, S. Kitagawa, M. Yamazaki, K. Uchida, Chem. Asian J. 2016, 11, 1348. Keywords: aldoxime · copper(II) acetate · dehydration · N-acyl amino nitrile · nitrile Keywords: aldoxime · copper(II) acetate · dehydration · N-acyl amino nitrile · nitrile [17] a) X.-Y. Ma, Y. He, T.-T. Lu, M. Lu, Tetrahedron 2013, 69, 2560; b) Y. Xu, X. Jia, J. Ma, J. Gao, F. Xia, X. Li, J. 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https://openalex.org/W4250562565	https://www.researchsquare.com/article/rs-655111/v1.pdf?c=1641600367000	English	null	Nucleotide Variation In The Phytoene Synthase (ClPsy1) Gene Contributes To Golden Flesh In Watermelon (Citrullus Lanatus L.)	Research Square (Research Square)	2,021	cc-by	11,986	Nucleotide Variation In The Phytoene Synthase (ClPsy1) Gene Contributes To Golden Flesh In Watermelon (Citrullus Lanatus L.) Shi Liu (  shiliu@neau.edu.cn ) Northeast Agricultural University https://orcid.org/0000-0003-1364-4043 Abstract Vitamin A deficiency is a worldwide public nutrition problem, and β-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, due to accumulated abundance of β-carotene) is an important germplasm resource. In this study, a genetic analysis of gf segregating populations indicated that gf was controlled by a single recessive gene. BSA-seq and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population with over 1,000 F2 plants narrowed this region to 39.08 Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines cosegregated with the gf trait only among individuals in the genetic population but were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs and one MYC2 exhibited expression trends similar as ClPsy1 in the parental lines, which may thus play roles in the regulation of ClPsy1 expression. Our research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in β-carotene accumulation in watermelon flesh. Research Article 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 Theoretical and Applied Genetics on October 11th, 2021. See the published version at https://doi.org/10.1007/s00122-021-03958-0. Page 1/27 Key Messages A gene controlling the golden flesh trait in watermelon was firstly discovered and fine-mapped to a 39.08- Kb region on chromosome 1 through a forward genetic strategy, and Cla97C01G008760 (annotated as phytoene synthase protein, ClPsy1) was recognized as the most likely candidate gene. Introduction Watermelon (Citrullus lanatus L.) is a commercial Cucurbitaceae crop that is cultivated worldwide and consumed fresh. The flesh color of watermelon is an important trait based on consumer demands and market requirements. The genetic diversity of multiple flesh colors (red, pink, orange, canary yellow, pale yellow, white and pale green) in watermelon is caused by genome domestication and human breeding selection (Guo et al., 2019). Carotenoids are the main pigment responsible for formation of the watermelon flesh color. Specifically, lycopene is the key pigment responsible for the red and pink flesh colors (Sun et al., 2018), whereas β-carotene, tetra-cis-lycopene, and ξ-carotene are responsible for the orange flesh color (Branham et al., 2017). In addition, violaxanthin and lutein are the two main pigments responsible for the formation of canary and pale yellow flesh, and violaxanthin is significantly more abundant in canary yellow than in pale yellow watermelon (Yuan et al., 2015 and Fang et al., 2020). White-fleshed watermelon contains only small amounts of lutein and violaxanthin (Lv et al., 2015). Carotenoids, which form part of the human diet, serve as precursors of vitamin A and substrates for many nutrients and also exhibit antioxidant activity. Although these compounds are indispensable for Page 2/27 Page 2/27 health, humans are incapable of de novo carotenoid synthesis (Rodriguez-Concepcion et al., 2018). The World Health Organization (WHO) estimates that approximately 190 million preschool children are deficient in vitamin A, and vitamin A deficiency is one of the major public health problems in the world. β- Carotene is the precursor for vitamin A synthesis, and the consumption of horticulture crops with high levels of β-carotene (golden flesh), which constitute an important germplasm resource, is an effective strategy for preventing vitamin A deficiency. Scholars began studying the inheritance of the watermelon flesh color as early as 1937(Porter, 1937). Canary yellow (C) is dominant to other colors (c) except white (Wf), which is epistatic to canary yellow (B) (Wehner, 2007). Coral red (Y) is more common than orange (yO) and salmon yellow (y), and orange (yO) dominates salmon yellow (y). In addition, canary yellow (C) is epistatic to Y and influenced by inhibitor of canary yellow (i-C), and a homozygous gene results in red flesh even in the presence of C (Henderson et al., 1998). Bang et al. (2010) reported that the py gene controls the formation of pale yellow-fleshed watermelon. Introduction To date, many scientists have summarized a series of QTLs or genes related to watermelon flesh color. In 2003, two red flesh-related QTLs, which were detected in groups 2 and 8, were first reported (Hashizume et al., 2003). The biosynthesis of carotenoids has been well studied in plants (Sun et al., 2018) and reported in watermelon. qFC.1 is reportedly related to β-carotene in a 2.4-Mb region on chromosome 1 that contains ClPsy1 (Branham et al., 2017). The pale green flesh color is also regulated by the major effective QTL qfc10.1 located on chromosome 10 spanning an approximately 519-Kb region, and Cla97C10G185970, annotated as plastid lipid-associated protein, was identified as the most candidate gene (Pei et al., 2021). The red flesh color gene ClLCYB (lycopene beta-cyclase) has been fine mapped to chromosome 4 (Liu et al., 2015; Wang et al., 2019). Zhang and colleagues found that the ClLCYB protein abundance, instead of the ClLCYB transcript level, is negatively correlated with lycopene accumulation (Zhang et al., 2020). Genes located in the carotenoid metabolic pathway are not the only factors responsible for flesh color formation. Yscr (a single dominant gene), which has been fine mapped to an approximately 40-Kb region on chromosome 6, generates the scarlet red flesh color rather than the common red flesh color in watermelon. Four genes encoding glycine-rich cell wall structural proteins are regarded as candidate genes (Li et al., 2020). ClPHT4;2 regulates chromoplast development, and the expression level of ClPHT4;2 in red-fleshed accessions is approximately 10-fold higher than that in white- fleshed accessions. In addition, the overexpression of ClPHT4;2 lead to a high accumulation of carotenoids (Zhang et al., 2017). The gf trait has been reported in some plants, such as cauliflower (Li et al., 2001), cassava (Welsch et al., 2010), melon (Tzuri et al., 2015), rice (Bai et al., 2016), wheat (Wang et al., 2014), potato (Mortimer et al., 2016) and banana (Paul et al., 2017), and the Psy and Or genes are involved in the formation of this trait. SlPsy1 is related to carotenoid accumulation in ripening tomato fruits (Fantini et al., 2013), and the silencing of SlPsy1 in tomato using CRISPR/Cas9 changes the flesh color from red to yellow (Dahan-Meir et al., 2018). In watermelon and citrus, the types and expression patterns of the Psy genes are similar to those found in tomato (Peng et al., 2013; Lv et al., 2015). Introduction Together with the DXS, LCYB, CHY and ZEP Page 3/27 genes, Psy always affects the gf trait at the transcriptional level (Pons et al., 2014, Zeng et al., 2015, Farre et al., 2016). The chromoplast is the main location for carotenoid accumulation, and enhancing the metabolic capacity would be beneficial for gf trait formation. The Or gene is known to play a role in carotenoid generation and chromoplast differentiation and exhibits chaperone activity for regulating Psy at the posttranscriptional level (Welsch et al., 2018). Or can also increase carotenoid accumulation by inhibiting hydroxylase and degradative reactions (Chayut et al., 2017), and overexpression of the Or gene in potato and white maize endosperm could enhance carotenoid synthesis by approximately 6- and 32-fold, respectively (Li et al., 2012, Berman et al., 2017). The carotenoid content cannot be enhanced by overexpression of the Or gene in tissues with abundant β-carotenoid accumulation (Shumskaya et al., 2012). Although the Psy gene plays an important role in carotenoid synthesis in many plants, molecular markers developed based on forward genetics results have more effective applications in breeding. In watermelon, the gf trait remains poorly understood. The objectives of our study were to understand the genetic basis of the golden flesh color trait in watermelon and identify the genes responsible for this important trait in segregating populations. Suggestions for marker-assisted selection (MAS) for the golden trait are also provided based on the results. Plant materials and phenotype evaluation “Cream of Saskatchewan” (COS) with a pale-yellow flesh color and the watermelon accession PI 192938 with the gf trait (orange flesh) were used as the parental materials for obtaining the F1, and F2 and backcross populations. COS was kindly provided by Angela R. Davis at the U.S. Department of Agriculture, Agricultural Research Service, South Central Agricultural Research Laboratory, and PI 192938 was obtained from the U.S. National Plant Germplasm System. All the experimental materials were grown in a greenhouse at the Xiangyang Experimental Agricultural Farm of Northeast Agricultural University, Harbin, China, from 2018 to 2020. The F2 populations were planted in 2018 and 2019 (93 and 297 plants, respectively) to verify the genetic segregation ratio and initial mapping, and BC1P1 (37 plants) and BC1P2 (84 plants) populations were planted in 2019 to further confirm their genetic inheritance. A total of 1,003 F2 individuals were planted in 2020 for recombinant selection and fine mapping. Fifteen days after planting, each plant was numbered with a tag, and young and disease-free leaves were sampled and freshly frozen for DNA extraction using the improved hexadecyl trimethyl ammonium bromide (CTAB) method. All the plants were artificially pollinated, and this date was recorded; and 35 to 40 days after pollination, the fruits were collected, cross-cut and photographed for flesh evaluation. BSA-seq and initial mapping BSA-seq and initial mapping Page 4/27 Page 4/27 Equal amounts of genomic DNA from 20 pale yellow and 20 gf individuals were selected from 297 F2 plants (in 2019) for flesh color gene pool construction. The two gene pools and genomic DNA of the parental lines were resequenced at the BGI Research Institute using the Illumina HiSeq Xten platform (at least 20× coverage genome sequencing depth for each sample) for BSA-seq analysis. The sequence data were aligned to the reference genome 97103 v2 Equal amounts of genomic DNA from 20 pale yellow and 20 gf individuals were selected from 297 F2 plants (in 2019) for flesh color gene pool construction. The two gene pools and genomic DNA of the parental lines were resequenced at the BGI Research Institute using the Illumina HiSeq Xten platform (at least 20× coverage genome sequencing depth for each sample) for BSA-seq analysis. The sequence data were aligned to the reference genome 97103 v2 (ftp://cucurbitgenomics.org/pub/cucurbit/genome/watermelon/97103/v2/) using Burrows-Wheeler Aligner (BWA, Li and Durbin, 2009) to obtain the snp.vcf file. Plant materials and phenotype evaluation The SNP sites were identified with SAMtools software (Li et al., 2009). Different homozygous sites in the two gene pools of the F2 population were extracted to calculate the SNP variation frequency and analyze the regions significantly associated with the gf trait. Subsequently, 500 bp before and after the SNP site sequences were extracted from the resequencing data for molecular marker exploitation. Candidate cleaved amplified polymorphic sequence (CAPS) lo (Thomas et al., 2004) with the snp.vcf file. According to the BS in the initial chromosome region were designed with nine rest MboI, TaqI, AluI, XhoI, DraI and BsaHI) using Primer Premier v6 amplification, mixing and enzyme digestion were previously d be converted into CAPS markers were designed as Kompetitiv genotyped at the Vegetable Research Center of the Beijing Aca Sciences. Individuals with recessive traits in the F2 population genotyped for initial mapping. Candidate cleaved amplified polymorphic sequence (CAPS) loci were detected using SNP2CAPS software (Thomas et al., 2004) with the snp.vcf file. According to the BSA-seq results, CAPS loci evenly distributed in the initial chromosome region were designed with nine restriction endonucleases (HincII, MspI, BsrI, MboI, TaqI, AluI, XhoI, DraI and BsaHI) using Primer Premier v6.24. The protocols used for PCR amplification, mixing and enzyme digestion were previously described. SNP sites that could not be converted into CAPS markers were designed as Kompetitive Allele Specific PCR (KASP) markers and genotyped at the Vegetable Research Center of the Beijing Academy of Agricultural and Forestry Sciences. Individuals with recessive traits in the F2 population planted in 2019 were selected and genotyped for initial mapping. Fine mapping and candidate gene sequence analysis A total of 1,003 F2 individuals were sown in a greenhouse in the spring of 2020, and the two flanking markers in the initial mapping region were used for recombinant selection. All recombinants were transplanted to the greenhouse at the Xiangyang Experimental Agricultural Station of Northeast Agricultural University, Harbin China, and self-pollinated to obtain F2:3 families. The flesh color of mature fruit was recorded, photographed and genotyped using new markers in the initial mapping segment to detect recombinant events for narrowing down the target region. The F2:3 seeds of recombinants with dominant traits were planted with 30 plants of each family in the autumn of 2020. The genotype of the recombinants with dominant traits was confirmed according to the flesh color segregation in their F2:3 families. Recombinants from the initial mapping panel were also selected to facilitate fine mapping. Candidate gene annotation in the fine-mapping region was performed with the reference genome (97103 v2). A coding sequence comparison was first performed with the resequencing data, and the results were further confirmed with the sequences from the gene cloning results to detect nonsynonymous SNPs and gene structure variations between COS and PI 192938. To examine the allele diversity of ClPsy1 among natural watermelon populations, resequencing data from 24 watermelon accessions (5 varieties resequenced in our previous study and 19 varieties resequencing data acquired from Guo et al., (2014) were used to extract the sequences of ClPsy1 for comparison. Page 5/27 Page 5/27 Page 5/27 Page 5/27 RNA-seq Flesh samples of COS and PI 192938 were collected from the center area of four parental fruits at 10, 18, 26, 34 and 42 days after pollination (DAP). The flesh tissues were immediately frozen in liquid nitrogen and stored at -80°C for RNA extraction. Three out of five watermelon fruits with similar growth conditions were collected per sample. Flesh samples with three replicates at each stage were sent to Biomarker Technologies for RNA-seq with an Illumina HiSeq 2000 system. RNA-seq library construction and data analysis were performed according to the protocols described by Zhong et al. (2011) and Guo et al. (2013). Bowtie and Trapnell (Langmead et al. 2009) software were used for identification of fragment mismatches and read alignment to the watermelon genome (Guo et al. 2013). Gene expression analysis The RNA Simple Total RNA Kit (Tiangen, China) and ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) were used for total RNA extraction and cDNA synthesis, respectively. Specific primers for candidate genes (Cla97C01G008760 and 196920), transcription factors (TFs; Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130) and carotenoid metabolic pathway genes [ClZDS (Cla97C06G118930), ClNCED-7 (Cla97C07G137260), ClCRTISO (Cla97C10G200950), ClCHYB (Cla97C05G090480), ClLCYB (Cla97C04G070940), ClPDS (Cla97C07G142100), ClNCED-1 (Cla97C01G024630), and ClCHXE (Cla97C01G002480)] were designed for gene expression analysis through quantitative real-time polymerase chain reaction (qRT-PCR) (Table 3). SYBR Green Master Mix (Novogene, Beijing) was used to perform the qRT-PCRs in the QTOWER Real-Time PCR System (Analytik Jena, Germany) according to the manufacturer’s instructions. qRT-PCR amplification and mixing were performed as previously described. Each experiment was performed with three biological repetitions and three technical repetitions, and relative gene expression levels were determined using the 2ΔΔCT method (Bustin et al 2009). Fine mapping and candidate gene sequence analysis The number of reads mapped to each watermelon gene model was obtained and then standardized to the number of reads per kilobase of transcript per million mapped reads (RPKM). The RNA-seq data of COS were sent to the NCBI database with the SRA number PRJNA587316, whereas the SRA number of the PI 192938 flesh RNA-seq data was PRJNA733842. The flesh tissue samples of COS and PI 192938 were collected at the same time. esh samples of COS and PI 192938 were collected from the center area of four parental fruits at 10, 18, 6, 34 and 42 days after pollination (DAP). The flesh tissues were immediately frozen in liquid nitrogen nd stored at -80°C for RNA extraction. Three out of five watermelon fruits with similar Promoter region cloning and candidate transcription factor prediction The promoter region of ClPsy1 was cloned with genomic DNA from five watermelon accessions with different flesh colors, namely, COS, PI 192938, LSW-177 (red flesh), PI 635597 (canary yellow flesh) and PI 186490 (white flesh), using the primers listed in Table 2. Cis-elements in each promoter were predicted using PlantCARE online software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and the five promoter sequences were compared to detect the variation. SNPs or structural alterations located in cis-elements were selected as important loci. The resequencing data of 24 other watermelon accessions were also used to extract the sequences of the ClPsy1 promoter and thus detect the variation diversity. Page 6/27 The RNA-seq data of COS and PI 192938 flesh tissues were used to preliminarily view the expression patterns of the TFs that may bind with the cis-elements located in the variation region. TFs exhibiting expression patterns similar to that of ClPsy1 between COS and PI 192938 may be regarded as important factors for further verification. Statistical analysis The genetic analysis and evaluations of the differences in gene expression were performed using SPSS v.21.0 software (SPSS Inc., Chicago, IL, USA). Prism 7.0 software was used (GraphPad Inc., La Jolla, CA, USA) for illustration preparation. The gf trait in watermelon is controlled by a simply inherited gene The carotenoid composition and content in the mature flesh of COS and PI 192938 were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in our previous study (Fang et al. 2020). β-carotene and violaxanthin appeared to be the two pigments showing major differences in content between the two parental materials, and the contents in PI 192938 (16.133 ± 0.952 µg/g) were approximately 53.2-fold higher than those in COS (0.033 ± 0.004 µg/g). Based on the flesh color associated with the two pigments, we speculated that the high accumulation of β-carotene in PI 192938 may be the main reason for the gf trait (Fig. 1a). The flesh color of F1 was canary yellow, similar to that of COS, and four flesh categories were segregated in the F2 generation: gf, pale yellow, canary yellow and gf mixed with canary yellow (Fig. 1b). F2 individuals can also be divided into gf and non-gf groups (pale yellow, canary yellow and gf mixed with canary yellow, Fig. 1c to e). According to these classification criteria, the COS×PI 192938-F2 population in 2018 (84 individuals in total) consisted of 17 gf and 67 non- gf plants and exhibited a 1:3 genetic ratio (χ2 = 1.016, p = 0.313), whereas in 2019, 279 F2 individuals consisted of 74 gf and 205 non-gf plants, which was also consistent with the 1:3 genetic ratio (χ2 = 0.345, p = 0.557). For generation of a backcross population between F1 and COS, none of the individuals exhibited the gf color. In the backcross population derived from F1 and PI 192938, 39 plants had the gf color, and 45 plants had a non-gf color, which corresponded to a ratio of 1:1 (χ2 = 0.429, p = 0.513, Table 1). Based on the above results, we conclude that the gf trait in watermelon flesh is controlled by a simply inherited gene and that pale yellow flesh is partly dominant to the gf color. BSA-seq and recombinant deletion of the gf locus in a 39‑Kb region identified ClPsy1 as a candidate gene After filtering low-quality and short reads, 125,549,810 and 60,739,330 clean read pairs were obtained for COS and PI 192938 with approximately 10.01 (28.76× depth coverage) and 8.56 (22.87× depth coverage) Gbp clean bases, respectively, and Q30 values above 89.89% were found. A total of 86.50% and 92.48% of these clean reads of COS and PI 192938, respectively, were successfully mapped to the reference genome, and 68,585,634 and 68,541,696 clean read pairs were generated from the gf pool (25.7× depth Page 7/27 Page 7/27 coverage and 92.35% properly mapped ratio) and pale-yellow flesh pool (25.88× depth coverage and 93.16% properly mapped ratio), respectively, through the Illumina high-throughput sequencing platform. A total of 366,358 and 374,417 SNPs were identified between the reference genome and the two gene pools, respectively. The SNP index for each identified SNP was calculated, and the average SNP index was computed in a 1-Mb interval using a 10-Kb sliding window. By combining the SNP index information from the gf color pool and pale-yellow flesh pool, the ΔSNP index was calculated and plotted against the genome positions. According to the ΔSNP index value, an obvious signal related to the gf color was detected on chromosome 1, spanning approximately 2.99 Mb (from 8,912,000 bp to 11,900,000 bp, Fig. 2a). A total of 30 CAPS and two KASP markers evenly distributed in BSA-seq chromosome segments were developed based on parental line resequencing data, and 10 markers (eight CAPS markers and two KASP markers) were used for initial mapping after polymorphism detection among COS, PI 192938 and their F1 generation. Individuals with a recessive phenotype (gf trait) from 2019 were selected for genotyping with the 10 polymorphic markers. The candidate region was narrowed to a physical distance of 290.214 Kb (from 9,272,322 bp to 9,562,536 bp) using 11 recessive-trait plants (including nine recombinants) between the CAPS markers Chr01_9272322 and Chr01_9562536 with one and two recombinants (Fig. 2b). To further narrow down the initial mapping region precisely, a larger COS×PI 192938-F2 segregating population including 1,003 individuals was subjected to genotyping of the primary flanking markers Chr01_9242322 and Chr01_9562536 in the spring of 2020. A total of 20 recombinants were screened for further fine mapping of the gf gene. Another nine polymorphic markers were developed to genotype the 20 recombinants. The target trait genotype of the dormant recombinants was confirmed based on phenotypic segregation in their F3 families. BSA-seq and recombinant deletion of the gf locus in a 39‑Kb region identified ClPsy1 as a candidate gene Finally, the gf locus was delimited between the CAPS markers Chr01_9440282 and Chr01_9479366 (physical distance of approximately 39.08 Kb) with two and nine recombinants, respectively (Fig. 2c). According to the watermelon reference genome, the 39.08-Kb region contained only two annotated candidate genes, Cla97C01G008760 and Cla97C01G008770. Cla97C01G008760 encodes a phytoene synthase protein (ClPsy1), and Cla97C01G008770 was annotated as a GATA zinc finger domain- containing protein. To identify the candidate gene for the gf locus, we first analyzed the genomic variations in the two candidate genes between the parental lines with resequencing data. The results identified no polymorphic sites in Cla97C01G008770, whereas one nonsynonymous SNP mutation, SNP9,448,870 (A→G, located in the first exon at the 9,448,870th bp position), was detected in the coding region of Cla97C01G008760 between COS and PI 192938. In COS, base A encodes glutamic acid (Glu), whereas in PI 192938, this base is mutated to base G, resulting in an amino acid change from Glu to lysine (Lys). To further confirm the sequence variation, we cloned the coding regions of the two candidate genes in COS and PI 192938. This SNP mutation was still found between the two parental lines. We further developed this nonsynonymous SNP into the KASP marker Chr01_9448870 and genotyped F2 individuals from 2018 to 2019. As a result, ClPsy1A:A exhibited the gf color, whereas ClPsy1G:A/G:G showed Page 8/27 a non-gf color, which indicated that Chr01_9448870 cosegregated with the phenotype in all the plants (Fig. 3a). a non-gf color, which indicated that Chr01_9448870 cosegregated with the phenotype in all the plants (Fig. 3a). Although no variation in the Cla97C01G008770 gene sequence was found between COS and PI 192938, we found some polymorphic sites in the promoter region. To further confirm this hypothesis, we analyzed the gene expression patterns of the two candidate genes in COS and PI 192938 flesh tissues collected from different stages of flesh color formation. The results showed that the two parental lines exhibited similar expression trends across the five developmental stages (10, 18, 26, 34 and 42 DAP), and no significant difference in Cla97C01G008770 was found (Fig. 3b). For Cla97C01G008760 (ClPsy1), the two parental lines also showed similar expression patterns (the expression level was upregulated gradually during flesh maturation), but at 26 DAP, the allele of ClPsy1 in PI 192938 exhibited significantly higher expression than that in COS, and this increase continued to be observed until 42 DPA (mature stage, Fig. 3c). BSA-seq and recombinant deletion of the gf locus in a 39‑Kb region identified ClPsy1 as a candidate gene Our previous research also showed that 26 DAP may be an important developmental stage for flesh color formation (Fang et al., 2020). At this stage, the colored COS and PI 192938 watermelon flesh started to abundantly accumulate carotenoids. Hence, we hypothesized that ClPsy1 is the most likely candidate gene for the gf locus and is responsible for high β-carotene accumulation in watermelon. Nucleotide variation in the ClPsy1 gene structure among natural watermelon accessions To examine the allelic diversity of the ClPsy1 gene in natural watermelon groups, we examined the nucleotide variation of the ClPsy1 locus in 26 resequenced accessions with different flesh colors (red, orange, canary yellow, pale yellow, light green and white), including 18 C. lanatus, four C. mucosospermus and four C. amarus accessions (Fig. 4). SNP9,448,870 was still present, but this mutation was not correlated with flesh color among the different watermelon accessions and exhibited no obvious difference between the cultivated and wild-type watermelon groups. These results indicated that this site may not affect carotenoid accumulation. In addition to SNP9,448,870, another nonsynonymous SNP mutation, SNP9,448,438 (C→T, located in the first exon at the 9,448,438th bp position), was also detected. Interestingly, SNP9,448,438 existed only in the C. amarus group, resulting in an amino acid substitution from proline (Pro in the C. lanatus and C. mucosospermus groups) to serine (Ser in the C. amarus group). To analyze the reason for the low ClPsy1 expression in COS, we cloned the 1,996-bp promoter sequence from four cultivated watermelon varieties: COS, PI 192938, LSW-177 (red flesh) and PI 635597 (canary yellow). The promoter sequences of 14 other cultivated watermelon accessions were extracted from their genome resequencing data. Interestingly, a total of six SNPs (SNP342, SNP598, SNP898, SNP1,257, SNP1,634 and SNP1,694) were detected in the COS promoter region compared with the other 17 watermelon accessions, which exhibited consistent promoter sequences. SNP598 and SNP1,257 were located in the MYC- and MYB-binding sites, respectively, whereas the other four SNPs were not located in the sequence of any cis-acting element (Fig. 5a). MYB and MYC2 may be important transcription factors regulating the expression level of ClPsy1 MYB and MYC2 may be important transcription factors regulating the expression level of ClPsy1 MYB and MYC2 may be important transcription factors regulating the expr Page 9/27 We then used the RNA-seq data of COS and PI 192938 flesh tissues (collected at 18, 26 and 42 DAP, data not shown in this manuscript) to obtain an overview of the expression patterns of all MYB and MYC TFs. A total of 65 MYB TFs with read per kilobase per million mapped reads (RPKM) values were detected, and only two MYB TFs (Cla97C10G196920 and Cla97C02G046390) exhibited expression tendencies similar to that of ClPsy1 in COS and PI 192938. An obvious significant difference in expression began to be observed at 26 DAP in PI 192938 compared with COS and continued to be observed at 42 DAP (mature stage). For all 22 MYC TFs, Cla97C06G112130 (annotated as a MYC2 transcription factor) also showed an obvious significant difference in expression between COS and PI 192938 at 26 DAP. We further examined the expression levels of Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130 in COS and PI 192938 flesh tissues collected at five developmental stages by qRT-PCR to verify the expression pattern. The results showed the same tendency as the RNA-seq data (Fig. 5b to d). LSW-177 was a red flesh-colored watermelon accession with the same genotype in the gene sequence and promoter region as PI 192938. The RNA-seq data between COS and LSW-177 (red flesh) flesh tissues (BioProject number PRJNA338036) were also used for analyzing the expression levels of Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130. The results showed that the three TFs also presented a higher expression level in LSW-177 than in COS (Fig. 8a to c). In watermelon fruit rinds, the expression levels of the three TFs were also clearly lower than those in the flesh of 97103 (red flesh), which indicated that these TFs may be expressed in tissues with high carotenoid accumulation according to the RNA-seq data of SRP012849 (Fig. 8d to f). Cla97C02G046390 and Cla97C06G112130. The results showed that the three TFs also presented a higher expression level in LSW-177 than in COS (Fig. 8a to c). In watermelon fruit rinds, the expression levels of the three TFs were also clearly lower than those in the flesh of 97103 (red flesh), which indicated that these TFs may be expressed in tissues with high carotenoid accumulation according to the RNA-seq data of SRP012849 (Fig. 8d to f). MYB and MYC2 may be important transcription factors regulating the expression level of ClPsy1 The conserved domains of Cla97C10G196920 (148 aa) and Cla97C02G046390 (110 aa) were extracted and compared to the Arabidopsis Information Resource (TAIR). The results showed that AtMYB21/AtMYB3, AtMYB24, AtMYB57, AtMYB59, and AtMYB48 were the first three (or two) homologs. Cla97C06G112130 has two conserved domains: the N-terminus of the bHLH-MYC and R2R3-MYB TFs and the N-terminus of a family of MYB and MYC transcription factors (156 aa). The other superfamily is the bHLH domain superfamily (70 aa), and AtMYC2 exhibited the highest homology. We speculated that ClMYB and ClMYC2 may be two important TFs regulating ClPsy1 expression due to variations in the binding sites in their promoter region between COS and PI 192938. Although MYB and MYC TFs have many functions, they have not been reported to play a role in flesh color formation in watermelon. The gene expression and genotype variations in carotenoid pathway genes between COS and PI 192938 provide insight into gf trait formation in watermelon flesh We examined the transcript abundances of ClPDS, ClZDS, ClCRTISO, ClLCYB, ClCHYB and ClNCED7 in COS and PI 192938 flesh tissues collected at five developmental stages (10, 18, 26, 34 and 42 DAP) by qRT-PCR (Fig. 6a to f). ClPDS, ClZDS and ClCRTISO exhibited the same expression trend as ClPsy1 between the two parental lines. The transcript abundance of PI 192938 was always higher than that of COS throughout all developmental stages, particularly at 26 DAP. It has been reported that ClLCYB regulates lycopene accumulation at the protein level (Zhang et al., 2020), and COS and PI 192938 have the same single-nucleotide mutation as red-fleshed watermelon accessions. The mutation of G676th to T676th altered the 226th amino acid from valine (Val) to phenylalanine (Phe), whereas the mutation of Page 10/27 Page 10/27 G1,305th to C1,305th altered the 435th amino acid from lysine (Lys) to asparagine (Asp). This finding indicated that the ClLCYB protein may have the same function in COS and PI 192938. The lycopene content in the mature flesh of the two parental lines was quite low compared with that in a red-fleshed variety identified in our previous study (Fang et al., 2020). The expression level of ClNCED-7 in both parental lines showed an increasing trend over time after pollination, and COS presented a significantly higher expression level than PI 192938, except at 26 DAP. The sequence variations in genes encoding enzymes at each step of the carotenoid pathway were also analyzed between COS and PI 192938 using resequencing data. Three SNPs were found in the coding region of ClZDS in PI 192938 compared with that in COS, and two of these SNPs led to amino acid substitutions. Mutation of the 161st base (G→A) resulted in a change in the 54th amino acid from serine (Ser) to asparagine (Asp), and mutation of the 480th amino acid (G→T) resulted in a change in the 160th amino acid from lysine (Lys) to asparagine (Asp). Only one nonsynonymous residue was detected in ClCRTISO, and mutation of the 526th base (T→C) changed the 176th amino acid from tyrosine (Tyr) to histidine (His). No variations were detected in the coding region of ClPDS or ClNCED-7. According to the above-described results, we speculated the cause of the high β-carotene content shown in Fig. 7. The gene expression and genotype variations in carotenoid pathway genes between COS and PI 192938 provide insight into gf trait formation in watermelon flesh High expression of ClPsy1 in PI 192938 contributed to increased phytoene accumulation, and the abundance of phytoene may upregulate the expression of ClPDS, ClZDS and ClCRTISO at each step of the carotenoid metabolism pathway to result in the synthesis of higher amounts of zeta-carotene and tetra-cis-lycopene in PI 192938. Tetra-cis-lycopene could be isomerized through ClCRTISO to generate lycopene, which is the carotenoid upstream of β-carotene. The same genotype of the ClLCYB protein may have a similar cyclization effect in PI 192938 and COS, and nearly all lycopene can be cyclized into β- carotene. High expression of ClCHYB increased violaxanthin accumulation, whereas low expression of ClNCED-7 may prevent β-carotene metabolism in PI 192938. These factors may be the main reasons for the high accumulation of violaxanthin and β-carotene in PI 192938 and thus its orange flesh color. Various patterns of ClPsy1 regulation may exist among different watermelon accessions The first committed and rate-controlling step is mediated by the phytoene synthase (Psy) protein, which catalyzes the conversion of two molecules of GGPP to phytoene (colorless) as the first carotenoid (Cazzonelli and Pogson, 2010; Welsch et al., 2010; Giuliano et al., 2017). As an important gene in the carotenoid metabolic pathway, Psy1 has been reported to function in carrot (Maass et al., 2009 and Massimo et al., 2016), banana (Paul et al., 2017), citrus (Lu et al., 2018), tomato (Xiong et al., 2019) and pepper (Jeong et al., 2019). Compared with white and bitter wild-type watermelon, modern cultivated watermelon (C. lanatus) exhibits various flesh colors. ClPsy1, located in a selective chromosome segment, may play an important role in watermelon flesh color formation during the domestication process (Guo et al., 2020). In this study, the sequence variations in both coding and promoter regions were analyzed in 26 watermelon accessions belonging to three subspecies with different flesh colors. No Page 11/27 Page 11/27 representative mutations were found between the C. mucosospermus and C. lanatus groups. However, multiple variations were detected in the C. amarus group compared with the C. mucosospermus and C. lanatus groups. A low gene expression level of ClPsy1 was also found in C. amarus (such as PI 296341- FR, Guo et al., 2015). SNP9,448,438 was detected only in the C. amarus group, but whether this SNP mutation alerts ClPsy1 function needs further verification. Based on published RNA-seq data or previous research, white-fleshed C. mucosospermus (such as PI 186490, Fang et al., 2020 and Wang et al., 2021) showed a significantly lower expression level than colored cultivated watermelon varieties, although the former presented the same genotype in coding and promoter regions as the colored accessions. This feature may be regulated by TFs or epigenetic factors, but further research is needed. Compared with other watermelon accessions, two SNP mutations in cis-acting element sites were only found in COS. Further qRT-PCR results implied that these two SNP mutations may be the main reason for the low ClPsy1 expression level. Enzyme activity is also an important factor affecting ClPsy1 gene function in carotenoid accumulation. The reduction in total carotenoids was consistent with the requirement of galactolipids for PSY protein activity in etiolated seedlings of Arabidopsis mutants (Fujii et al., 2018). Various patterns of ClPsy1 regulation may exist among different watermelon accessions In loquat (Eriobotrya japonica Lindl.), a mutant EjPSY2Ad lacking the C-terminal region (694-bp segment) in the fifth exon and thus no corresponding catalytic activity resulted in the absence of carotenoids and thus an inability to form white flesh (Fu et al., 2014). The aspartic acid-rich region (DXXXD) is an important functional domain of the PSY protein (Zhai et al., 2016). We also compared the mutation locations among watermelon accessions with different flesh colors, but none of the SNPs were located in this domain region. Among the varieties belonging to the C. mucosospermus and C. lanatus groups, SNP9,448,870 was also not correlated with flesh color in the natural watermelon panel. These two lines of evidence may imply that the function of ClPsy1 in flesh color formation is dependent on expression rather than a difference in enzymatic activity. These results showed that the expression pattern of ClPsy1 was regulated in different ways in different watermelon subspecies. Regulatory factors in carotenoid accumulation and flesh color formation The function of Psy is affected by many regulatory factors at both the transcriptional and protein translation levels. In tomato, ripening inhibitors (RINs), through their interaction with the SlPsy1 promoter, have been shown to regulate fruit carotenoid concentrations (Martel et al., 2011). B-box zinc- finger transcription factor (SlBBX20) can activate the expression of SlPsy1, leading to dark-green fruits and leaves and higher levels of carotenoids, by directly binding to a G-box motif in its promoter (Xiong et al., 2019). A transcriptomic analysis of AdMYB7-infiltrated N. benthamiana leaves showed upregulation of NbPSY (Ampomah-Dwamena et al., 2019). The overexpression of CsMADS6 in citrus calli can increase the carotenoid content by directly binding to the CsPSY, CsPDS and CsCCD1 promoters (Lu et al., 2018). Two ClMYB and one ClMYC2 TFs exhibited expression trends similar to that of ClPsy1 in COS and PI 192938 and were thus hypothesized to be regulatory factors of ClPsy1. Compared with the Arabidopsis genome, AtMYB21/AtMYB3, AtMYB24, AtMYB57, AtMYB48 and AtMYB59 showed high identity in conserved domains with the two ClMYBs (Cla97C10G196920 and The function of Psy is affected by many regulatory factors at both the transcriptional and protein translation levels. In tomato, ripening inhibitors (RINs), through their interaction with the SlPsy1 promoter, have been shown to regulate fruit carotenoid concentrations (Martel et al., 2011). B-box zinc- finger transcription factor (SlBBX20) can activate the expression of SlPsy1, leading to dark-green fruits and leaves and higher levels of carotenoids, by directly binding to a G-box motif in its promoter (Xiong et al., 2019). A transcriptomic analysis of AdMYB7-infiltrated N. benthamiana leaves , ) p y showed upregulation of NbPSY (Ampomah-Dwamena et al., 2019). The overexpression of CsMADS6 in citrus calli can increase the carotenoid content by directly binding to the CsPSY, CsPDS and CsCCD1 promoters (Lu et al., 2018). Two ClMYB and one ClMYC2 TFs exhibited expression trends similar to that of ClPsy1 in COS and PI 192938 and were thus hypothesized to be regulatory factors of ClPsy1. Compared with the Arabidopsis genome, AtMYB21/AtMYB3, AtMYB24, AtMYB57, AtMYB48 and AtMYB59 showed high identity in conserved domains with the two ClMYBs (Cla97C10G196920 and Page 12/27 Page 12/27 Cla97C02G046390), whereas bHLH-MYC/R2R3-MYB and AtMYC2 showed the highest homology to ClMYC2 (Cla97C06G112130). Regulatory factors in carotenoid accumulation and flesh color formation With the exception of the two mutation sites, some other MYB- and MYC2- binding sites were located in the promoter regions of COS and PI 192938, which could partly explain why obvious expression levels of Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130 in COS could be detected at 26 DAP, even though the binding sites participate in the variation. The MYB TF family is very large and functionally diverse in plants. MYB members have been reported to participate in carotenoid metabolism. Chili pepper fruits synthesize and accumulate carotenoid pigments, which are responsible for the yellow, orange and red colors. CaCCS (capsanthin-capsorubin synthase), CaBCH (β-carotene hydroxylase), and CaPSY were clustered with six MYB-related genes (CaDIV1, CaDIV3, CaMYBR13, CaTRF2, CaMYBC1, and CaPHR9) and an atypical MYB (CaMYB5R) based on a coexpression analysis (Arce-Rodriguez et al., 2021). The R2R3-MYB transcription factor CrMYB68 directly regulates the α- and β-branches in the carotenoid pathway in Citrus reticulata. Reduced expression of CrBCH2 and CrNCED5 is responsible for the delay in α- and β-carotene synthesis. The expression of these genes is negatively correlated with the expression of CrMYB68 in green fruit (Feng et al., 2017). A coexpression network and transient expression analysis suggested a potential direct link between flavonoid and carotenoid biosynthesis pathway genes (PSY, ZDS and CYP97C) through MYB TF regulation in Primula vulgaris (Li et al., 2020). SlMYB72 directly binds to the SlPSY, SlZISO, and SlLCYB genes and regulates carotenoid biosynthesis in tomato. The downregulation of SlMYB72 decreases the lycopene content and promotes β-carotene production and chromoplast development (Wu et al., 2020). CpMYB1 (MYB44-like) and CpMYB2 are transcriptional repressors that can bind to the CpPDS2, CpPDS4, and CpCHY-b promoters and suppress the activities of these genes in papaya (Fu et al., 2020). In Medicago truncatula, an R2R3-MYB protein (WHITE PETAL1, WP1) functions as a transcriptional activator that modulates floral carotenoid pigmentation by directly regulating the expression of multiple carotenoid biosynthesis-related genes (Meng et al., 2019). The silencing of strigolactone (SL, carotenoid- derived phytohormone) biosynthesis-related genes results in the upregulation of MYC2 (Xu et al., 2019). To date, the regulation of flesh color by MYB or MYC2 TFs in watermelon has not been studied. The binding effect and regulatory mechanism of Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130 need further verification and research. Regulatory factors in carotenoid accumulation and flesh color formation In Medicago truncatula, an R2R3-MYB protein (WHITE PETAL1, WP1) functions as a transcriptional activator that modulates floral carotenoid pigmentation by directly regulating the expression of multiple carotenoid biosynthesis-related genes (Meng et al., 2019). The silencing of strigolactone (SL, carotenoid- derived phytohormone) biosynthesis-related genes results in the upregulation of MYC2 (Xu et al., 2019). To date, the regulation of flesh color by MYB or MYC2 TFs in watermelon has not been studied. The binding effect and regulatory mechanism of Cla97C10G196920, Cla97C02G046390 and Cla97C06G112130 need further verification and research. The Or gene (encoding a plastid-targeted protein containing a cysteine-rich zinc finger domain) is also another important gene for golden trait formation. In melon (Tzuri et al., 2015) and cauliflower (Li et al., 2001), the Or gene reportedly regulates β-carotene accumulation based on forward genetic research. CmOr exerts only a slight effect on CmPsy1 expression but strongly affects the CmPsy1 protein levels and enzymatic activity in melon (Chayut et al., 2017). In Arabidopsis and sweet potato, the PSY protein is affected by OR/OR-like proteins (Zhou et al., 2015; Park et al., 2016). The AtOR protein enhances AtPSY protein stability and increases the enzymatically active proportion of AtPSY in Atclpc1 in Arabidopsis (Welsch et al., 2018). Page 13/27 Page 13/27 Molecular breeding suggestions for golden-fleshed watermelon This study provides the first report of the gf gene in watermelon. Although the Psy gene reportedly functions in gf trait formation in some plants, genetic evidence remains lacking. According to the results, abundant ClPsy1 transcripts may lead to a rich β-carotene content in PI 192938. Red-colored flesh always has a higher ClPsy1 expression level than flesh with other colors (Wang et al., 2016), starting at the color turning stage and ending at the mature stage. Interestingly, the ClPsy1 content in the red- fleshed watermelon accession LSW-177 was higher than that in PI 192938 (Fang et al., 2020), but the β- carotene content in LSW-177 flesh was significantly lower than that in mature PI 192938 flesh (2.605±0.375 versus 16.133±0.952 μg/g), whereas the lycopene content in LSW-177 was significantly higher than that in PI 192938 (1.523±0.199 versus 25.950±0.390 μg/g). This finding indicated that the high expression of ClPsy1 was not the only main reason for β-carotene accumulation. The upstream gene of β-carotene is ClLCYB, which encodes a cyclase that can catalyze the conversion from lycopene to β- carotene. The downregulation of ClLCYB causes lycopene accumulation, whereas the overexpression of ClLCYB in the red-fleshed line causes the flesh color to change to orange (β-carotene) (Zhang et al., 2020). Among watermelon accessions, two nonsynonymous SNP sites generate three haplotypes (ClLCYBred, ClLCYBwhite, and ClLCYByellow). COS and PI 192938 contained the same haplotype of ClLCYByellow, which indicated that the ClLCYB protein had the same function in the two lines. In PI 192938, nearly all lycopene was cyclized into β-carotene to form a gold flesh. This finding may indicate that ClLCYB is also an important gene for β-carotene accumulation and partly explains the accumulation of β-carotene observed in LSW-177, COS and PI 192938. In tomato, the accumulation of tetra-cis-lycopene (the upstream carotenoid of lycopene) can also lead to the formation of orange flesh by SlCRTISO (Jayaraj et al., 2021, Dahan-Meir et al., 2018). In watermelon, the genotype of ClCRTISO also cosegregates with an orange flesh color (Jin et al., 2019). Visual observation alone does not allow differentiation of the two types of carotenoids. Although they have the same color, tetra-cis-lycopene is not converted into the precursor of vitamin A without β-cyclization. Molecular breeding suggestions for golden-fleshed watermelon The ld i l f fl h l b l id h h i l i l f i f In tomato, the accumulation of tetra-cis-lycopene (the upstream carotenoid of lycopene) can also lead to the formation of orange flesh by SlCRTISO (Jayaraj et al., 2021, Dahan-Meir et al., 2018). In watermelon, the genotype of ClCRTISO also cosegregates with an orange flesh color (Jin et al., 2019). Visual observation alone does not allow differentiation of the two types of carotenoids. Although they have the same color, tetra-cis-lycopene is not converted into the precursor of vitamin A without β-cyclization. The golden trait not only focuses on flesh color but also considers the physiological function of carotenoids. For the gf trait, the MAS process depends only on the expression level and the genotype of ClPsy1, and visual observation is not sufficient or accurate. The genotypes of ClPsy1, ClCRTISO and ClLCYB should also be considered at the same time to confirm carotenoids. For the gf trait, the MAS process depends only on the expression level and the genotype of ClPsy1, and visual observation is not sufficient or accurate. The genotypes of ClPsy1, ClCRTISO and ClLCYB should also be considered at the same time to confirm the carotenoid composition. The genotypes of ClPsy1, ClCRTISO and ClLCYB should also be considered at the same time to confirm the carotenoid composition. “Push” and “block” are the two main strategies for gf trait molecular breeding and biofortification (Watkins et al., 2020). The “push” strategy involves enhancing the metabolic flux of carotenoids synthesized upstream of β-carotene and is regarded as the most effective method. The overexpression of Psy or 1-deoxy-D-xylulose-5-phosphate synthase (DXS) increases the synthetic amounts of total carotenoids or β-carotene. Cassava (Welsch et al., 2010), rice (Bai et al., 2016), wheat (Wang et al., 2014), potato (Mortimer et al., 2016) and banana (Paul et al., 2017) show up to 100- to 1,000-fold higher contents of total carotenoids. Silencing the expression of genes downstream of β-carotene to prevent the degradation of target products is another strategy for regulating the gf trait, and this method Page 14/27 is called “block”. In potato, the inhibition of zeaxanthin epoxidase (ZEP), lycopene epsilon-cyclase (LCYE) and carotene hydroxylase (CHY) could lead to approximately 10-fold increases in the zeaxanthin and β- carotene contents (Pons et al., 2014). Molecular breeding suggestions for golden-fleshed watermelon “Push” and “Block” have always been used together to obtain better results: the overexpression of CrtB (a homologous gene of Psy in bacteria) accompanied by the silencing of CHY could produce a large amount of β-carotene in the endosperm of wheat (Zeng et al., 2015). Significantly high accumulation of zeaxanthin could be induced by the overexpression of Psy1 and the silencing of LCYE in the maize endosperm (Farre et al., 2016). Significantly high accumulation of zeaxanthin could be induced by the overexpression of Psy1 and the silencing of LCYE in the maize endosperm (Farre et al., 2016). Declarations Acknowledgments This work was supported by funding from the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province [Grant# UNPYSCT-201816]. Author contribution statement Shi Liu designed the experiment, analyzed the data and wrote the manuscript. Zhongqi Gao performed the major experiments with the help of Shi Liu. Xuezheng Wang and Feishi Luan revised the manuscript. Zuyun Dai, Zhongzhou Yang, Qian Zhang provided part of materials used in this study and participated in phenotypic data collection. Shi Liu and Zhongqi Gao contributed equally to this work. Feishi Luan, Xuezheng Wang and Shi Liu were as the co-corresponding authors. Data availability All data pertinent to the reported work have been provided in the manuscript or in the supplemental online materials. Conflict of interest The authors declare that there is no conflict of interest. Conflict of interest The authors declare that there is no conflict of interest. References 1. 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Wang N, Liu S, Gao P, Luan FS, Davis AR (2016) Developmental changes in gene expression drive accumulation of lycopene and beta-carotene in watermelon. J Am Soc Horti Sci 141:1–10 53. Wang N, Liu S, Gao P, Luan FS, Davis AR (2016) Developmental changes in gene expression drive accumulation of lycopene and beta-carotene in watermelon. J Am Soc Horti Sci 141:1–10 54. Watkins JL, Pogson BJ (2020) Prospects for carotenoid biofortification targeting retention and catabolism. Trends in Plant Sci 25(5):501–512 55. Wehner T (2007) Gene list for watermelon. Cucurbit Genet Coop Rep 30:9 56. Welsch R, Arango J, Bar C, Salazar B, Al-Babili S, Beltran J et al (2010) Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. Plant Cell 22:3348–3356 57. 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Zeng J, Wang XT, Miao YJ, Wang C, Zang ML, Chen X et al (2015) Metabolic engineering of wheat provitamin A by simultaneously overexpressing CrtB and silencing carotenoid hydroxylase (TaHyd). Tables Table 1. Segregation of golden flesh color in six generations from COS × PI 192938 cross over 2 years Populations Plants Orange Non- orange Expected segregation ratio P value of Chisquare tests PI 192938 15 15 0 N/A N/A COS 15 0 15 N/A N/A F1 15 0 15 N/A N/A 2018 F2 84 17 67 1:3 0.313 2019 F2 279 74 205 1:3 0.557 2019 BC1P1 37 0 37 0:1 0.513 2019 BC1P2 84 39 45 1:1 0.563 N/A: Not Applicable Table 2 and 3 are not available with this version. cultivars. Food Chem 174:52–59 Zhu F, Luo T, Liu CY, Wang Y, Yang HB, Yang W et al (2017) An R2R3-MYB transcription factor represses the transformation of alpha- and beta-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate. New Phytol 216(1):178–192 Table 2 and 3 are not available with this version. cultivars. Food Chem 174:52–59 J Agr Food Chem 63:9083–9092 62. Zeng J, Wang XT, Miao YJ, Wang C, Zang ML, Chen X et al (2015) Metabolic engineering of wheat provitamin A by simultaneously overexpressing CrtB and silencing carotenoid hydroxylase (TaHyd). J Agr Food Chem 63:9083–9092 63. Zhai SN, Li GY, Sun YW, Song JM, Li JH, Song GQ et al (2016) Genetic analysis of phytoene synthase 1 (Psy1) gene function and regulation in common wheat. BMC Plant Biol 16:228 63. Zhai SN, Li GY, Sun YW, Song JM, Li JH, Song GQ et al (2016) Genetic analysis of phytoene synthase 1 (Psy1) gene function and regulation in common wheat. BMC Plant Biol 16:228 64. Zhang J, Guo SG, Ren Y, Zhang HY, Gong GY, Zhou M et al (2017) High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon. New Phytol 213:1208–1221 64. Zhang J, Guo SG, Ren Y, Zhang HY, Gong GY, Zhou M et al (2017) High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon. New Phytol 213:1208–1221 65. Zhang J, Sun HH, Guo SG, Ren Y, Li MY, Wang JF et al (2020) Decreased protein abundance of lycopene β-cyclase contributes to red flesh in domesticated watermelon. Plant Physiol 183(3):1171– 1183 Page 19/27 66. Zhong S, Joung JG, Zheng Y, Chen YR, Liu B, Shao Y et al (2011) High-throughput illumine strand- specific RNA sequencing library preparation. Cold spring harbor protocols, (8), pdb-prot5652 66. Zhong S, Joung JG, Zheng Y, Chen YR, Liu B, Shao Y et al (2011) High-throughput illumine strand- specific RNA sequencing library preparation. Cold spring harbor protocols, (8), pdb-prot5652 67. Zhou XJ, Welsch R, Yang Y, Alvarez D, Riediger M, Yuan H et al (2015) Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis P Natl Acad Sci USA112:3558–3563 67. Zhou XJ, Welsch R, Yang Y, Alvarez D, Riediger M, Yuan H et al (2015) Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesi P Natl Acad Sci USA112:3558–3563 68. Zhu F, Luo T, Liu CY, Wang Y, Yang HB, Yang W et al (2017) An R2R3-MYB transcription factor represses the transformation of alpha- and beta-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate. New Phytol 216(1):178–192 68. Figures Page 20/27 Figure 1 Flesh color segregated in the genetic populations and carotenoid composition of the parental lines. a. Carotenoid composition of COS and PI 192938. The carotenoid data were obtained from our previous publication (Fang et al., 2020). b. From left to right, flesh color of mature fruits of COS, PI 192938 and their F1 generation. c to e. Three flesh colors in the F2 generation derived from COS and PI 192938. Figure 1 Flesh color segregated in the genetic populations and carotenoid composition of the parental lines. a. Carotenoid composition of COS and PI 192938. The carotenoid data were obtained from our previous publication (Fang et al., 2020). b. From left to right, flesh color of mature fruits of COS, PI 192938 and their F1 generation. c to e. Three flesh colors in the F2 generation derived from COS and PI 192938. Flesh color segregated in the genetic populations and carotenoid composition of the parental lines. a. Carotenoid composition of COS and PI 192938. The carotenoid data were obtained from our previous publication (Fang et al., 2020). b. From left to right, flesh color of mature fruits of COS, PI 192938 and their F1 generation. c to e. Three flesh colors in the F2 generation derived from COS and PI 192938. Page 21/27 Page 21/27 Page 21/27 Figure 2 BSA-seq analysis and fine mapping of the golden flesh color trait. a. BSA-seq results and chromoso egion related to the golden flesh color trait. b. Initial mapping of the golden flesh color trait. c. Fine mapping of the golden flesh color trait and candidate genes in the fine-mapping region. Figure 2 BSA-seq analysis and fine mapping of the golden flesh color trait. a. BSA-seq results and chromosome region related to the golden flesh color trait. b. Initial mapping of the golden flesh color trait. c. Fine mapping of the golden flesh color trait and candidate genes in the fine-mapping region. Page 22/27 Page 22/27 Figure 3 Genotyping of the individuals in the F2 generation and expression patterns of two candidate genes in COS and PI 192938. Genotyping results of the SNP at the 9,448,870th bp in the F2 generation. Gene expression patterns of the two parental materials at different flesh color formation stages. Figure 3 Genotyping of the individuals in the F2 generation and expression patterns of two candidate genes in COS and PI 192938. Genotyping results of the SNP at the 9,448,870th bp in the F2 generation. Gene expression patterns of the two parental materials at different flesh color formation stages. Figure 4 Page 23/27 Nucleotide variation in the ClPsy1 gene structure among natural watermelon accessions. a. Gene structure of ClPsy1. b. Nucleotide variations and amino acid substitutions in the ClPsy1 gene among 26 watermelon accessions. Figure 5 SNP variations in the promoter region and expression of candidate transcription factors. a. SNPs located in the promoter regions of COS and PI 192938. b to d. Gene expression patterns of the three candidate transcription factors in flesh tissues at different flesh color formation stages. Figure 8 Gene expression patterns of the three candidate transcription factors based on published RNA-seq data. a to c, BioProject number PRJNA338036. d to f, BioProject number SRP012849. Figure 5 SNP variations in the promoter region and expression of candidate transcription factors. a. SNPs located in the promoter regions of COS and PI 192938. b to d. Gene expression patterns of the three candidate transcription factors in flesh tissues at different flesh color formation stages. Page 24/27 Page 24/27 Page 24/27 Figure 6 Differentially expressed genes involved in the carotenoid metabolic pathway. ifferentially expressed genes involved in the carotenoid metabolic pathway. Differentially expressed genes involved in the carotenoid metabolic pathway. Page 25/27 igure 7 nferred golden flesh trait formation based on gene expression and gene structure variations in gen nvolved in the carotenoid metabolic pathway. The yellow and orange arrows indicate pale yellow f olor and gf color formation, respectively. The blue arrows indicate that genes in the carotenoid me athway located in the α-band did not show any gene expression difference between the two paren nes. Figure 7 Inferred golden flesh trait formation based on gene expression and gene structure variations in genes involved in the carotenoid metabolic pathway. The yellow and orange arrows indicate pale yellow flesh color and gf color formation, respectively. The blue arrows indicate that genes in the carotenoid metabolic pathway located in the α-band did not show any gene expression difference between the two parental lines. Page 26/27 Page 26/27 Figure 8 Gene expression patterns of the three candidate transcription factors based on published RNA-seq dat to c, BioProject number PRJNA338036. d to f, BioProject number SRP012849. Figure 8 Tables.xlsx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 27/27
https://openalex.org/W4206746402	https://figshare.com/articles/thesis/The_Role_of_Misrule_in_the_Practice_of_Performance/16999471/3/files/31449055.pdf	English	null	The Role of Misrule in the Practice of Performance	null	2,021	cc-by	61,990	The Role of Misrule in the Practice of Performance Gregor Cameron Abstract In the theatre we are familiar with how rehearsal and collaboration can lead to a number of unintended discoveries that can cause a production to change tack. At the heart of this thesis is discovery- of oneself as an artist, as part of community collaboration and as part of a story told on stage. Using carnival as a frame for the space we work in, accepting that the ʻtricksterʼ cultural figure can be a manifestation of the carnival, it should be possible for the role of trickster to be taken on by someone in the rehearsal process. This is often but not always the director. These ʻhappy accidentsʼ can in fact arise through the directorʼs deliberate disruption of the community. At times, problems that raise their heads are solved through this creative process. Misrule is my definition of this and I offer my experience of this within the process of bringing Saint Punch, a carnivalesque/ Grand Guignol show, to the stage. I compare this with some other practitionersʼ experience of this process of disruptive imagination as defense of my position. By recognizing it in both theory and practice through a critical analysis my aim is to seek a synthesis intended to enrich the theatrical experience of the audience. 2 MA Thesis: The Role of Misrule in the practice of Performance Acknowledgements First and foremost for being such a provocateur in my life for over twenty years, My Supervisor: John Downie, My Supervisor: John Downie, My Cast and Crew, without whom nothing would have reached playing on the floor, you know who you are and hopefully also know how grateful I am, My Mentors: Alex Funke and Horst Sarubin, Annie Forbes and Tim Denton, Damien Brell, and especially Tom McCrory, My co-conspirators for their generous advice: Kerry Glamsch, Chris Ryan, Ralph Johnson, Sean Redmond, Doug Van Belle, and Matthew Wagner My co-conspirators for their generous advice: Kerry Glamsch, Chris Ryan, Ralph Johnson, Sean Redmond, Doug Van Belle, and Matthew Wagner Friends and Staff who stepped up to help: Dawa Devereaux, Paul Wolffram, Jim Davenport, William Franco, and Bernard Blackburn Family & Friends always: Shane Roberts, Roc Travers, Morna Lorden, Claire My co-conspirators for their generous advice: Kerry Glamsch, Chris Ryan, Ralph Johnson, Sean Redmond, Doug Van Belle, and Matthew Wagner Friends and Staff who stepped up to help: Dawa Devereaux, Paul Wolffram, Jim Davenport, William Franco, and Bernard Blackburn Family & Friends always: Shane Roberts, Roc Travers, Morna Lorden, Claire OʼLoughlin and the rest of the academic and general staff of SEFTMS, Martin Payne and the Payne Whanau, Joan and Robbie Cameron (Our little group of three). Friends and Staff who stepped up to help: Dawa Devereaux, Paul Wolffram, Jim Davenport, William Franco, and Bernard Blackburn Family & Friends always: Shane Roberts, Roc Travers, Morna Lorden, Claire OʼLoughlin and the rest of the academic and general staff of SEFTMS, Martin Payne and the Payne Whanau, Joan and Robbie Cameron (Our little group of three). Gregor Cameron 198272670 3 Gregor Cameron 198272670 Table of Contents Table of Conten Abstract Acknowledgements Table of Contents List of Figures Introduction. Part One: Inspiration The Script Part Two: The Path to Performance; an exegesis Context Workshops Thoughts Pre-‐Production Casting Rehearsals Thoughts Mentors Design Show and Tell Thoughts Part Three: Critical Analysis of Misrule and its part Abstract 2 Conclusion 5 Workshops Thoughts MA Thesis: The Role of Misrule in the practice of Performance Bakhtin and the Carnivalesque. 129 The Grotesque Body and the Simulacra. 140 The Dark Clown and the Trickster 156 Circling the Question of Misrule 177 Sacred/ Profane 184 Conclusion 189 Works Cited. 196 Appendix One 203 Appendix Two 204 Appendix Three 206 Appendix Four 207 129 Gregor Cameron 198272670 5 Gregor Cameron 198272670 List of Figures Figure 1. Gilles de Rais- Des Morgan 7 Figure 2. Saint Punch Finale: Punch hangs the Hangman 85 Figure 3. Elliott and Adnan; discussion of The Whipping of the Quaker Women 86 Figure 4. Patrick, Patricia and Thomas, with Toy Soldiers and Barbies, playing out The Whipping of the Quaker Women. 88 Figure 5. Audrey begins to bring her puppet, Judith, to life 93 Figure 6. Judith, Audrey's Puppet, threatens to open the forbidden cabinet 98 Figure 7. In rehearsal the Inquisitor condemns Gilles to death 104 Figure 8. The television, showing the climax of the Bluebeard story, in a kitchen. 115 Figure 9. The Stage: TV Screen, Curtain, Punch Booth and Grille. 118 Figure 10. Looking down the Grille 122 Figure 11. The image in front of the rape 175 All photos by Gregor Cameron or Maria Becerro Casero, 2011, used with permission. 6 6 MA Thesis: The Role of Misrule in the practice of Performance Figure 1. Gilles de Rais- Des Morgan MA Thesis: The Role of Misrule in the practice of Performance Figure 1. Gilles de Rais- Des Morgan Introduction. Gregor Cameron 198272670 7 How often have I experienced that creative moment when everything can change? Not as much as I would like, as this process is rare and needs careful nurture as it arises out of a collaborative community. Certainly when this has arisen it has managed to provide a solution to a problem, which has been very helpful. I have realised that the theatre space we work in is not unlike the Carnivalesque space described by Mikhail Bakhtin. It occurs that the trickster figure that engages so many differing cultures also belongs to this space. As well as appearing in our stories I wonder whether it can also become a role in the practice of theatre and performance. Certainly I can see how, as a director, I sometimes use a trickster-like presence to inspire the rest of our community, hopefully bringing them into a creative space that allows them to dream, 7 engage and adventure. These ʻhappy accidentsʼ have, in my experience, brought solutions where there were only problems and inspiration where there was a block. In bringing Saint Punch to performance I have tried to map out both the physical journey of the process with the theoretical frame of the carnival and its use of the trickster. I have chosen to call this misrule. In order to investigate this I have thought about the cultural theory frame, both in its “constative” and its “performative” forms1. Matt Hills (2005) describes how the constative refers to words that make statements and tend towards an observation and report position on cultural theory. The performative position acknowledges the constative but also demands that theory produce or has an affect upon the observed object or behaviour. In fact Hills suggests that even in its constative form cultural theory can have an affect upon those interacting with it. In a sense reporting about the workshop performance is a constative position while the work on the play itself can be observed to be performative, the cultural object as representative of the carnivalesque culture it belongs in. Thus, since my work is in itself misrulic, it demands a link between what can be seen/ experienced in the performance and then in the work that surrounded bringing it to the stage. 1 Matt Hills cites J.L. Austinʼs work, How to do things with words (1976) as source for the terms ʻconstativeʼ and ʻperformativeʼ. Introduction. I will introduce a series of elements that will trace this idea in a large historical frame through popular culture in order to try and define the concept of misrule and its purpose in the social spectacle of the theatre. 8 MA Thesis: The Role of Misrule in the practice of Performance In order to do this I have split this thesis into three parts. In the first part I offer some insight into where the project came from, its inspiration. I describe its literary and historical origin and how the three stories may be linked. There are a number of texts that contributed to my thinking. Then there is a consideration of how oneʼs own experience contributes to the work. Misrule as a project sprang from some of these experiences and the knowledge gained. After this comes an examination of the cultural frame of the Carnival. This, in turn, leads to the dark clown and how this icon fits within the history and performance of Saint Punch. Finally I pose the question whether our consideration of this ritual space also belongs to the ʻtricksterʼ figure. Then I have included the final performance script of Saint Punch. Secondly an exegesis of the development of the system of rehearsal, design, mentorship and performance of Saint Punch is included. I discuss the summer workshops and the difficulties that were imposed upon the process by both my own lack of confidence and the external limitations that arose. Then I move on to the pre-production and casting process where this difficult stage of opportunity and challenge collide and how the casting decisions were made. Then the rehearsals begin and the show begins to take form. I describe the system I wanted to maintain and the offers to the cast for them to participate and contribute to the formation of the play. The three differing stage environments are discussed and described, as the three stories contained in the play are each given an environment; Punch his booth, Bluebeard upon the masked stage and Gilles de Rais and Joan DʼArc the television screen. To Secondly an exegesis of the development of the system of rehearsal, design, mentorship and performance of Saint Punch is included. I discuss the summer workshops and the difficulties that were imposed upon the process by both my own lack of confidence and the external limitations that arose. Then I move on to the pre-production and casting process where this difficult stage of opportunity and challenge collide and how the casting decisions were made. Then the rehearsals begin and the show begins to take form. MA Thesis: The Role of Misrule in the practice of Performance I describe the system I wanted to maintain and the offers to the cast for them to participate and contribute to the formation of the play. The three differing stage environments are discussed and described, as the three stories contained in the play are each given an environment; Punch his booth, Bluebeard upon the masked stage and Gilles de Rais and Joan DʼArc the television screen. To Gregor Cameron 198272670 9 9 further disrupt this, the characters exchange their native environments and play upon, within the particular constrains of their story, the other two environments as well. Thus, for instance, the audience meets not just the puppet Punch but also his mask and his ʻrealʼ self as mediated through visual media. An example of a moment of misrule is introduced as scene four is described and its development discussed. I then offer some thoughts on the way the rehearsal proceeded and my reflection upon them. The mentors that I interacted with are introduced with a particular focus on how a conversation with Tom McCrory influenced my thinking on Saint Punch and, in fact, my whole approach to my work within the performance area. As the production nears its performance dates I consider the implications rehearsals and the venue had upon the design that developed and how talking with my mentors offered opportunities that were introduced into the design. Part two concludes with some insights into the show and tell performances and my thoughts about how it felt to successfully bring the text to stage. 10 In the final part of the thesis I bring, I hope, a more conventional academic eye to the critical theory surrounding my object, the text of Saint Punch. I frame this part with an introduction that suggests that in order to beget misrule, one must be operating within a system, using Stanislavski as my first practitioner exemplar. Then I begin with a consideration of the work of Mikhail Bakhtin and his description of the carnivalesque. As his work concentrates around the 10 10 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance novel I use a number of thinkers that have followed his lead and expanded his work into the performative art arena. Of particular interest is the suggestion that the carnival exists within a liminal state, a borderland where things may and can change. I begin to find a number of contemporary examples before applying this frame to Saint Punch. This leads to a discussion of the context of the play and what I am attempting to do. The discussion continues with the elements of the grotesque body and its role in producing a simulacrum of the reality it is critiquing. I consider the Grand Guignol of the early twentieth century. I then turn to a consideration of Brecht and his dialogue with the problem of empathy and alienation. In turn this leads back to the carnivalesque and how system and misrule can both be present on the ʻstageʼ or classroom. A number of thinkers raise their heads here as the nature of the reality we encounter in the theatre is considered. The hoodie is offered as an example of how an object can be perceived in a number of ways depending on oneʼs cultural and real experience. Then the figures of the Commedia DellʼArte are connected to Mr. Punch, and out of this arises the dark clown of the present era. The simulacra of the text are then described and some sense of their significance is discussed in terms of the different mediated images that the play offers. The context of the various stories is also interrogated for the reasons that the stories are offered onstage. As the Dark Clown is introduced it is also compared with the role the trickster has played in the cultural literacy that many societies share. Clowns are also linked into the work of Bakhtin. This trickster can be seen to play a very Gregor Cameron 198272670 11 11 11 significant role in stories but more importantly Lewis Hyde (2008) argues that the trickster is a role that artists and creatives take up in order to spark the disruptive imagination that jumps their art into something new- a moment I would argue is a manifestation of misrule. Harlequin and, in turn, Punch provide something of this disruptive spirit which still exists within our modern culture. MA Thesis: The Role of Misrule in the practice of Performance To illustrate this I choose to use a television performance that features a ʻmodernʼ trickster. Matt Hills contributes to the picture of the Doctor in the BBC series Doctor Who (1963- ) as a manifestation of the trickster figure. Punchʼs presence as a trickster figure is examined and this leads to a discussion of how artists can inhabit this role. The trickster I will illustrate to be a figure that does not so much do, as one that causes or offers opportunity for things to change- a role that can also be present in the carnival. Theatre is described as a community of collaborators and within this context an example is drawn from the rehearsal process of Saint Punch of how misrule manifested itself. Finally in part three the question of a link between theory, story and practice is explored. Using contemporary practitioners such as Anne Bogart, Mike Alfreds, Baldwin and Bicât, Judith Weston and thinkers such as Bella Merlin and Alison Hodge who comment on a range of twentieth century practitioners I attempt to draw together a number of ʻmomentsʼ where I think the misrulic is observable. Using the writing of Jacque Lecoq I try to signal how this practice 12 MA Thesis: The Role of Misrule in the practice of Performance is also tied to the trickster figure and what it can offer in the project of performance. MA Thesis: The Role of Misrule in the practice of Performance is also tied to the trickster figure and what it can offer in the project of performance. In this way, rather than be exhaustive upon the topic, I wish to set in motion a project of thinking, a drawing together of history and culture that captures an essence of anarchy within the spirit of social spectacle. I have deliberately set my tonal approach towards the work with an eye both to the subjectivity of the artistʼs relationship with his work, and as I move outside into critical theory and practice I have chosen to foreground a more neutral position in the writing. In this sense to draw the constative closer to the performative and suggest that object and practice are much closer in co-existence than I previously thought. 13 Gregor Cameron 198272670 Gregor Cameron 198272670 Part One: Inspiration Part One: Inspiration Part One: Inspiration 14 Saint Punch has its beginnings with a book that fell into my path some years ago, at the Wellington Public Library. For some forgotten reason I was seeking information about Joan of Arc and the catalogue pointed out this book, The Saint and the Devil by Frances Winwar. Published in 1948 it seemed too old to be relevant and yet I took it home. From that moment my path was forever changed (said in a melodramatic but ironic way). The book compared the journey of Joan to sainthood with that of her Companion-at-Arms, Gilles de Rais, towards a life of devilry and death. It pointed out the travesty of Joan being put to death for the crime of wearing menʼs clothing with that of Gilles, about ten years later for the crime of conjuring demons. The irony was underlined by the fact that de Rais was also known to have raped and murdered over 144 children after his mission with Joan at Orleans and her 14 MA Thesis: The Role of Misrule in the practice of Performance subsequent death, but for which crimes he was not condemned. In fact reports came out that he would do these things purely for pleasure and often at evenings of entertainment for his noble neighbours. He took as his mandate the difference between noble and peasant as permission to do as he pleased. Grimly enough his being a noble allowed him to ask for a mercy denied Joan, that he would be suffocated before the fires were lit so that no naked flame should touch his still living body. I was aware however that there are a number of different versions and that the ʻhistoryʼ was open to some negotiation about what may or may not have happened. It is not my purpose to debate these arguments here, but it was this first forage that fed the imagination. MA Thesis: The Role of Misrule in the practice of Performance This story/ ʻhistoricalʼ event was to stay within me for some time. It came up again when I was flicking my way through a friendʼs edition of Women Who Run With The Wolves by Clarissa Pinkola Estes (1992). I found myself taken by the reference to the story of Bluebeard. Subsequent reading would connect these two narratives as one, Bluebeard serving as a mediated, through folk tale, version of Gilles de Rais, who reportedly did colour his beard blue. Estes used this particular story to allow an exploration of a womanʼs rise into her sacred power- spotting predators, trusting instincts, seeing through the appearance to a truth beyond and thus navigating the path of her life under her own direction. Bluebeard offers the external threat that Estes later is able to compare with the internal. Much later, in the midst of rehearsal, I discovered Angela Carterʼs version, The Bloody Chamber (1979), where the young protagonist is attracted to the cunning ʻBluebeardʼ. She also acknowledges her inner complicity in this descent into the pit. Carter has been aligned with the Gregor Cameron 198272670 15 feminist-centred readings of Perrault and de Sade, however she has disputed such a simplistic labeling preferring to see the work as deeply rooted within the subconscious2. Food for thought perhaps, but not essential to my point. Not only are these stories popular but also offer a gateway into cultural significance. Bluebeard can be found as a reference or a topic in cultural artifacts as diverse as opera (Bartokʼs Bluebeardʼs Castle) to Heavy Metal (Cradle of Filthʼs 2008 Godspeed on the Devilʼs Thunder) through to appearances in plays (Shawʼs Saint Joan) to movies. Most close to home is Jane Campionʼs The Piano (1993) where his story is being played out as the childrenʼs shadow puppets. Which is a good place to start looking at why Saint Punch revolved around the characters of the traditional Punch and Judy of English seaside holiday fun of the Victorian and early to mid-Twentieth century eras. Of course the tradition is much older than that. Some indication has been given that it may have connection to the trickster traditions that many cultures share. In 1994 Neil Gaiman followed up his success in The Sandman series of comics and graphic novels with The Tragical Comedy or Comical Tragedy of Mr. Punch. Rogue Artists Ensemble mounted a stage adaptation of the graphic novel in 20073. Books \| The Guardian http://www.guardian.co.uk/books/2006/jun/24/classics.angelacarter accessed Tue. 19 July 2011 3 http://www.rogueartists.org/index.php?option=com_content&task=view&id=34&Itemid=92 accessed Wed 24 Aug 2011 2Helen Simpson on Angela Carter's Bloody Chamber \| Books \| The Guardian Helen Simpson on Angela Carter's Bloody Chamber \| Books \| The Guardian http://www.guardian.co.uk/books/2006/jun/24/classics.angelacarter accessed Tue. 19 July 2011 3 2Helen Simpson on Angela Carter's Bloody Chamber \| Books \| The Guardian Helen Simpson on Angela Carter's Bloody Chamber B k \| Th G di h // di k/b k /2006/j /24/ l i l d T 19 J l 2011 Helen Simpson on Angela Carter s Bloody Chamber \| Books \| The Guardian Helen Simpson on Angela Carter s Bloody Chamber \| Books \| The Guardian http://www.guardian.co.uk/books/2006/jun/24/classics.angelacarter accessed Tue. 19 July 2011 3 \| Books \| The Guardian http://www.guardian.co.uk/books/2006/jun/24/classics.angelacarter accessed Tue. 19 J 3 http://www.rogueartists.org/index.php?option=com_content&task=view&id=34&Itemid=92 accessed Wed 2 16 \| p g j g y http://www.rogueartists.org/index.php?option=com_content&task=view&id=34&Itemid=92 accessed Wed 24 Aug 2011 4 http://www.punchandjudy.org/mainframesethistory.htm accessed 24/8/11 h // h dj d h h d / / Gregor Cameron 198272670 17 p p j y p p j y g y http://www.punchandjudy.comwho.htm accessed 24/8/11 Gregor Cameron 198272670 p p j y 4 http://www.punchandjudy.org/mainframesethistory.htm accessed 24/8/11 http://www.punchandjudy.comwho.htm accessed 24/8/11 http://www.punchandjudy.org/mainframesethistory.htm accessed 24/8/11 http://www.punchandjudy.comwho.htm accessed 24/8/11 5 http://www.punchandjudy.comwho.htm accessed 24/8/11 6 P M V l 2 N 3 65 68 d f h // ild k/di i d hi h 6 Puppet Master Volume 2, No 3 pp. 65-68 accessed from http://www.puppetguild.org.uk/digivendpmarchive.htm 2011-09-12 16:27:00 MA Thesis: The Role of Misrule in the practice of Performance Punch one cannot but see how allied they seem to be when considering the dark underlying motif of violence that they provide. In the nineteenth century the more familiar Judy emerged. During this century the characters and tragical story evolved even as the string puppets lost ground with the upper classes and the glove puppet gained ground with those lower down. It was this lower class version that was recorded in London Labour and the London Poor by Henry Mayhew, published in 1851. By this time the Joey clown and the hangman Jack Ketch are still part of the company of puppets but the Negro servant, or Darkie as he was known have been supplanted by the crocodile (perhaps there is a link with the one in Peter Pan by J. M. Barrie).5 Also the Devil no longer features in the cast, possibly also replaced by the crocodile. Judy is now playing the Ghost and it is she who is first resurrected as the cycle reconstitutes itself. J.R. Cleland writing in 1948 likens Mr. Punch to the morality plays of the mediaeval era.6 He suggests that by considering the characters as allegories one might see Mr. Punch as an Everyman seeking to travel from his baser earth-bound existence to one more closely allied with God- a carnivalesque journey. He acknowledges that truly tracking Punchʼs origins is always going to involve a certain amount of conjecture but I think there is certainly worth in entertaining the idea that significantly, Joan. While it is conjecture to link the other two stories with that of Mr. Punch one cannot but see how allied they seem to be when considering the dark underlying motif of violence that they provide. In the nineteenth century the more familiar Judy emerged. During this century the characters and tragical story evolved even as the string puppets lost ground with the upper classes and the glove puppet gained ground with those lower down. It was this lower class version that was recorded in London Labour and the London Poor by Henry Mayhew, published in 1851. By this time the Joey clown and the hangman Jack Ketch are still part of the company of puppets but the Negro servant, or Darkie as he was known have been supplanted by the crocodile (perhaps there is a link with the one in Peter Pan by J. M. p p j y 6 Puppet Master Volume 2, No 3 pp. 65-68 accessed from http://www.puppetguild.org.uk/digivendpmarchive.htm 2011-09-12 16:27:00 5 http://www.punchandjudy.comwho.htm accessed 24/8/11 6 Puppet Master Volume 2, No 3 pp. 65-68 accessed from http://www.puppetguild.org.uk/digivendpmarchive.htm 2011 09 12 16:27:00 MA Thesis: The Role of Misrule in the practice of Performance In this graphic novel the nature of loneliness and dreams are interrogated and exposed using the character of Mr. Punch as provocateur. I was reading Which is a good place to start looking at why Saint Punch revolved around the characters of the traditional Punch and Judy of English seaside holiday fun of the Victorian and early to mid-Twentieth century eras. Of course the tradition is much older than that. Some indication has been given that it may have connection to the trickster traditions that many cultures share. In 1994 Neil 16 MA Thesis: The Role of Misrule in the practice of Performance Annalisa Di Liddo (2009) who, in writing about the work of Alan Moore (Watchmen, Lost Girls and other graphic novels), makes the argument to look at these objects not only as literature but also as a form of performative text. Following her logic it is not surprising how easily these texts adapt to film, television or the stage considering their similarity to the storyboarding technique used in these industries at times. She also uses Bakhtinʼs terms including the chronotope, to describe these forms. Moore like Gaiman uses a wide knowledge of history, mythology and culture to bring his works to life. I was fascinated to read this innovative use of the Punch character and began to find out more about him. On May 9, 1662, Samuel Pepys notes that he is ʻmighty pleasedʼ by an Italian puppet show encountered near St Paulʼs Church in Londonʼs Covent Garden.4 It is likely that Pepysʼ Punch was a marionette rather than a hand puppet and probably named Punchinello which allies with the names Polichinella, Pollicinello, Polichinelle, and the commedia import, Pulcinella. Hence then a link to the Commedia DellʼArte companies traveling Europe and Mr. Punch, as the name became, would in all likelihood have offered a harlequinade relief to even the most serious of stories, in much the same way as clowns were used in the plays of Shakespeare. This dark clown, it seems, was introduced to England with the restoration of Charles II, the Merry Monarch. In terms of my work I was amazed to find out that in the eighteenth century Mr. Punch had acquired a wife to fight and bicker with. At that time she was called, 17 significantly, Joan. While it is conjecture to link the other two stories with that of Mr. MA Thesis: The Role of Misrule in the practice of Performance Barrie).5 Also the Devil no longer features in the cast, possibly also replaced by the crocodile. Judy is now playing the Ghost and it is she who is first resurrected as the cycle reconstitutes itself. J.R. Cleland writing in 1948 likens Mr. Punch to the morality plays of the mediaeval era.6 He suggests that by considering the characters as allegories one might see Mr. Punch as an Everyman seeking to travel from his baser earth-bound existence to one more closely allied with God- a carnivalesque journey. He acknowledges that truly tracking Punchʼs origins is always going to involve a certain amount of conjecture but I think there is certainly worth in entertaining the idea that 18 MA Thesis: The Role of Misrule in the practice of Performance Punch represents a spirit of some consequence rather than just being dismissed as a puppet. Two further interruptions in my life were to influence the birth of Saint Punch. Firstly I was hospitalised with appendicitis and spent over three weeks in a drug-induced nightmare of visions and delusions, often involving giant, man- sized, and, for some reason, yellow ants. These nightmares were probably an indication of how hard my body was trying to fight the post-operative infection that had necessitated the doctors to give me what they described as a toxic level of antibiotics. On my recovery the first draft of Saint Punch was born. Secondly came the impression left on me of my travels the following year, where my passion for theatre was reborn. In circling the world I was able to seek out and experience many different theatrical styles that are not normally available here in New Zealand. In particular was a commedia show called Carnivale I encountered in Venice, which brought together a number of commedia characters and a formidable audio-visual component to tell the history of Venice. With the easy humour of the clowns offsetting the more documentary feel of the slides and music this was one of the best examples of how theatre can engage in educating and entertaining that I have ever seen. A few days later I was in Berlin and took a day trip to Sachsenhausen Concentration Camp (see appendix 4). This stark reminder of the atrocities of the Nazis was very affecting. Particularly when I saw a picture of Harlequin on the wall of victims. MA Thesis: The Role of Misrule in the practice of Performance A young gay dancer, who had loved playing this role, had been incarcerated in the camp and there met his end. It occurred to me that Concentration Camp (see appendix 4). This stark reminder of the atrocities of the Nazis was very affecting. Particularly when I saw a picture of Harlequin on the wall of victims. A young gay dancer, who had loved playing this role, had been incarcerated in the camp and there met his end. It occurred to me that 7 See Appendix Five Gregor Cameron 198272670 19 only the rigidity of the Nazi rule could have killed the misrule of Harlequin/Arlecchino. Also of import was the poem by prisoner/ Pastor Martin Niemöller7. This is where I am first aware that my mind had begun to engage in the problem of misrule. only the rigidity of the Nazi rule could have killed the misrule of Harlequin/Arlecchino. Also of import was the poem by prisoner/ Pastor Martin Niemöller7. This is where I am first aware that my mind had begun to engage in the problem of misrule. Misrule, it seemed to me, was a process or an opportunity. It was not the opposite of rule and didnʼt quite fit with ideas of order and chaos since to my mind order at its most extreme would tend to exclude the possibility of misrule. If misrule was some sort of opportunity then how did it manifest itself, what affect might it produce and how could one both observe it and exploit it in creative practice. My thinking began to be influenced by readings about the frame of the carnivalesque. I began looking at Mikhail Bakhtinʼs work, which centres on the novel. That brought me to the writings on the carnivalesque that have expanded his work to include the theatre. Added to this is the consideration that Mr Punch has some aspects of the dark clown and that sent me looking at the development and history of the Buffo/ Bouffon. The Dark Clown is a very interesting alternative to the white-faced/ sad/ angry Clown and the red-nosed/glad/ foolish clown which Eric Idle writes about in The Road to Mars (1999). Idle writes this post-modern novel as an 'academic's' attempt to reconstruct a thesis of an android who in turn is trying to serve his comedian owners while researching into the nature of comedy. 20 20 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Idle seems to be saying that in order to be funny one has to be disturbed, that the funniest comedians are also the saddest personalities on the planet. He cites such pairings as Laurel and Hardy or Martin and Lewis. Oliver Hardy and Dean Martin are examples of the white face clown and are often the instigators of self-serving action- possibly action that is at odds with what society would consider acceptable. In counterpoint Stan Laurel and Jerry Lewis are examples of the red nose Clown, the fool whose innocence and naïveté constantly complicates and defeats the white face clownʼs efforts to control things. Other partnerships (which I have added to) fit these descriptions: Abbot and Costello, Penn and Teller, French and Saunders & Flight of the Conchords Bakhtin acknowledges these two forms of clown- the rogue and the fool. He also adds a third category simply known as the clown- which can be seen in dramatic terms as the clown in some of Shakespeareʼs plays who is able to comment upon high and low alike. This 'dark' clown however has a very different agenda. Often allied with the ʻkiller clownʼ genre the dark clown is more likely to be looking outward into the world than these two inward reflecting clowns. In carnivalesque terms these clowns have a relationship to both high culture and the low. They are often located within the low culture but through their vision, as the Fool in Shakespeare's King Lear, may be located quite close to the centre of power. Set as commentators upon these cultural centres they either observe or participate. The clowns seem to be assuming very specific roles. Bakhtin, according to Vice, sees the three behaving according to these roles thus: The rogue parodies high languages; the fool is 'naively in-comprehending' Gregor Cameron 198272670 of the high languages s/he comes into contact with; the [dark] clown 21 'unmasks' high languages and has license to utter the otherwise unacceptable. (Vice, 1997:69) The appellation 'dark' is appended to the Bakhtin description to differentiate this clown from the others. This clown most closely combines the elements of carnival within their appearance and behavior. 8 A term applied to the companions of Hal, who as he becomes Henry V distances himself from them. Quoted as a word on Page 445 of the notes in A New Variorum Edition of Shakespeare see Bibliography. I use it here to connect the concept of misrule with the behaviour of the clowns and to identify the element that needs to enter practice to allow it to exist in the theatrical endeavor. 8 A term applied to the companions of Hal, who as he becomes Henry V distances himself from them. Quoted as a 8 A term applied to the companions of Hal, who as he becomes Henry V distances himself from them. Quoted as a word on Page 445 of the notes in A New Variorum Edition of Shakespeare see Bibliography. I use it here to connect the concept of misrule with the behaviour of the clowns and to identify the element that needs to enter practice to word on Page 445 of the notes in A New Variorum Edition of Shakespeare see Bibliography. I use it here to connect the concept of misrule with the behaviour of the clowns and to identify the element that needs to enter practice to allow it to exist in the theatrical endeavor. MA Thesis: The Role of Misrule in the practice of Performance Puck in A Midsummer Night's Dream might be an essence of this misrulic8 clown- it is not chaotic since that would necessitate no regard for the rules- Puck is subversive of the rules but not transgressive. The Joker out of the Batman comic mythos is perhaps a more chaotic clown. However both these examples are emblematic of the Dark Clown through which the status quo, the normative might be interrogated and judged. They challenge the way things are -one through magic and the other through acts of insanity and crime. Thus, Mr. Punch can be viewed through the lens of the dark clown. He upsets the status quo and holds no respect for high or low within his grasp. He is the grotesque body alive and laughs in the face of consequence. He is reborn within each story as are the other characters. We, as audience, may be shocked at his behaviour and yet is he not acting, as sometimes we ourselves are tempted to do. He brings misrule to the fore so that we may seek, through reflection, that which is good within the actions of the bad. 22 9 http://www.sacred-texts.com/bos/bos071.htm accessed Sun 18/9/11 Gregor Cameron 198272670 23 p g Stage THE PROFESSOR Isn't it nice when we gather...(improvise).... I want to tell you a story. It's a story of a young woman, a beautiful woman, constantly chased by the boys, and as she grew she is then chased by the men, in fact, she draws attention from a couple of the women too... heh! One day her father calls her, he wishes.... A phone rings. A At the back is a large Curtain through which the characters come. To the right Punch's stage/ booth is empty except for a large 'brick' phone lying to one side. The Booth is in the classic pseudo-pros arch but larger than a normal punch-man's booth. It includes the flies at the back that allow a change of scene. Centre stage is an open trap with a grille to look through. At the back directly opposed to the booth is a large old-fashioned Television screen where the historical 'real' black and white images appear. The phone continues ringing. A voice is heard from off-stage. Nothing changes on the stage. MA Thesis: The Role of Misrule in the practice of Performance This ritualism of the sacred and the profane is also a fascinating aspect of the carnivalesque. I wonder if there is not something of the trickster myth embroiled in Mr. Punch and the dark clown. The trickster is often the catalyst of action rather than the ʻactorʼ himself. In Anansi, Coyote, Maui and Loki he, most often, causes something to happen or draws action towards himself that requires others to seek truth, and the power to solve the problem. This action is often transgressive or grotesque, for example Maui trying to capture the Sun or how Coyote stole fire. These figures are often seen as vulgar unfinished types. “Trickster is a creator, a transformer, a joker, a truth teller, a destroyer.”9 The exploration of the transgressive and grotesque can offer insight into the sacred and aspirational. And here I again face my questions. If the carnivalesque exists as a framework for our story telling, is it also a manifestation of our developing work? What is the relationship between the carnivalesque and misrule in terms of creative endeavour? How can we approach a work that is itself an example of the carnivalesque with an open hand to the ʻhappy accidentʼ? As you can see with the following script of the workshop performance, some of these questions are very pertinent. It is important to note that the first draft of this text was submitted to the scriptwriting course run by John Downie in 2006. Since that time and especially as rehearsals developed the piece was altered and updated as discoveries were made on the floor. The script is included, not 23 as part of the marking, but to acknowledge how important a part of the creative practice it was, and as a way of recording, along with the enclosed DVD, the final shape of the show. 24 24 The Script SCENE ONE (Punch throws the baby out the window, Gilles 'rapes' a child and Bluebeard scares his wife. ) Gregor Cameron 198272670 THE PROFESSOR Mr. Punch? (Silence) THE PROFESSOR Mr. Punch? (Silence) THE PROFESSOR Punch? Aren't you going to answer the phone? Silence. The phone continues ringing- more Insistently THE PROFESSOR Mr. Punch? (Silence) THE PROFESSOR Mr. Punch? (Silence) 25 25 PUNCH. (O.S.) Can't I hear what? THE PROFESSOR The one that's ringing! PUNCH. I can't hear it. PUNCH. (O.S.) No. I can't hear it. THE PROFESSOR Why not? 26 THE PROFESSOR Mr. Punch? regor Cameron 198272670 THE PROFESSOR Answer the phone. He leaves shaking his head. The phone is now the most annoyingly loud sound. Punch stands on the phone. PUNCH. Hello? Silence. The phone rings again. PUNCH. Hello? Hello, hello, hello, hello? THE PROFESSOR (Pausing at the exit)) Push the button, Punch. Punch looks over the audience side of the phone. PUNCH. What button? 27 27 Gregor Cameron 198272670 PUNCH. Ohhhhh. PUNCH. Ohhhhh. PUNCH. Hello, hello, hello, hello. GILLES Bring me another one. GILLES Bring me another one. Judy's voice comes out of the phone. Judy's voice comes out of the phone. JUDY. Now what's all the noise Mr. Punch? You'll wake the baby. PUNCH. Hello, Hello, Hello, Hello. JUDY. Really Mr. Punch, don't make so much noise. I do believe you've woken the baby. The PROFESSOR shakes his head and quietly leaves the stage. JUDY. Really Mr. Punch, don't make so much noise. I do believe you've woken the baby. The PROFESSOR shakes his head and quietly leaves the stage. PUNCH. I haven't. JUDY. Oh yes you have. PUNCH. Oh no I haven't. 28 PUNCH. Judy. JUDY. Yes Mr. Punch. PUNCH. Give-us-a-kiss. JUDY. What. Over the Phone? Punch leers down the phone. PUNCH. Oo-kay. What are you wearing? JUDY. What? Tell you that- out there in front of the audience? You'll not get round me like that. PUNCH. Oh yes, I will. JUDY. Oh no you won't. PUNCH. Oh yes I will- You're wearing a pink dress with a head scarf and sensible shoes. Silence. Judy enters the stage. She is dressed just as Punch described her. She is talking into a little cell phone of her own. JUDY. How did you know that? PUNCH. (as though it is obvious) I sat across from you at breakfast. JUDY. You are a rascal Punch. Now I will make you look after the baby. JUDY. What? Tell you that- out there in front of the audience? You'll not get round me like that. Gregor Cameron 198272670 Oh no you won't. PUNCH. Oh yes I will- You're wearing a pink dress with a head scarf and sensible shoes. Silence. Judy enters the stage. She is dressed just as Punch described her. She is talking into a little cell phone of her own. JUDY. How did you know that? PUNCH. (as though it is obvious) I sat across from you at breakfast. JUDY. You are a rascal Punch. Now I will make you look after the baby. She exits. PUNCH. 29 29 JUDY. There you are, Punch. Now, look after him, the little darling. (To audience.) Now 'boys and girls', I want you to make sure Mr. Punch looks after the baby properly. If he doesn't treat it properly will you all call me? 021 TELL ON PUNCH GILLES GILLES And almost able to suckle a child. A miracle, for such as you would all-too-soon find yourself with sucklings mewling at your ankles, a farmer's too rough hands bruising flesh too soon brought to its age. Never mind, my lovely, tonight we will not worry at such things, we will instead look forward into the distance, we will instead provide the night with calls to entertainment. See, above even now, the crowd does gather, their faces eager, wondering what the nature of the evening is going to bring before them. GILLES And almost able to suckle a child. A miracle, for such as you would all-too-soon find yourself with sucklings mewling at your ankles, a farmer's too rough hands bruising flesh too soon brought to its age. Never mind, my lovely, tonight we will not worry at such things, we will instead look forward into the distance, we will instead provide the night with calls to entertainment. See, above even now, the crowd does gather, their faces eager, wondering what the nature of the evening is going to bring before them. The child tries to make herself as small as she can. Gilles runs his hand sinuously down her back. A ripple of excited laughter echoes from above as the women coo and bid their lord to get quickly up to action. GILLES From behind the child he quickly translates to the front and pulls open the shift, looking down. The women giggle and whisper to each other. GILLES GILLES I said- bring me another one! The two women giggle and peep over to see the scene below. To their consternation they don't see Poitou/Professor. A child/teen is thrown into the room. It is confused and afraid. The two women 30 GILLES So what do we have here? A lonely goat boy or the dutiful daughter lost upon her way to Grandmama's. I Cannot tell, it is so dirty and badly dressed. Is this a dress or is it some hand down shirt from older brothers now left home? He roughly seeks to determine the gender of the child, pulling at the child's clothes- each lunge of which is met by fear and cringing from the child. He works his way around the child. His hands disappear up the back of the shift. GILLES See how it cringes, seeking about for some form of salvation. A little dalliance upon the path to little death. A girl? They were running low. Ah, my lovely- it seems you are a girl! From behind the child he quickly translates to the front and pulls open the shift, looking down. The women giggle and whisper to each other. The child sobs and tries to crawl away. The child sobs and tries to crawl away. GILLES Ah, I see you misunderstood. It grieves me not to provide another taste. GILLES Ah, I see you misunderstood. It grieves me not to provide another taste. He kicks her again. GILLES And doesn't all this oh- so soft and yet, so firm flesh excite the hackles of those too jaded by what they may not have, who's only redress is to watch, to revel in 31 Gregor Cameron 198272670 the pain of one so vulnerable. He kicks her. GILLES Let that serve as an object lesson. My audience has all too much appetite for such melodrama that to cheat them of it would prove to both our tastes a bitterness. The child sobs and tries to crawl away. GILLES Ah, I see you misunderstood. It grieves me not to provide another taste. He kicks her again. GILLES So, now. Upon your knees, peasant, and open up the path. For to me, it matters not whether, a he or a she, you be. The two women signal their enjoyment of the sport. Gilles prepares to enter the girl from behind. Gilles and his victim slow to slow motion as counterpoint to Punch's song. The girl whimpers Punch unfreezes and carries the baby to the edge of his booth. Booth PUNCH. (Sits down and sings to the baby) 'Hush-a-by, baby, upon the tree-top, When the wind blows the cradle will rock, When the bough breaks the cradle will fall, Down comes the baby and cradle and all.' The Baby cries. Gilles reaches over and takes the throat of the girl in two hands- her whimpering is cut off PUNCH. (Shaking it ) the pain of one so vulnerable. the pain of one so vulnerable. He kicks her. GILLES Let that serve as an object lesson. My audience has all too much appetite for such melodrama that to cheat them of it would prove to both our tastes a bitterness. GILLES PUNCH. (Shaking it.) What a cross boy! 32 A Thesis: The Role of Misrule in the practice of Performance He lays it down on the booth's stage, and rolls it backwards and forwards, as if to rock it to sleep, and sings again. Punch continues rocking the child. It still cries, and he takes it up in his arms. PUNCH. What a cross child! I can't a-bear cross children. Then he vehemently shakes it, and knocks its head up against the side of the proceedings several times, representing to kill it, and he then throws it out of the booth. Screen Gilles has all but throttled the girl when her face comes up and takes on a strength that throws him off her and across the room. She has transformed into Joan of Arc. Gilles is terrified. JOAN So it seems, 'monsieur champion', that the years have set your devilry upon those who in the past you were full sworn to protect with all the strength of both your heart and that of your arm. Gilles cowers again at the bottom of the stairs. GILLES (terrified beyond reason) Jeanne? La Pucelle? The screen goes dark. Stage. Above the two women turn and cower as a huge man stamps across to them. BLUEBEARD Didn't I tell you not to, didn't I tell you? Didn't I say to you- all my worldly goods I gift you, if only this one small room belongs to me? And how do you pay me? What have you done? The moment my back is turned so I fail to see, You break my heart, you take it away, You prove to be, it seems, the very thing I wished you not, He lays it down on the booth's stage, and rolls it backwards and forwards, as if to rock it to sleep, and sings again. CLARA Judith, Be Careful! CLARA Judith, Be Careful! CLARA Judith, Be Careful! GILLES BLUEBEARD Didn't I tell you not to, didn't I tell you? Didn't I say to you- all my worldly goods I gift you, if only this one small room belongs to me? And how do you pay me? What have you done? The moment my back is turned, so I fail to see, You break my heart, you take it away, You prove to be, it seems, the very thing I wished you not, 33 Gregor Cameron 198272670 Judith stands in front of a terrified Clara stands before the other. Judith stands in front of a terrified Clara stands before the other. JUDITH It is not what you think, my love, it is not what you think it. My sister and I, my love, just wanted to peek. We did no harm, my love, we did no harm- We sport with keys, my love, and we run the house. It wasn't no trouble , my love, no trouble we seek. The huge man towers over the terrified women, they shriek and run off. Booth JUDY. What's a pity Punch? Where's the baby? PUNCH. Gone to bed. JUDY. Gone to bed? 34 34 MA Thesis: The Role of Misrule in the practice of Performance PUNCH. Yes. JUDY. (To audience.) Is that true? AUDIENCE (responding to her?) Thrown him out of window, etc. JUDY. What? Thrown my beautiful baby out of the window. Oh you cruel man. PUNCH. Didn't. JUDY. Oh yes you did. PUNCH. Oh no I didn't, etc. JUDY. Oh you cruel horrid man. How could you? PUNCH. (Wheedling.) Judy. JUDY. Yes. PUNCH. Give-us-a-kiss. JUDY. Yes, I'll give you a kiss. She bobs below to get slapstick with which she be-labours Punch. JUDY. H ' i ki (b ) d h ' h (b ) 35 PUNCH. Thank you Judy for your kisses, thank-you (he hits her.) Thank-you, thank-you. JUDY. Enough Mr. Punch. No more kisses. PUNCH. Just one more. He hits her especially hard and she falls motionless on to the booth stage. He pokes her experimentally - then rolls her to one side of booth. JUDY. Enough Mr. Punch. No more kisses. PUNCH. Just one more. He hits her especially hard and she falls motionless on to the booth stage. He pokes her experimentally - then rolls her to one side of booth. PUNCH. That's the way to do it. Poor Judy. What-a-pity, what-a-pity: There is the sound of men's voices. A little group of drunken soldiers appear upon Punch's booth/ stage. They speak in muted 'foreign' voices obviously familiar with each other/ comradely. They begin to gamble. One man seems to be keen to join in but is at the back of the group. He constantly wishes to gamble. There is the sound of approaching hooves. Joan and Gilles puppets appear in the booth riding small horses. Joan is resplendent in shining white armour and Gilles in darkest matte black. GILLES 36 p JOAN Come now, sirrah, you must know what they do. GILLES I do know, but little good can come from you Interfering. JOAN Tell me! GILLES They gamble... JOAN What? GILLES They gamble, a game played by drunks for the sole purpose of losing all they have- leaving their families to starve... JOAN They gamble? GILLES It is oft the way with such as these. Surely you must have come across such fools in that little mud puddle that you hark from. JOAN Take care with your impertinence, de Rais, never forget that the Dauphin placed you in MY service. GILLES I never forget that, la pucelle, you never allow me to forget that. Joan indicates the men gambling JOAN And so... GILLES They do no harm. JOAN But do they not understand that they stand for the Maid's army- the holy army of France! JOAN Come now, sirrah, you must know what they do. 37 Gregor Cameron 198272670 GILLES The maid's army? JOAN Don't fool with me, sirrah, I lead the Dauphin's forces. Gilles stops as Joan steps forward. If he were human an eyebrow might raise. GILLES Indeed. JOAN Indeed. Now deal with these sinners. GILLES Leave them alone, girl, please, can you not see they do no harm. JOAN Harm? I'll show them harm. Joan leaps off her horse. She draws her sword- a large white version of Punch's stick. GILLES La Pucelle, I beg of you, do not provoke these men. It can only lead to trouble He seeks about as if hoping for some help from others. JOAN If you are unwilling to stand up for me - then I guess I must do for myself- they shall rue the day they stepped across the line between grace and sin. I will not leave this alone- the Dauphin's army shall be walking in the eyes of God- they must be ready, sinless. I will drive out the sinners, the whores. These men shall neither gamble nor swear, they shall not steal and they shall look to their brothers as they would look to themselves. Don t fool with me, sirrah, I lead the Dauphin s forces. Gilles stops as Joan steps forward. If he were human an eyebrow might raise. GILLES Indeed. JOAN Indeed. Now deal with these sinners. GILLES Leave them alone, girl, please, can you not see they do no harm. GILLES JOAN Harm? I'll show them harm. Joan leaps off her horse. She draws her sword- a large white version of Punch's stick. GILLES JOAN Indeed. Now deal with these sinners. JOAN If you are unwilling to stand up for me - then I guess I must do for myself- they shall rue the day they stepped across the line between grace and sin. I will not leave this alone- the Dauphin's army shall be walking in the eyes of God- they must be ready, sinless. I will drive out the sinners, the whores. These men shall neither gamble nor swear, they shall not steal and they shall look to their brothers as they would look to themselves. As she says this she approaches the group of men swinging her sword. She begins hitting out at 38 A Thesis: The Role of Misrule in the practice of Performance anyone near enough to feel the bite of the flat of her sword. Most of the men manage to escape but one poor fellow falls at her feet and she just keeps on hitting him. He screams GILLES Enough, girl, enough. Whatever he may have done he does not deserve to die. JOAN He does not? GILLES No, Jeanne. He does not. She gives the man one final wallop. He lies there unmoving. JOAN Well, at least this way he will serve a purpose, a lesson to others who would dishonour the maid, and thus go against the will of god. GILLES (aside) Some God. They freeze. Stage There is a huge scuffle going on off stage. Suddenly Judy's body lands on the stage and lies there as if dead. Punch enters fighting a black crocodile. Both are now masques. Punch hits the crocodiles nose with his cudgel. The crocodile reels and turns to Punch. CROCODILE. Why ever did you do dat fer! I dint cause no harm to you. You'll no doubt break ma dose! PUNCH. H bl d I b d I did t t h l it Stage There is a huge scuffle going on off stage. Suddenly Judy's body lands on the stage and lies there as if dead. Punch enters fighting a black crocodile. Both are now masques. Punch hits the crocodiles nose with his cudgel. The crocodile reels and turns to Punch. egor Cameron 198272670 CROCODILE. Why ever did you do dat fer! PUNCH. Sausages? What Kind? PUNCH. Sausages? What Kind? GILLES I dint cause no harm to you. You'll no doubt break ma dose! PUNCH. Humbly do I beg your pardon, I did not go to help it. CROCODILE. Nebber yo' mind, Sir Punch, come and sit down, and den we'll hab a song. 39 PUNCH. I'll not sit and sing with you' Sirrah, for the 'notes' within thy smile, do to me, look far too sharp. I'll not be the melody in case it too soon becomes 'harm-to-me'. The crocodile follows Punch around stage, begging - until turning suddenly Punch comes face to face with it. CROCODILE. In that case will you share my stock of Sausages? PUNCH. Sausages? What Kind? CROCODILE. Pork and Garlic? PUNCH. With Cheese? CROCODILE. Three kind of Cheeses, tasty, mozzarella , and go-to-church holy cheese! PUNCH. Holy cheese? You wouldn't be wanting to Swiss me would you? CROCODILE. What, me? Crocodile grabs Punch's nose and there is a violent tussle. Finally he breaks free and drives the Crocodile off with a shower of blows. He kills the crocodile just beside Judy's body PUNCH. Oh dear, Oh dear, my poor nose, my poor beautiful nose. What-a-pity, what-a-pity, ditty-ditty-do. Punch freezes. POITOU M'lord. JUDITH I'm no lady, M'lord. CROCODILE. Pork and Garlic? PUNCH. Oh dear, Oh dear, my poor nose, my poor beautiful nose. What-a-pity, what-a-pity, ditty-ditty-do. Punch freezes. 40 POITOU (O.S.) Messire, messire, she comes, she comes. BLUEBEARD And she waits where? JUDITH Without, m'lord, your man told us she may quietly listen from there and yet give us some privacy. BLUEBEARD Ah, Poitou, where would I be with out him. I shall of course make sure your sister sups as we take our own refreshment. JUDITH M'lord, you are too kind. BLUEBEARD Nonsense, your company is fair exchange for any kindness I may show. Judith flushes and begins to look around for a way to change the subject. JUDITH You live here alone, m'lord JUDITH You live here alone, m'lord BLUEBEARD As alone as one can be surrounded by servants, my Judith. Do you appreciate my home, Judith? JUDITH It's beautiful, m'lord, it puts my father's house to shame. BLUEBEARD 41 41 Gregor Cameron 198272670 And Poitou's no judge of character. And yet, tonight I think he may be right. Few ladies would hold a light to the shine I see hovering about your head, the jewels within your eyes and the blush upon your lips. You came here alone? JUDITH No, m'lord, my sister, Clara, rode as chaperone, if it please you. BLUEBEARD And she waits where? Judith indicates the entrance she came in from. JUDITH Without, m'lord, your man told us she may quietly listen from there and yet give us some privacy. BLUEBEARD Ah, Poitou, where would I be with out him. I shall of course make sure your sister sups as we take our own refreshment. JUDITH M'lord, you are too kind. BLUEBEARD Nonsense, your company is fair exchange for any kindness I may show. Judith flushes and begins to look around for a way to change the subject. JUDITH You live here alone, m'lord BLUEBEARD As alone as one can be surrounded by servants, my Judith. Do you appreciate my home, Judith? JUDITH It's beautiful, m'lord, it puts my father's house to shame. And Poitou's no judge of character. And yet, tonight I think he may be right. Few ladies would hold a light to the shine I see hovering about your head, the jewels within your eyes and the blush upon your lips. You came here alone? JOEY Joey the Clown keeps bobbing up and down, and Punch tries to hit him all the time till Punch is nearly exhausted. Maybe Punch is fooling so Joey is careful, he's too cunning for him though, and knows him too well. The dance continues. Eventually falling into a heap laughing the clown turns to Punch and they both shake hands and make it up. BLUEBEARD 42 MA Thesis: The Role of Misrule in the practice of Performance But he is rich in daughters is he not? JUDITH Not, m'lord, as much as if we three were sons, m'lord BLUEBEARD Will you not call me Guy, my Judith? Judith stares at him for a moment. She makes a decision. JUDITH Clara! Come here. Clara enters from the same entrance as Judith CLARA Judith? JUDITH Are you there my sister? What think you? CLARA He's very forward, this one. Do we feel safe? JUDITH I believe we are. CLARA Then it will do no harm for you to enjoy a little company, I know that is what you wish. I shall go back to my embroidery. Judith smiles as Clara leaves. JUDITH You do that. But keep an ear out- I know how you can get after eating too much and too well. Bluebeard smiles. BLUEBEARD Poitou! The lights dim. Stage. Joey the Clown enters. Joey dances round Punch in 43 43 determined to catch him if possible. JOEY No bono, allez tooti sweet, Mounseer. Look out sharp! Make haste! Catch 'em alive! Here we are! How are you? Good morning! Don't you wish you may get it? Ah! Coward, strike a white man! Joey the Clown keeps bobbing up and down, and Punch tries to hit him all the time till Punch is nearly exhausted. Maybe Punch is fooling so Joey is careful, he's too cunning for him though, and knows him too well. The dance continues. Eventually falling into a heap laughing the clown turns to Punch and they both shake hands and make it up. JOEY Now it's all fair; ain't it, Punch? PUNCH. Yes. Punch devises a bit of business involving getting Joey to hold up his leg so that he might rest. Joey is angry. JOEY Now I shall begin again. They begin trading blows again till it begins to really look like a dance. Finally Joey uses his finger to so insult Punch knocks him down, apparently dead. Punch then begins to count the bodies - Judy, Crocodile and Joey. But the Clown is not dead and every time Punch moves a body to centre stage, the Joey Clown moves it back to the side when Punch is not looking. Punch gets more and more excited. Then the Clown removes bodies JOEY No bono, allez tooti sweet, Mounseer. Look out sharp! BLUEBEARD Make haste! Catch 'em alive! Here we are! How are you? Good morning! Don't you wish you may get it? Ah! Coward, strike a white man! OEY No bono, allez tooti sweet, Mounseer. Look out sharp! Make haste! Catch 'em alive! Here we are! How are you? Good morning! Don't you wish you may get it? Ah! Coward, strike a white man! JOEY Now it's all fair; ain't it, Punch? Booth Sounds of battle come from the booth. Gilles and Joan are back on their horses watching the battle. regor Cameron 198272670 Joan are back on their horses watching the battle. JOAN (praying)) Holy lord, heed thy servant, listen to the entreaty of thy humble servant. For surely the battle doth turn away from thy face and towards the English. If this truly be thy will tell me and we may withdraw. But if thy will is for us to carry the day show thy strength, bolster our wills and send us clear sign that your will be done. GILLES How much good will this do, girl, god doesn't seem to be here this day. Keep up your entreaty if you will but until the wind changes the tide of battle will flow with the English. JOAN Then the wind must change! Oh Lord hear thy servant! Let the wind turn against the tide of the battle. Let your servant carry her pennant victorious into Orleans this day. Let not the night come before french feet encircle and break the siege before us. There is a turning in the sounds of the battle as the men begin cheering. Joan turns to Gilles JOAN You see? The battle turns with the wind. GILLES Some god. JOAN I must get down there. My pennant must fly. She gets off her horse, unfurls her flag and makes for the battle. GILLES La pucelle! Stop, you must not go there! You know not JOEY They begin trading blows again till it begins to really look like a dance. Finally Joey uses his finger to so insult Punch knocks him down, apparently dead. Punch then begins to count the bodies - Judy, Crocodile and Joey. But the Clown is not dead and every time Punch moves a body to centre stage, the Joey Clown moves it back to the side when Punch is not looking. Punch gets more and more excited. Then the Clown removes bodies one at a time till only he is left. Punch is puzzled and looks very closely - the Clown suddenly comes to life and hits Punch, then runs away. JOEY JOEY Good! You can't catch me. 44 GILLES How much good will this do, girl, god doesn't seem to be here this day. Keep up your entreaty if you will but until the wind changes the tide of battle will flow with the English. (praying)) Holy lord, heed thy servant, listen to the entreaty of thy humble servant. For surely the battle doth turn away from thy face and towards the English. If this truly be thy will tell me and we may withdraw. But if thy will is for us to carry the day show thy strength, bolster our wills and send us clear sign that your will be done. what you do. what you do. JOAN You think me a fool, De Rais? God is with us, we can not be defeated. JOAN You think me a fool, De Rais? God is with us, we can not be defeated. JOAN You think me a fool, De Rais? God is with us, we can not be defeated. JOAN JOAN My champion! The Maids champion?! Crying like a village girl. Get hard, De Rais, the battle's hardly over. God would not allow the English to be disappointed in their enemy- the Maid of France. She climbs back up on her horse and gallops off, G S Aye, but can we not still be killed- I see them e'en now being cut down by the English archers. She acts the fool- running among those whose role is to die, holding her pennant high. A stray arrow is all it would take, a stray- and there it is, she falls. GET HER OUT OF THERE, YOU FOOLS, IT IS THE MAID, BRING HER HERE, FOR GOD'S SAKE!!! Some men carry in Joan's body aloft. An arrow sticks out of her shoulder. She does not move as they lay her down. Gilles gets off his horse and moves over to her. Aye, but can we not still be killed- I see them e'en now being cut down by the English archers. She acts the fool- running among those whose role is to die, holding her pennant high. A stray arrow is all it would take, a stray- and there it is, she falls. GET HER OUT OF THERE, YOU FOOLS, IT IS THE MAID, BRING HER HERE, FOR GOD'S SAKE!!! Some men carry in Joan's body aloft. An arrow sticks out of her shoulder. She does not move as they lay her down. Gilles gets off his horse and moves over to her. JOAN 45 45 GILLES Ah, Jeanne. Too soon taken, foolish girl. All your talk of god and voices avails you nothing- I can not believe in your god if all he does is allow his servants thus to die. I can not believe this- I care. A foolish peasant girl and I care, I feel a sadness that I've never felt before- and this a girl! A girl? Where are your miracles now girl? G S Aye, but can we not still be killed- I see them e'en now being cut down by the English archers. She acts the fool- running among those whose role is to die, holding her pennant high. A stray arrow is all it would take, a stray- and there it is, she falls. GET HER OUT OF THERE, YOU FOOLS, IT IS THE MAID, BRING HER HERE, FOR GOD'S SAKE!!! G S Aye, but can we not still be killed- I see them e'en now being cut down by the English archers. She acts the fool- running among those whose role is to die, holding her pennant high. A stray arrow is all it would take, a stray- and there it is, she falls. GET HER OUT OF THERE, YOU FOOLS, IT IS THE MAID, BRING HER HERE, FOR GOD'S SAKE!!! Some men carry in Joan's body aloft. An arrow sticks out of her shoulder. She does not move as they lay her down. Gilles gets off his horse and moves over to her GILLES Damn girl. End of Scene two JOAN 46 MA Thesis: The Role of Misrule in the practice of Performance pennant high. Gilles peels off and glumly follows her. GILLES Damn girl. End of Scene two Gregor Cameron 198272670 47 Gregor Cameron 198272670 SCENE THREE ( Punch's arrest, The Demon's inquisition and the beginning of the folk tale. ) Booth The booth becomes the inside of a forest- there are the sounds of men beating the bush in the distance. Three puppet girls come into the glade singing. They are dressed, very flowery, as simple folk and walk hand in hand as sisters do. JUDITH Clara, Esmeralda, look at the flowers, this place is so beautiful. CLARA Indeed it is, little sister, indeed it is. Wouldn't it be fine to live within these lands as ladies, free to walk these paths as if they were our own. ESMERALDA But they are not our own, sisters, we must remember we are here without permission. I for one admit to not a little fearfulness. JUDITH Oh, stop it Esme, you are forever worrying- look at this day, look at these flowers, can there be a better time to be alive. ESMERALDA I have heard tales, Judith... JUDITH And tales are all they are. We can hear the Gamesmen, they are far away, no one will bother us in this part of the wood. CLARA And besides Esme- their lord is never home, he travels constantly on business, trade, whatever it is that men do. We are perfectly safe... ESMERALDA SCENE THREE ( Punch's arrest, The Demon's inquisition and the beginning of the folk tale. ) folk and walk hand in hand as sisters do. JUDITH Clara, Esmeralda, look at the flowers, this place is so beautiful. CLARA Indeed it is, little sister, indeed it is. Wouldn't it be fine to live within these lands as ladies, free to walk these paths as if they were our own. ESMERALDA JUDITH Clara, Esmeralda, look at the flowers, this place is so beautiful. CLARA I saw him do murder, I saw him take blood, I saw him take pleasure, my witness to god. They freeze Screen As the lights come up on the screen Mr. Punch is seated at a table. He appears to be in a police interview room. A door opened and a detective's shadow stands in the light. POLICEMAN Now then, now then, what's going on here? PUNCH. (To camera) The Bill? POLICEMAN Now Mr. Punch, I've a warrant for your arrest. PUNCH. You've left your wallet in your vest? POLICEMAN I haven't left my wallet in my vest - I've a warrant for your arrest PUNCH. You want a rest? POLICEMAN No I don't want a rest. I've an order to take you in. PUNCH. And I've an order to knock you down. (Does so.) CLARA CLARA Indeed it is, little sister, indeed it is. Wouldn't it be fine to live within these lands as ladies, free to walk these paths as if they were our own. ESMERALDA But they are not our own, sisters, we must remember we are here without permission. I for one admit to not a little fearfulness. JUDITH Oh, stop it Esme, you are forever worrying- look at this day, look at these flowers, can there be a better time to be alive. ESMERALDA I have heard tales, Judith... JUDITH And tales are all they are. We can hear the Gamesmen, they are far away, no one will bother us in this part of the wood. CLARA And besides Esme- their lord is never home, he travels constantly on business, trade, whatever it is that men do. We are perfectly safe... ESMERALDA 48 BLUEBEARD (O.S.) And yet your sister may be right. They freeze. Stage The Inquisitor walks out onto the stage. Gilles is lead out in chains. They follow a stylized grid pattern on the floor which could suggest a 'court'. INQUISITOR My Lord, De Rais, You stand accused, you stand accused of crimes so odious, Odious crimes for which you stand condemned. Condemned to die through your own confession, Confessed before these lords so pious. GILLES I tell my tale to you, most pious judges, I tell my tale, I tell it voluntarily, freely and grievously, I committed these crimes, so odious to your ears, according to my imagination and idea, suffering no one's counsel and following my own feelings, solely for my pleasure and it's carnal delight. I had no other intention, nor any other end. GILLES I tell my tale to you, most pious judges, I tell my tale, I tell it voluntarily, freely and grievously, I committed these crimes, so odious to your ears, according to my imagination and idea, suffering no one's counsel and following my own feelings, solely for my pleasure and it's carnal delight. I had no other intention, nor any other end. Prelati the "sorcerer" is lead out in chains. GILLES Francois? PRELATI My lord. 49 Gregor Cameron 198272670 INQUISITOR Your word? PRELATI I came as a mendicant, was made a supplicant, conformed as an applicant, what followed I recant. Milord would have devils, Milord would have demons, as into the children he would go place his semens. PRELATI PRELATI I came as a mendicant, was made a supplicant, conformed as an applicant, what followed I recant. Milord would have devils, Milord would have demons, as into the children he would go place his semens. I saw him do murder, I saw him take blood, I saw him take pleasure, my witness to god. Screen POLICEMAN I haven't left my wallet in my vest - I've a warrant for your arrest PUNCH. You want a rest? POLICEMAN No I don't want a rest. I've an order to take you in. 50 POLICEMAN Now then Mr. Punch, striking an officer of the law is a very - serious offense. He punctuates his remarks with blows from his phone book, he finally pins Punch against the wall. POLICEMAN (CONT'D) Can I see your driving license? PUNCH. (Wriggles free and biffs Policeman) Here you are then. POLICEMAN (Hits Punch.) Punch, you are a villain. PUNCH. So are you (hits him.) POLICEMAN That's a good'un. PUNCH. That's a better. PUNCH. That's a better. POLICEMAN That's a topper. PUNCH. That's a whopper. The fight becomes violent and Punch finally kills the policeman. His blood is covering the table. PUNCH. That's a whopper. PUNCH. That's the way to do it. Oh what-a-pity, what-a-pity. Punch leaves. Stage. The inquisitor turns to the entrance. 51 51 Gregor Cameron 198272670 POITOU POITOU M'lord? I say these things not to hurt you, but to save myself. POITOU M'lord? I say these things not to hurt you, but to save myself. GILLES Ah, Poitou, Would I could just say I forgive you, it must suffice to say that into the next world shall we stride together, hand in hand. Gregor Cameron 198272670 Poitou walks in, chained as the others. GILLES Poitou? POITOU M'lord? GILLES Et tu, Brute? POITOU (confused.) I'm sorry, m'lord? GILLES Poitou? POITOU M'lord? GILLES Et tu, Brute? POITOU (confused.) I'm sorry, m'lord? The inquisitor raises himself to new heights. INQUISITOR INQUISITOR The prisoners will remain silent! This servant here has told a tale, a tale told of evil deeds. His deeds do paint a disagreeable picture, a tapestry of vile crime, against man, against god. Throughout the country-side, he tells, of going, he speaks of foul means and base spells, of luring the peasantry into giving up their children. Children gone missing over fourteen years, One hundred and forty-four children used in sacrifice. INQUISITOR The prisoners will remain silent! This servant here has told a tale, a tale told of evil deeds. His deeds do paint a disagreeable picture, a tapestry of vile crime, against man, against god. Throughout the country-side, he tells, of going, he speaks of foul means and base spells, of luring the peasantry into giving up their children. Children gone missing over fourteen years, One hundred and forty-four children used in sacrifice. INQUISITOR INQUISITOR I think you mad, m'lord, I think you mad, As for your boons I think to grant you. Your men they shall accompany thee, subject, under pain of torture, to their recantation. Recantation such as yours moves me thus, I, personally, shall then make sure that as you stand upon the pyre, a rope be placed around your throat, to give respite to your suffering and, as such, will your humble contrition gift you with our mercy. However for the third, I must confess, I do not understand, the Maid is dead, and has been, lo, these past ten years. I can not, as I know not, how to grant you this poor boon, but if it pleases you I shall leave you to your own devices, no visitor to succour you within your cell. Gregor Cameron 198272670 Gilles is left spread eagle on the stage. Booth The three girls cower as the sound of hooves is heard. Bluebeard, resplendent on a black roan enters the glade. BLUEBEARD I heard your singing. It brightened up a day when the boars have gone to ground and the deer are too clever by half. Who do I have the pleasure of addressing? CLARA My name is Clara, oldest daughter of the village Miller, these two are my sisters, Esmeralda and the youngest, Judith. BLUEBEARD Poitou and Prelati are led off. GILLES (CONT'D) Goodbye, Francois, my friend! As like never again shall we see each other in this world; I pray that God gives you plenty of patience and understanding, and to be sure, provided you have plenty of patience and trust in God, we shall meet again in the Great Joy of Paradise. And so I face my fate and while I have to face God, to whom I bow my head, I find myself with little to 52 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance regret, with little to forget, except the maid. I admit my fault as I,unbridled, applied myself to whatever pleased me, and pleased myself with every illicit act. I would ask my lords, most humbly, for three boons, first, I would request that I and my two men are taken off together. Then I ask that, as a noble tween two commons, no naked flame be allowed to touch my living body. And final boon, I humbly ask, as God doth fix the devil, that as I do await my fate no man of god nor man at arms will allow the maid entrance into my cell. She knocks Gilles to the ground and puts him in the position of acquiescence as she speaks. Booth g BLUEBEARD I heard your singing. It brightened up a day when the boars have gone to ground and the deer are too clever by half. Who do I have the pleasure of addressing? CLARA My name is Clara, oldest daughter of the village Miller, these two are my sisters, Esmeralda and the youngest, Judith. BLUEBEARD BLUEBEARD I heard your singing. It brightened up a day when the boars have gone to ground and the deer are too clever by half. Who do I have the pleasure of addressing? CLARA BLUEBEARD 53 Clara, whose beauty would tame the heart of the wildest boar. ((Sotto voce)) and bore me stupid, she would methinks, both child and mind. (She giggles) Esmeralda, proud keeper of the rules, I see you ready and able to defend your sisters, against all comers. (Sotto voce) and holds her maidenhead against all cum-mers- I'll find no pleasure there. (She harumphs.) And youngest Judith, the fairest flower, blossoms true all smiles. (Sotto voce) If I'm not too mistaken, this one sets herself a man- just as well then, that here, a man sets. He gets off his horse and saunters over to the Clara, whose beauty would tame the heart of the wildest boar. ((Sotto voce)) and bore me stupid, she would methinks, both child and mind. (She giggles) Esmeralda, proud keeper of the rules, I see you ready and able to defend your sisters, against all comers. (Sotto voce) and holds her maidenhead against all cum-mers- I'll find no pleasure there. (She harumphs.) And youngest Judith, the fairest flower, blossoms true all smiles. (Sotto voce) If I'm not too mistaken, this one sets herself a man- just as well then, that here, a man sets. He gets off his horse and saunters over to the women. Clara, whose beauty would tame the heart of the wildest boar. He gets off his horse and saunters over to the women. BLUEBEARD (CONT'D) BLUEBEARD (CONT'D) I see you admire the great extent of my lands. That pleases me greatly. Little did I realise that when I left my home to rove about my woods today that instead of the mangy boar I expected, I would be dazzled by the beauty I behold before me. Prithee, am I unlucky? Are all of you married? CLARA So please your grace, I must report that today you are unlucky- I am not married but am betrothed to a fine blacksmith's apprentice. My sister here is also tak'n, tho' by God rather than by man- she's for a nunnery. BLUEBEARD And Judith.... If I may be so bold to ask. CLARA Alas. M'lord, she is too young- no man has claimed her. My father has been traveling the nearby villages but as yet can find no suitable suitor. CLARA Alas. M'lord, she is too young- no man has claimed her. My father has been traveling the nearby villages but as yet can find no suitable suitor. 54 BLUEBEARD No suitor to suit 'er? How sad. I wonder how that might be put right. May I suggest that you pretty ladies accompany to my home for some refreshment? ESMERALDA Oh, no, sirrah. That would not be right (she frowns at Judith) I could not possibly be party to leaving with an unknown man- who are you, that you feel so willing to be bold? BLUEBEARD My name is Bluebeard, noble of this parish, owner of these woods and all you see around you. JUDITH Sisters! Do you hear? He is the owner of these woods. He can give us leave to enjoy these paths. BLUEBEARD My name is Bluebeard, noble of this parish, owner of these woods and all you see around you. JUDITH CLARA And does that mean, sister, that you would go with this man? What would our father say? I think he would little approve of your excitement, however much this man may mean us no harm. Your pardon m'lord. Gregor Cameron 198272670 mean us no harm. Your pardon m'lord. BLUEBEARD No pardon needed, my dear, I would that ev'ry father's daughter could be as faithful. And thus I take my leave- I will be sure to call upon your father, as time arises, so that I may enjoy your company anon. He jumps upon his horse and rides off. The girls begin to walk off. JUDITH Clara! ESMERALDA Judith! CLARA Sisters! We must get home and tell our father of these things, Oh, what news! ESMERALDA It's he I have heard tales of. I like it not. JUDITH Oh, Esme! But didn't you think him handsome.... 55 End of Scene three End of Scene three 56 56 SCENE FOUR (Punch's grand larceny, the night of Joan's escape and the running game.) Stage The stage features an old time Tavern sign. It is night-time. Punch enters laughing. PUNCH. That was a jolly lark, wasn't it? (Sings,) 'I'd be a butterfly in a bower. Making apple-dumplings without any flour.' Punch disappears and re-enters with a bell. PUNCH. This is my pianner-sixty: it plays fifty tunes all at one time. He goes up to the public-house painted on the side-scene, or cottage, represented as a tavern, or hotel. Punch plays up a tune and solicits for money. Publican wakes up in a passion through the terrible noise; pokes his head out and tells him to go away. PUNCH. Go away? Yes, play away! Oh, you means, O'er the hills and far away. Punch keeps on ringing his bell violently. egor Cameron 198272670 SCENE FOUR (Punch's grand larceny, the night of Joan's escape and the running game.) Stage The stage features an old time Tavern sign. It is night-time. Punch enters laughing. PUNCH. That was a jolly lark, wasn't it? (Sings,) 'I'd be a butterfly in a bower. Making apple-dumplings without any flour.' Punch disappears and re-enters with a bell. PUNCH. This is my pianner-sixty: it plays fifty tunes all at one time. He goes up to the public-house painted on the side-scene, or cottage, represented as a tavern, or hotel. Punch plays up a tune and solicits for money. Publican wakes up in a passion through the terrible noise; pokes his head out and tells him to go away. PUNCH. Go away? Yes, play away! Oh, you means, O'er the hills and far away. Punch keeps on ringing his bell violently. PUBLICAN ( in a violent passion, opens the door, and pushes him away) Be off with you PUNCH. I will not. (Hits him over the head with the bell.) You're no judge of music. (Plays away.) Publican exits to fetch a cudgel to pay him out. Punch no sooner sees the cudgel than he exits, taking his musical instrument with him. Punch returns again with his bell. Publican pretends, as he stands beside the curtain, to be fast asleep, PUNCH. 57 Gregor Cameron 198272670 PUBLICAN If he comes up here, I'll put one upon his tibby. Punch comes out from behind the opposite side, and rings his bell violently. Publican makes a blow at him with his cudgel, and misses, PUBLICAN How dare you intrude upon my premises with that nasty, noisy bell! Punch, while publican is watching at this side-scene, appears over at the other, with a artful dodge, and again rings his bell loudly, and again the publican misses him; and while publican is watching at this side-scene, Punch re-enters, and draws up to him very slowly, and rests his pianner-sixty on the board, while he slowly advances to him, and gives him it whack on the head with his fist. Punch then disappears, leaving his bell behind, and the Landlord in possession of his music. PUBLICAN How dare you intrude upon my premises with that nast noisy bell! Punch, while publican is watching at this side-scene, appears over at the other, with a artful dodge, and again rings his bell loudly, and again the publican misses him; and while publican is watching at this side-scene, Punch re-enters, and draws up to him very slowly, and rests his pianner-sixty on the board, while he slowly advances to him, and gives him it whack on the head with his fist. Punch then disappears, leaving his bell behind, and the Landlord in possession of his music. PUBLICAN (collaring the bell) Smuggings! prusession is nine points of the law! So this bell is mine, ((guarding over it with a stick)) Smuggings! This is mine, and when he comes up to take this bell away, I shall have him. Smuggings! It's mine. Punch re-enters very slowly behind the publican as he is watching the bell, and snatches up the bell. PUNCH. That's mine! He runs away Publican sadly returns inside to his bed. Booth Enter Judith followed by Clara who is puffing. PUBLICAN (collaring the bell) Smuggings! prusession is nine points of the law! So this bell is mine, ((guarding over it with a stick)) Smuggings! This is mine, and when he comes up to take this bell away, I shall have him. Smuggings! It's mine. PUBLICAN 58 MA Thesis: The Role of Misrule in the practice of Performance They have been chasing about Bluebeard's house and are finally standing before the cupboard they are not allowed to look in. JUDITH He loves me, can't you see he loves me. He gives me his keys and the run of his house. CLARA Oh, sister, please, sister, can't you run more slowly. To run about and check things out does not to me seem seemly. JUDITH But Clara dear, you heard him dear, Just before he left us, This house is mine dear, all mine dear, to do with as I pleases. CLARA Yes, sister, so I heard, sister, with but one exception. His keys, dear sister, all his keys, sister, will unlock the doors. But, sister, remember, dear sister, all but one are yours. JUDITH Yes, sister, no doubt sister, we must do this for our master. But look here Clara, dearest Clara, doesn't it your interest pique? CLARA No. JUDITH No? CLARA No. What you have, dear, is what you have, and this you should not dispute. For ev'ry man, dear, just ev'ry man, will have his little secret. JUDITH But why, dear, I ask you why, when he's safely married. For surely dear what's yours is his and should be JUDITH He loves me, can't you see he loves me. He gives me his keys and the run of his house. CLARA Oh, sister, please, sister, can't you run more slowly. To run about and check things out does not to me seem seemly. JUDITH But Clara dear, you heard him dear, Just before he left us, This house is mine dear, all mine dear, to do with as I pleases. CLARA Yes, sister, so I heard, sister, with but one exception. His keys, dear sister, all his keys, sister, will unlock the doors. But, sister, remember, dear sister, all but one are yours. JUDITH Yes, sister, no doubt sister, we must do this for our master. But look here Clara, dearest Clara, doesn't it your interest pique? CLARA No. JUDITH No? CLARA No. What you have, dear, is what you have, and this you should not dispute. For ev'ry man, dear, just ev'ry man, will have his little secret. JUDITH But why, dear, I ask you why, when he's safely married. For surely, dear, what's yours is his, and should be vice- versa. PUBLICAN CLARA What should and what is, dear sister, are almost always diff'rent. A woman's place, dear, is to keep quiet, dear, and in all things acquiescent. CLARA Oh, sister, please, sister, can't you run more slowly. To run about and check things out does not to me seem seemly. JUDITH But Clara dear, you heard him dear, Just before he left us, This house is mine dear, all mine dear, to do with as I pleases. CLARA What should and what is, dear sister, are almost always diff'rent. A woman's place, dear, is to keep quiet, dear, and in all things acquiescent. 59 Gregor Cameron 198272670 They Freeze. They Freeze. Screen. On the screen a low light fades up with a soundscape that suggests a dungeon, though not a terrible one. Joan sits in the best light hugging her knees close to her. She is dressed on in a sack with a rope too small to actually tie around her waist. The crudely cut head and arm holes display more than they conceal. She weeps. Above her at the top of the steps Gilles appears. He is dressed in a bloodied English soldiers garb. He pauses to check she is alone. GILLES Psst! Jeanne? Jeanne D'Arc, La Pucelle? You are there? JOAN I am here. Who is this? GILLES Tis' I, Gilles de Rais, thy champion and Marshal of France. JOAN Go away. GILLES I come to rescue you JOAN I told you. Go away. GILLES But.... He comes down to the bottom of the stairs. JOAN Leave me, De Rais, I have nothing left to give. GILLES I understand. You have given so much. JOAN GILLES JOAN I told you. Go away. 60 MA Thesis: The Role of Misrule in the practice of Performance And yet God has abandoned me. I no longer hear his voice. GILLES (aside- sotto voce) Did you ever... JOAN I did GILLES What? JOAN I heard his voice, Perhaps through his angels but no matter twas His voice. GILLES And as such you should not give up. To give in to your enemies.... JOAN Enemies who sit beside the throne of France as much as live across the channel. GILLES Granted and yet you sit here despondent, ready to give in to their demands. JOAN I shall not give in to their demands- not while there is life in my body. GILLES Then come with me! JOAN And how does that help? GILLES The people are with you. voice. GILLES (aside- sotto voce) Did you ever... JOAN I did GILLES What? JOAN I heard his voice, Perhaps through his angels but no matter twas His voice. GILLES And as such you should not give up. To give in to your enemies.... JOAN Enemies who sit beside the throne of France as much as live across the channel. GILLES Granted and yet you sit here despondent, ready to give in to their demands. JOAN I shall not give in to their demands- not while there is life in my body. GILLES Then come with me! JOAN And how does that help? GILLES The people are with you. JOAN With me? Here in this cell? Where I am constantly visited by their soldiers and their demands? They have even taken away my clothes, my soldiers garb- the only protection a woman may possess in a place such as this. GILLES Why do they place you here? JOAN Enemies who sit beside the throne of France as much as live across the channel. GILLES Granted and yet you sit here despondent, ready to give in to their demands. Gregor Cameron 198272670 GILLES Why do they place you here? 61 61 JOAN Cauchon would have me in whatever state he can. I fear this is no longer about me or in fact about the English, it is about the throne of France- and who controls it GILLES Ah, it was always about that. Nothing changes! JOAN But I heard the voice of God. GILLES Of course you did. No. Something goes on. GILLES No thanks by the looks of it. We should leave, girl. GILLES But that means nothing in the ears of men. You must now look to yourself. JOAN Myself- look at myself, a peasant in sacking, an easy target to any man who would. GILLES You remain the maid of France! JOAN And where in this is the King? Did he send you? GILLES No (he hangs his head) I came myself, for my own reasons. JOAN And what would they be? GILLES You are the maid of France. JOAN Enough! De Rais, I think you are lying. Why are you here? GILLES Isn't it enough that I am here? 62 62 Thesis: The Role of Misrule in the practice of Performance No. Something goes on. GILLES It's just not right. That you should be here. In this prison rather than a place of Nuns. You, who gave so much, who led the army of France to victory. I could not stand still and not act. JOAN And that action brings you what, De Rais? GILLES No thanks by the looks of it. We should leave, girl. JOAN Ah, its back to 'girl', is it? GILLES I know not what else to call you. La Pucelle? Or have the English already had their way? JOAN No. They have attempted but so far respond to the lashing of my tongue- that drives them off almost as well as the two dozen cords that firmly lace and tie together my manly garb. And that Cauchon uses to abuse the law against me. LA HIRE (O.S.) M'lord, a soldier comes! JOAN That is la Hire? GILLES You did not think I would venture this alone? He calls upstairs GILLES (CONT'D) La Hire. Hold them off. Tell them an 'English lord' doth take his pleasure. That should hold off their queries. LA HIRE (O.S.) JOAN I thank God for him then. JOAN I thank God for him then. GILLES As do I. But he remains right, Jeanne, We must be quick. JOAN Then indeed, M'lord, take thy pleasure. JOAN GILLES (CONT'D) La Hire. Hold them off. Tell them an 'English lord' doth take his pleasure. That should hold off their queries. LA HIRE (O.S.) I shall, M'lord. But be quick my Lord. LA HIRE (O.S.) I shall, M'lord. But be quick my Lord. GILLES I do the best I can, La Hire. Thank you. 63 Gregor Cameron 198272670 JOAN He remains a good man? GILLES As good a man as any. Better for having met you, I believe. JOAN I thank God for him then. GILLES As do I. But he remains right, Jeanne, We must be quick. Joan stands and walks over to him. JOAN Then indeed, M'lord, take thy pleasure. GILLES What? JOAN Take thy pleasure, M'lord, better to give it to a Frenchman to have it tak'n by an English. GILLES No! I intended that we flee! We must get you out of here. JOAN I shall not flee, De Rais, I told you that. My voices have told me, back before they abandoned me, that I shall not leave Rouen. GILLES But, as you say, they have abandoned you. JOAN Aye, so they have, but I have not abandoned God. So now, to business, allow me to die a french-woman not an English whore. I would you were quick- I care not for your gentility. They freeze. JOAN He remains a good man? GILLES As good a man as any. Better for having met you, I believe. GILLES What? JOAN Take thy pleasure, M'lord, better to give it to a Frenchman to have it tak'n by an English. GILLES No! I intended that we flee! We must get you out of here. JOAN I shall not flee, De Rais, I told you that. My voices have told me, back before they abandoned me, that I shall not leave Rouen. GILLES But, as you say, they have abandoned you. JOAN Aye, so they have, but I have not abandoned God. So now, to business, allow me to die a french-woman not an English whore. I would you were quick- I care not for your gentility. They freeze. 64 MA Thesis: The Role of Misrule in the practice of Performance Booth Judith and Clara stand before the Cupboard. A large Key is in the lock- it is part of a huge key-ring with a multitude of Keys. MA Thesis: The Role of Misrule in the practice of Performance Booth Judith and Clara stand before the Cupboard. A large Key is in the lock- it is part of a huge key-ring with a multitude of Keys. JUDITH This key, sister, so you see, Clara, does fit the lock. And so, sister, all it needs, Clara, is but one small turn. So if you please, Clara, check my arm, Clara, As I fear that it may do so on it's own CLARA Judith! Do not touch it! Do not do this thing you know to be so wrong. For if you do, sister, you lose all, sister, And I can not see it ever coming home. JUDITH But you can see, sister, how unfair, sister, that he should have something I do not. CLARA But is it not, sister, the true nature, sister, of a man to hold back. Where we,sister, in our nature, sister, share with all, that which we have? CLARA JUDITH So will he change, sister, will he change,and thus share what he has told me not. Or should I push, sister, for this right, sister, to know all, as I should. CLARA I can not answer, little sister, this fair question, that you pose, for to do so, little sister. Would do more harm than it solves. JUDITH And so it falls, sis, to this move, sis, That I take now, of great size. She turns the Key. The cupboard opens to reveal seven bottles on two shelves each containing a woman's head. Gregor Cameron 198272670 BLUEBEARD (O.S.) And would you stake, love, you're very lives now, I see you have, love, now met my wives. Blackout The lights come up on the stage. 65 Screen Gilles now kneels astride Joan who lies with her head turned away from him. GILLES I'm not sure about this, It seems not proper. JOAN Tis' but a moment's pain or so I am told- a moment twix woman and maid, between longing and belonging. GILLES Belonging to whom? JOAN That is the question. GILLES I can not do this (he withdraws away from her) I feel no desire for you. JOAN What? You! Marshall of France. No desire? GILLES No desire for you. JOAN Why not? GILLES Your God didn't make me that way. Silence. Joan sits up. JOAN You are saying... GILLES Your god chose another way for me. JOAN The way of the devil? Screen Gilles now kneels astride Joan who lies with her head turned away from him. GILLES Your God didn't make me that way. Silence. Joan sits up. JOAN You are saying... GILLES Your god chose another way for me. JOAN The way of the devil? 66 MA Thesis: The Role of Misrule in the practice of Performance GILLES Perhaps.... Yes. The devil make me do it. JOAN I still wish for you to offer this small mercy to me. Should I turn this way? She turns round rising to her knees. GILLES No, Jeanne, It is too much. I am not going to do this. JOAN You will! I wish it! I would rather die a woman than be wronged by these damned English. Do it!!! GILLES (near tears) Jeanne! JOAN De Rais! He moves behind her, his hand brushing her back. GILLES I...I... JOAN I already forgive you, de Rais. He unfastens his trousers. Moves into position. JOAN (CONT'D) Forgiveness, De Rais. No more maidenhead. The Screen begins to fade JOAN (CONT'D) Forgiveness. He thrusts. They freeze. Stage The Joey CLOWN re-enters with Punch 67 67 PUNCH. I'm very glad to see you. Oh, Joey, my friend, how do you do? JOEY Here, Punch, are you a mind for a lark? (Peeping in at the cottage window, represented as a public house.) Are you hungry, Punch would you like something to eat? PUNCH. Yes. JOEY What would you like? PUNCH. Not peculiar. JOEY I'll go up, to the landlord, and see if he's got anything to eat. (Goes into the tavern, then pokes his head out.) Here, Punch; here's a lot of sausages hanging up here. Would you like some for supper, eh, Punch? PUNCH. I'm very glad to see you. Oh, Joey, my friend, how do you do? JOEY Here, Punch, are you a mind for a lark? (Peeping in at the cottage window, represented as a public house.) Are you hungry, Punch would you like something to eat? PUNCH. Yes. JOEY What would you like? PUNCH. Not peculiar. JOEY I'll go up, to the landlord, and see if he's got anything to eat. (Goes into the tavern, then pokes his head out.) Here, Punch; here's a lot of sausages hanging up here. Would you like some for supper, eh, Punch? PUNCH. Yes, to be sure. JOEY Make a noise; wake the landlord. PUNCH. (Whispering) Hand' em out here. Screen JOEY Nay, I have an idea. Do you pretend as I did to be dead over there. Can you do this? PUNCH. Maybe JOEY I'll go up, to the landlord, and see if he's got anything to eat. (Goes into the tavern, then pokes his head out.) Here, Punch; here's a lot of sausages hanging up here. Would you like some for supper, eh, Punch? JOEY Make a noise; wake the landlord. PUNCH. (Whispering) Hand' em out here. JOEY Nay, I have an idea. Do you pretend as I did to be dead over there. Can you do this? PUNCH. Maybe. 68 MA Thesis: The Role of Misrule in the practice of Performance Would you like something hot, Punch? PUNCH. Yes, to be sure. JOEY Then do you make like a deadman, sleeping. - Here's a lark - Make haste - Here's the landlord a coming. Punch lies down and seems to be sleeping. Enter the Publican scratching and yawning JOEY Hey now, good man, I would buy a drink for the house! PUBLICAN At this hour? away, go to bed! JOEY Nay, be more accommodating. A drink I say and something to eat, I have coin. PUBLICAN Very well. One drink and that'll be all right. JOEY And one for my friend, out here, sleeping. PUBLICAN Very well, very well, I'll take one out to him. JOEY He sleeps, like as not you'll need to wake him Joey enters the tavern. Publican brings a drink for Punch. PUBLICAN Heigh-ho, mister, here's your drink. Punch doesn't move. PUBLICAN Heigh ho MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Would you like something hot, Punch? PUNCH. Yes, to be sure. JOEY Then do you make like a deadman, sleeping. - Here's a lark - Make haste - Here's the landlord a coming. Punch lies down and seems to be sleeping. Enter the Publican scratching and yawning JOEY Hey now, good man, I would buy a drink for the house! PUBLICAN At this hour? away, go to bed! Would you like something hot, Punch? JOEY Nay, be more accommodating. A drink I say and something to eat, I have coin. JOEY And one for my friend, out here, sleeping. PUBLICAN Very well, very well, I'll take one out to him. 69 What is this? Dead? Is this a wake? JOEY (entering) Awake? have you given my friend his drink? PUBLICAN I-I-I can not. It appears he's dead. JOEY Dead? No he was alive mere minutes ago. You're lying (He shakes Punch) Oh Punch, what has happened, are you dead, has this rascal kilt you? PUBLICAN No, I.... JOEY You have killed him! I shall have justice. I shall tell all (shouting) Help! Murder! Send for the Beadle, my friend is dead! PUBLICAN I did not do this. Please ? Do not shout, do not! JOEY What should I do, my friend is dead and you were the one nearest him. What am I to do now? How do I see my friend off properly. A wake you say, a wake. I have not coin for that. How am I to feed the mourners? PUBLICAN That I can help with. I have sausages, I have bread, I have both wine and Beer for you to drink, only please be quiet, I did not do this, please don't say I did. P h H th bli i i JOAN Gilles? Is it? Is it supposed to hurt like this? GILLES Only.... A ...moment's..... Hurt. JOAN It feels funny (pause) I'm not sure.... Gilles? ....I'm not sure that this is right..... Gilles? GILLES For.... Give..... -ness. For ....give ....-ness. JOEY PUBLICAN That I can help with. I have sausages, I have bread, I have both wine and Beer for you to drink, only please be quiet, I did not do this, please don't say I did. Punch opens one eye. He sees the publican giving Joey the sausages and the beer and begins to giggle. Joey is caught red-handed. Punch and Joey run off leaving the Publican to curse his fate and set off to tell the Beadle. S Screen 70 MA Thesis: The Role of Misrule in the practice of Performance The screen lights up but to blackness. There is just the sound. Gilles resumes thrusting, each one punctuated by a syllable. GILLES For... give.... -ness. For....give.... -ness. JOAN Gilles? Is it? Is it supposed to hurt like this? GILLES Only.... A ...moment's..... Hurt. JOAN It feels funny (pause) I'm not sure.... Gilles? ....I'm not sure that this is right..... Gilles? GILLES For.... Give..... -ness. For ....give ....-ness. JOAN Gilles! Stop! This isn't right. Gilles! GILLES For ....give ....-ness. FOR........GIVE.........NESS!!! He withdraws. Breathing. JOAN Gilles! Stop! This isn't right. Gilles! GILLES For ....give ....-ness. FOR........GIVE.........NESS!!! He withdraws. Breathing. GILLES For ....give ....-ness. FOR........GIVE.........NESS!!! He withdraws. Breathing. Gregor Cameron 198272670 JOAN That.... Wasn't what I thought it was. I feel no different. Are you sure that was it- should I feel more? GILLES I.... Beg your forgiveness, maid. Stage Joey and Punch enter puffing, and laughing. They begin to share out there loot JOEY That was a lark was it not, Mister Punch, I do believe his face went through three shades of red. JOAN That.... Wasn't what I thought it was. I feel no different. Are you sure that was it- should I feel more? 71 Gregor Cameron 198272670 PUNCH. Indeed it was, Joey indeed it was. And to end up with all this loot. I do believe I shall eat well tonight. (He suddenly looks up) What's that? He points up and away. Joey turns to look. Punch hits him over the head. Joey collapses dead. That's-the-way-to-do-it. what-a-pity, what-a-pity. He smiles at the audience. Mr Punch leaves the stage dragging his body. Screen The screen fades up to show Gilles and Joan lying together. JOAN Maid no more. GILLES Aye- still a maid. (There is a long pause. They look at each other.) I could not do it. I could not take it from you, but I could not say nay to you either. I must beg your forgiveness. JOAN You...? GILLES I stayed with what I know. JOAN You stayed..... You buggered me? GILLES Aye. To my shame. I gave in to my basest pleasure. Joan throws herself away from him. JOAN You beast! You blackguard! Get away from me! Go, get Screen JOAN You beast! You blackguard! Get away from me! Go, get out! 72 72 MA Thesis: The Role of Misrule in the practice of Performance GILLES I beg your forgiveness. JOAN To the devil with you. Ask his forgiveness, for you'll get none of mine- nor of my God's. GILLES Jeanne? You offered forgiveness... JOAN Not for this. Gilles stands. He looks at her for a time but she ignores him. With her presence gone he has nothing to do but leave. He begins up the stairs. JOAN Gilles. GILLES Yes, Jeanne? JOAN At least leave me your trousers. Gilles hesitates and then removes his trousers. He throws them over. Then head down he walks up the stairs. Joan takes the trousers and holds them close. JOAN De Rais. Tomorrow I go to my death- we shall not meet again before then. But mark my words- WE WILL MEET AGAIN. The lights fade down. JOAN At least leave me your trousers. Gilles hesitates and then removes his trousers. He throws them over. Then head down he walks up the stairs. Joan takes the trousers and holds them close. JOAN De Rais. Tomorrow I go to my death- we shall not meet again before then. But mark my words- WE WILL MEET AGAIN. The lights fade down. End of Scene four 73 Gregor Cameron 198272670 Gregor Cameron 198272670 SCENE FIVE (Jack Ketch and the Ghost, The burning of Gilles and Joan and the woodsman's chimney flue.) h GHOST OOOOooooohhhhhhh! Boooooooo! Ppppppuuuuunnnccchhhh! (She eggs on the audience to help get Punch's attention) AUDIENCE Mr Punch, look out behind you! A ghost etc.etc. PUNCH. What's the matter? ... You've seen a ghost? I don't believe in ghosts. Ghost appears again - Punch turns, sees it, is terrified and chased around the stage but eventually he finds his slapstick and beats the ghost. PUNCH. That's the way to do it, etc. Diddy-diddy-doo. Enter the Beadle. Punch and the Ghost still struggling together. BEADLE. (Closing with them.) Hi, hi! This is him; behold the head of a traitor! Come along! Come to gaol! PUNCH. (A-kicking.) I will not go, help! PUNCH. What's the matter? ... You've seen a ghost? I don't believe in ghosts. Ghost appears again - Punch turns, sees it, is terrified and chased around the stage but eventually he finds his slapstick and beats the ghost. PUNCH. That's the way to do it, etc. Diddy-diddy-doo. Enter the Beadle. Punch and the Ghost still struggling together. BEADLE. (Closing with them.) Hi, hi! This is him; behold the head of a traitor! Come along! Come to gaol! PUNCH. (A-kicking.) I will not go, help! 74 BEADLE. (Shouting.) More help! More help! More help! More help! Come alo to gaol! Come along! Come along! More help! More he More help! Be quick! Be quick! Alas there is no more help but between them the Beadle and the Ghost haul Punch off to Jail. Screen On the screen the table is back, but this time a kitchen scene. Judith runs in screaming. She is alone. Suddenly the door bursts open. She cowers but it is not Bluebeard. Instead there are two people there- Clara stands behind a huge woodsma wielding an Axe. JUDITH Clara! CLARA Sister Judith! Judith looks to the big man. JACK My name is Ketch, Jack Ketch, they calls me that because I catch'em. I catch'em then dispatch'em. They share the joke at Jack's cheesiness. JACK Excuse me, Ladies, Duty calls. I shall be back. He heads off up to the booth. Bluebeard enters BLUEBEARD My Love? I come to find you. Come out and play. Freeze Booth PUNCH. What, am I to die three times? PUNCH. What, am I to die three times? Screen Gregor Cameron 198272670 BLUEBEARD My Love? I come to find you. Come out and play. Freeze Booth Enter Hangman Jack Ketch, or Mr. GRABALL the puppet version. The booth is now the town. As he speaks to Punch, Jack is constructing the gallows. 75 JACK Now, Mr. Punch, you are going to be executed by the British and Foreign laws of this and other countries, and you are to be hung up by the neck until you are dead - dead - dead. MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance JACK Now, my boy, here is the coffin, and here is the gibbet. PUNCH. There's the coffee-shop, there's giblets, and over there St. Paul's. JACK Get out, young foolish! Now then, place your head in here. PUNCH. What, up here? JACK No; a little lower down. A little comic business PUNCH. (Dodging the noose.) What, here? JACK (showing the noose again.) No, no; in there PUNCH. This way? JACK No, a little more this way; in there. Punch falls down, and pretends he's dead. Jack stops looking down at him. He worries for he may not be able to do his job. JACK Punch, Are you dead? They Freeze Stage. PUNCH. (Dodging the noose.) What, here? JACK No, no; you're only to die once. PUNCH. How is that? You said I was to be hung up by the neck till I was dead - dead - dead? You can't die three times. JACK Oh, no; only once. PUNCH. Why, you said dead - dead - dead. y y JACK Yes: and when you are dead - dead - dead - you will be quite dead. PUNCH. Oh! I never knowed that before. JACK. Now, prepare yourself for execution. PUNCH. What for? JACK For killing your wife, throwing your dear little innocent baby out of the window, for killing a policeman and striking the Beadle unmercifully over the head with a slap-stick. Come on. Hangman moves behind Scene, and re-enters, leading Punch slowly forth to the foot of the gallows. Punch comes most willingly, having no sense. 76 GILLES Are they not the same? JOAN Do you not know? GILLES Did you not? JOAN Aye, I did. I faced my God. As you will face yours. GILLES Are they not the same? GILLES Are they not the same? JACK 77 JOAN Do you not know? GILLES I do. My lord is far more unforgiving than yours. JOAN So, do you really recant your sins? GILLES Do you of yours? JOAN Aye, I did. And served my time. As you'll serve yours. GILLES With little hope of redemption. JOAN Which little do you deserve. GILLES Aye, I know. And yet I have with dignity met my maker. JOAN And that makes all the difference? 78 JUDITH That's him! The monster! The things he's done. She breaks down in tears. She breaks down in tears. JACK Step forward you. I would see the man I'm about to kill. BLUEBEARD Kill, is it? This is the man you would bring before me, Judith, You think he is enough? Perhaps this will change his mind. He throws some coins onto the stage. Jack ignores them. Gregor Cameron 198272670 79 JACK More than enough for the likes of you. Judith, Clara, open the fire. The two women begin to move to either side of the Fireplace. BLUEBEARD No, don't! JACK Ladies, do it! Clara and Judith hurry over to the fire place to open it. Jack takes up his Axe and begins to close on Bluebeard. (**As the fireplace opens many blackened skulls fall and roll across the Gregor Cameron 198272670 JACK More than enough for the likes of you. Judith, Clara, open the fire. The two women begin to move to either side of the Fireplace. BLUEBEARD No, don't! JACK Ladies, do it! Clara and Judith hurry over to the fire place to open it. Jack takes up his Axe and begins to close on Bluebeard. (As the fireplace opens many blackened skulls fall and roll across the 79 floor.) floor.**) JACK And so the sins of the miscreant stand revealed. Back away, ladies, it seems fitting that he ends up the same way his poor victims met their end. Bluebeard backs up until he is on the threshold of the fireplace. JACK Any last words? BLUEBEARD You can't kill me. I must go on. It is for others to.... He backs in amongst the 'skulls', Jack strikes Bluebeard with his axe and the chimney fills with flames. The two women close the door on Bluebeard's screams. Jack straightens up and gathers Clara up into a kiss. She swoons. JACK Excuse me, ladies! I see another opportunity to ply my trade. Fare thee well! He leaves the two shaken women to stare after him as the screen goes dim. Stage Jack enters the stage putting on his executioners mask. He stands behind Gilles. As he speaks he begins to twist and tighten the rope around Gilles throat. JACK The church has spoken, my Lord, the charges found against you do condemn thee to death. However for thy 80 MA Thesis: The Role of Misrule in the practice of Performance true contrition thou art gifted a peaceful death. Gilles struggles to speak. JACK JACK Very well; as you're so kind and condescending, I will certainly oblige you by showing you the way. Here, my boy! Now, place your head in here, like this. (Jack puts his head in the noose) This is the right and the proper way; now, you see the rope is placed under my chin; I'll take my head out, and I will place yours in and when your head is in the rope, you must turn round to the ladies and gentlemen, and say - Good-by; fare you well. JACK Very well; as you're so kind and condescending, I will certainly oblige you by showing you the way. Here, my boy! Now, place your head in here, like this. (Jack puts his head in the noose) This is the right and the proper way; now, you see the rope is placed under my chin; I'll take my head out, and I will place yours in and when your head is in the rope, you must turn round to the ladies and gentlemen, and say - Good-by; fare you well. Jack speaks very slowly then - a stop between each of the words; for he's not for driving the people out of the world in quick haste without giving 'em time for repentance. He's far too hopeful. PUNCH. (Quickly pulling the rope and swinging the hangman out into space) Good-bye; fare you well. (Hangs the hangman.) Now, I'm free again for frolic and fun. (yelling off for his wife) Judy, Judy, here's a man hung hisself JACK PUNCH. Oh, yes I am. PUNCH. Please, sir, (bowing to the hangman) Do show me the way, for I never was hung before, and I don't know the way. Please, sir, do show me the way, and I'll feel extremely obliged to you, and return you my most sincere thanks. She breaks down in tears. GILLES La Pucelle? La Pucelle? I would still have your forgiveness. JOAN I can not give it. It is not within me. GILLES No, it was not any of the other times either. But all in all I must confess... He dies. JOAN Confess to your lord. And wish him greeting from the maid of heaven, for all too soon he may rue... JACK Light the fires! Gilles and Joan's masques are engulfed in flames. Jack heads back up to the booth. Booth JACK Hi! Punch, are you dead? Are you dead? Are you dead? PUNCH. (Hitting him with his right hand, knocking him back.) Yes. JACK (Rubbing his nose with his hand.) I never heard a dead man speak before. Punch, you are not dead! PUNCH. Oh, yes I am. JACK JOAN I can not give it. It is not within me. JOAN I can not give it. It is not within me. GILLES No, it was not any of the other times either. But all in all I must confess... He dies. JOAN Confess to your lord. And wish him greeting from the maid of heaven, for all too soon he may rue... JACK Light the fires! Gilles and Joan's masques are engulfed in flames. Jack heads back up to the booth. Booth JACK Hi! Punch, are you dead? Are you dead? Are you dead? PUNCH. (Hitting him with his right hand, knocking him back.) Yes. JACK (Rubbing his nose with his hand.) I never heard a dead man speak before. Punch, you are not dead! PUNCH. Oh, yes I am. JACK How long have you been dead? PUNCH. About si eeks GILLES No, it was not any of the other times either. But all in all I must confess... He dies. 81 81 PUNCH. PUNCH. (Quickly pulling the rope and swinging the hangman out into space) Good-bye; fare you well. (Hangs the hangman.) Now, I'm free again for frolic and fun. (yelling off for his wife) Judy, Judy, here's a man hung hisself Enter Judy doffing the ghost costume, in quick haste, bobbing up against the gallows. 82 MA Thesis: The Role of Misrule in the practice of Performance JUDY. Why, dear Mr. Punch, you've hung a man! Do take him down! How came you to do it? PUNCH. He got wet through, and I hung him up to dry. JUDY. Dear me! Why you've hung him up till he's dried quite dead! PUNCH. Poor fellow! Then he won't catch cold with the wet. Let's put him in this snuff-box. He Points to the coffin. Judy takes the figure down and gives it to Punch to hold, so as the body does not turn away, and then proceeds to remove the gallows. In doing so she by accident hits Punch on the nose. PUNCH. Mind what you are about! JUDY Make haste, Mr Punch, everybody's a-waiting! They hustle his legs and feet in; but they can't get his head in, the undertaker not having made the coffin large enough. PUNCH. We'd better double him up, and take the man to the grave, - not the grave, but the brave: 'cos he's been a brave man in his time maybe. They quickly finish the business checking too see who might be coming. Judy helps but extols Punch to hurry up as they are coming. Finally Punch lifts the coffin with Ketch in it, he checks around him before walking off while with the coffin he bobs Judy on the head. They leave. The Stage is empty. Silence. End of Scene five. C i ll Then the curtain call. The Professor enters with a long piece of chalk. He walks to the front of the 83 Gregor Cameron 198272670 stage and draws a line across the front. He goes right up the wall of the booth and across Punch's play- stage. Then he walks back and stands at his door. The other players enter. Punch and Judy enter and stand behind but close to the line. Bluebeard and Judith enter and stand behind the line but not as close as P & J appear to be. PUNCH. Finally Gilles and Joan appear, walk to the centre and place their toes over the line. All others appear arranged around them. As a chorus they say the final line and wait to receive the audiences applause. There is no bow. THE PROFESSOR In times to come, mark my words, this story will be told, to entertain the children. FIN Acknowledgments. Various histories of Gilles De Rais and Joan of Arc. Most particularly The Saint and the Devil by Frances Winwar 1948. The Folk tradition and tale of Bluebeard. A Punch and Judy Script as it first appeared in London Labour and the London Poor by Henry Mayhew, published in 1851 I appreciate that these three sources gave up major inspiration to the above work which is while somewhat truthful but is in fact complete bollocks. Any truth found herein is completely coincidental and not the intention of the author. THE PROFESSOR In times to come, mark my words, this story will be told, to entertain the children. THE PROFESSOR In times to come, mark my words, this story will be told, to entertain the children. FIN I appreciate that these three sources gave up major inspiration to the above work which is while somewhat truthful but is in fact complete bollocks. Any truth found herein is completely coincidental and not the intention of the author. 84 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Figure 2. Saint Punch Finale, Punch hangs the Hangman. Figure 2. Saint Punch Finale, Punch hangs the Hangman. Workshops The first manifestation of process was in calling together some workshops to investigate choices for the play. One of the first design choices in writing the play had been to choose puppets as a mode of story telling. The first workshop was an opportunity to tell a story using one of Johnʼs provocations. He knew that I was considering puppets and encouraged me to think across a wider field, suggesting dolls. I wanted my process to be open and therefore willing to explore offers became an important component in how I would approach this text. I arranged a Saturday workshop and called for collaborators. Several expressed an interest but the challenge here was the timing of the workshop outside of the University term. This limited how many responses I received. text. I arranged a Saturday workshop and called for collaborators. Several expressed an interest but the challenge here was the timing of the workshop outside of the University term. This limited how many responses I received. This meant a smaller group of people who were not all actors but who brought interesting insight to the process.10 I used a story from my Quaker background that seemed to me to reflect some of what I wanted to say in Saint Punch. The story, The Whipping of the Quaker Women11, comes from the Dover (NH) Public Library and is a true story of a very young America. It describes three young women on Quaker ministry who were punished by their more puritan neighbours. They were to be paraded through the villages where they had been delivering ministry and there the priest ordered them “to be stripped naked from the middle upwards, and tied to a cart, and after awhile cruelly whipped them, whilst the priest stood and looked and laughed at it.” The priest and deputy then travelled with them from Dover to Hampton. However, at Hampton the constable refused to take their clothes off. Further, he would This meant a smaller group of people who were not all actors but who brought interesting insight to the process.10 I used a story from my Quaker background that seemed to me to reflect some of what I wanted to say in Saint Punch. The story, The Whipping of the Quaker Women11, comes from the Dover (NH) Public Library and is a true story of a very young America. MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance 10 See Appendix One 11 http://www.dover.lib.nh.us/doverhistory/whippingofthequakerwomen.htm accessed 21/9/11 10 See Appendix One 11 Gregor Cameron 198272670 ttp do e b us do e sto y pp go t equa e o e t accessed 9 Context Gregor Cameron 198272670 85 The path from page to performance involved a number of elements that interconnected with each other. Although they seem separate elements, it is important to acknowledge that each one has had an affect upon the others. The script of Saint Punch arose from the specific provocation of John Downieʼs 2006 Honours course in Scriptwriting. As I approached this Creative Practice component of my course, John, now serving as Supervisor, offered a number of provocations that were very helpful (if a little challenging). 2010 had been a The path from page to performance involved a number of elements that interconnected with each other. Although they seem separate elements, it is important to acknowledge that each one has had an affect upon the others. The script of Saint Punch arose from the specific provocation of John Downieʼs 2006 Honours course in Scriptwriting. As I approached this Creative Practice component of my course, John, now serving as Supervisor, offered a number of provocations that were very helpful (if a little challenging). 2010 had been a 85 year of gathering knowledge across a number of theories to form a framework for the creative practice. John suggested trying to utilise the whole space when we had secured 93 Kelburn Parade, Old Drama House as our venue. His provocation was to think in terms of using the whole space. Old Drama House, 93 Kelburn Parade is just that, an old house which has been redesigned to offer a clear stage space on the top floor where chairs can be set up in a number of different ways, with a lighting box in one corner, an alcove opposite and a small backstage area. Downstairs is a green room and a studio space, which can be used for wardrobe/, dressing room. Off that space is a locked wardrobe space and a sunroom that stores furniture and is used as an office. Upstairs and Down are linked by a stairway at the back. Figure 3. Elliott and Adnan; discussion of The Whipping of the Quaker Women Figure 3. Elliott and Adnan; discussion of The Whipping of the Quaker Women 86 Workshops It describes three young women on Quaker ministry who were punished by their more puritan neighbours. They were to be paraded through the villages where they had been delivering ministry and there the priest ordered them “to be stripped naked from the middle upwards, and tied to a cart, and after awhile cruelly whipped them, whilst the priest stood and looked and laughed at it.” The priest and deputy then travelled with them from Dover to Hampton. However, at Hampton the constable refused to take their clothes off. Further, he would 87 have whipped them before day to save their embarrassment. However, the three young women insisted that the punishment be carried out just as it had been set. The constable, unable to find anyone willing to step in for him, set about the execution of his duties, shaking and trembling. He carried them through the other villages, dirt and snow, until in Salisbury he was convinced by a doctor to swear him in as a deputy who cared for their ripped bodies and then turned them over the Picataqua River away from those who would punish them. Such was the strength then of their witness that in time they returned, formed a meeting and over a third of Doverʼs citizens became Quakers. This subversive use of the puritan punishment seemed to me to be reminiscent of Figure 4. Patrick, Patricia and Thomas, with Toy Soldiers and Barbies, playing out The Whipping of the Quaker Women. Figure 4. Patrick, Patricia and Thomas, with Toy Soldiers and Barbies, playing out The Whipping of the Quaker Women. Figure 4. Patrick, Patricia and Thomas, with Toy Soldiers and Barbies, playing out The Whipping of the Quaker Women. Joanʼs conundrum at Rouen. This was the text that I wanted to play out during the workshop. Having gathered together we began to explore first the telling of stories and how these become a focus for performance. 88 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance The beginnings of the workshop involved a process of introductory and trust exercises. Together, this diverse group of people split into groups, would be able to work collaboratively. They included musicians and filmmakers, singers and actors, however most had very little experience onstage. This limited how far I could expect the work to develop at this time. However, we decided to have fun together with the exercises and this, for me, was key. I needed to be able to go with the offer, saying yes without expectation and receiving whatever this produced as an opportunity for me to learn. After a while I introduced the Quaker story and then provided the participants with the objects and dolls they were to use to perform the story. They took the elements and using the toys, dolls and other objects and spent about 30 minutes devising a presentation of the story. They worked generously together however and the stories gave me clear insight into the possibilities of using such props onstage. A couple of weeks later I put out a call for another workshop but timing got the better of me and I ended up working with a smaller though much more focused group. We decided that as we were such a small group that the most effective way of using the time would be to do a reading of the text of Saint Punch. This allowed me to hear the words for the first time. I loved it. Not perfect but it did give me confidence that I had something to say. If only it were not so hard to find people to play with. Gregor Cameron 198272670 89 Pre-Production Gregor Cameron 198272670 91 With some trepidation, I agreed to move my dates to the half-year break and started seeking actors with which to collaborate. This process was quite time consuming and challenging. It was important to me that those who chose to enter into the world of Saint Punch do so with their eyes open and with a sense of what my vision for the play was. I held auditions and interviews and finally ended up with a cast drawn from the university and from outside. They were a mixture of those with solid experience behind them and some who had very little experience of the stage. Fortunately, for the role of Gilles de Rais, I was able to invite an old acquaintance back to the stage after a decade of working professionally in film and television and this collaboration would turn out to be key to Saint Punchʼs success. I also had at this stage some key collaborators in Sound and Video. There were times however when I experienced some large doses of self- doubt. In discussion with my supervisor, John Downie, and Theatreʼs Jim Davenport, senior Technical Officer, it was decided to look towards a workshop performance of Saint Punch around Queenʼs Birthday in the mid Year break of the university. It was also made clear that the main stage at 77 Fairlie Terrace was not the right venue and that I could not finish the performance with real fire. Both of these decisions while they felt like a lack of faith proved to be the right ones and opened up my opportunities rather than closing them down. I spent the rest of the summer making design and look With some trepidation, I agreed to move my dates to the half-year break and started seeking actors with which to collaborate. This process was quite time consuming and challenging. It was important to me that those who chose to enter into the world of Saint Punch do so with their eyes open and with a sense of what my vision for the play was. I held auditions and interviews and finally ended up with a cast drawn from the university and from outside. They were a mixture of those with solid experience behind them and some who had very little experience of the stage. Thoughts Confidence is a key aspect of the creative process. It is at once the most fragile and your strongest ally. If it is not present then there is a great temptation to stall and not move on with a project. The workshops that occurred over the summer break 2010/2011 were a difficult pill to swallow. They seriously challenged my confidence in my own voice. It also felt very depressing as I had to renegotiate my timetable from the beginning of the first semester to the mid year break for putting on the showcase workshop performances. Confidence is a fragile thing that constantly straddles the edge of too much or too little. Despite putting a number of calls for people to become involved there were simply too many other priorities out there for most of the students I was contacting. They were either rehearsing for Summer Shakespeare or the Fringe Festival, or saving costs by having the summer at home, or working their butts off in one of the decreasing number of summer jobs. None of these things was a reflection on me or upon them but it did mean that only a few close friends became the players at the workshops, and most of them were more interested in crew work rather than acting. Of the people that turned up Thomas would go on to play Punch and Patrick to serve as Bluebeard, Elliott would take over the video studio and Patricia would do sound. It was these peoples faith that held this difficult time together for me. The workshops themselves served to put some ideas on their feet and to give me an 90 MA Thesis: The Role of Misrule in the practice of Performance appreciation of what the words sounded like. This was invaluable information and set my mind formatting what worked and what did not. This offered me the stimulus to write the final draft of the play. MA Thesis: The Role of Misrule in the practice of Performance appreciation of what the words sounded like. This was invaluable information and set my mind formatting what worked and what did not. This offered me the stimulus to write the final draft of the play. Pre-Production Fortunately, for the role of Gilles de Rais, I was able to invite an old acquaintance back to the stage after a decade of working professionally in film and television and this collaboration would turn out to be key to Saint Punchʼs success. I also had at this stage some key collaborators in Sound and Video. There were times however when I experienced some large doses of self- doubt. In discussion with my supervisor, John Downie, and Theatreʼs Jim Davenport, senior Technical Officer, it was decided to look towards a workshop performance of Saint Punch around Queenʼs Birthday in the mid Year break of the university. It was also made clear that the main stage at 77 Fairlie Terrace was not the right venue and that I could not finish the performance with real fire. Both of these decisions while they felt like a lack of faith proved to be the right ones and opened up my opportunities rather than closing them down. I spent the rest of the summer making design and look 91 91 decisions. This included beginning work on creating the Mr Punch Puppet. I would have loved to be able to get the cast to have worked upon their own puppets but unfortunately; time and space did not allow this. Rehearsals began on the second week of Trimester One 2011. We were lucky enough to be able to rehearse in the venue itself though this would also present challenges later on in the process. Casting Finding the actors, to fill up the cast proved to be another challenge. The summer had been dry of potential collaborators as people were either working, back at home or in other shows, for example planning for the fringe Festival in March or for the Summer Shakespeare. As the new first trimester began, I had Thomas Pepperell in place for Punch and Patrick Coelho in place for Bluebeard but needed another six or seven actors. I visited the theatre courses, put up notices and created a Facebook page for FatCircus to show up as an activity/production page. I received some response from the first year actors but in meeting them some were unable to commit, some unwilling to engage in the darkness of the piece. Finally I found Rosie Tapsell and Audrey Martin who would turn out to be an absolute gift in the way they whole- heartedly entered into the process. Audrey was perfect to play Judith and Rosie was great for Joan. I was talking to fellow postgrad, Kerry Glamsch, about the difficulty of casting and he was kind enough to promote my show at his Meisner class downtown. Out of that I met with three of his pupils, Emma about the difficulty of casting and he was kind enough to promote my show at his Meisner class downtown. Out of that I met with three of his pupils, Emma 92 MA Thesis: The Role of Misrule in the practice of Performance Smith seemed good casting as Judy, Piotr Wołodźko as Joey/The Policeman and Maria Becerro Casero seemed to fit Clara, Judithʼs sister. Martin Quicke appeared out of the second year theatre class in response to a notice and was keen to take on a number of challenges, Innkeeper, Jack Ketch, Poitou and the Professor/old man. Crucially I still did not have casting for Gilles de Rais but this is where Facebook came up trumps. Being on the Social Media site had meant that I had been getting back in touch (ʻfriendingʼ) with the wider Theatre community. One such person from my wider professional contacts was Des Morgan. Des and I had known each other for over a decade but had been travelling somewhat different roads. Getting back in touch with him served both of us as he was able to return to the stage after a decade mostly Figure 5. Casting Audrey begins to bring her puppet, Judith, to life Figure 5. Audrey begins to bring her puppet, Judith, to life Figure 5. Audrey begins to bring her puppet, Judith, to life doing film and television and I had a very strong experienced actor to take on the darkness that Gilles de Rais could have been. I finally had a cast that would travel with me generously and competently. Gregor Cameron 198272670 93 Rehearsals Rehearsals ran on Sunday afternoons (1-3 pm), Wednesday and Thursday evenings (6-8 pm) and a Saturday workshop (1-4 pm). This four-session breakdown was a deliberate choice to allow me to work on each of the three stories separately before bringing the scene together on the Saturday afternoon. It was also informed by the cast's own timetables. Thus Sunday began the week with Punch and Judy, Wednesday became home for the Gilles de Rais/ Joan story, Thursday the Bluebeard story. The first five weeks of rehearsal then allowed us to concentrate on one scene at a time and then begin putting it together on Saturdays. In order to prepare the soil for the framework I wanted to work within, I, very early on, decided that I would run rehearsals as consistently as I could. Early on in my experience, back in 1984 while studying at Wellington College of Education, I worked in workshop with Dorothy Heathcote, one of the worldʼs leading teachers of role and drama in the classroom, with the patients and staff at Porirua Hospital. Dorothy never seemed to change, each day she appeared exactly as she had the day before. My college tutor, Ralph McAllister, explained that Dorothy believed that in order to build trust and expectation in children and others that she worked with she wished to build into her work an image of who she was and how consistent she could be. To this end, she would only ever wear one outfit during a multi- day workshop, each night 94 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance washing it and then laying it out to dry overnight. Thus, the next day, she would appear to be just as the workshoppers had seen her the day before. She felt this would bring a consistency and a quicker entry into the next phase of the workshop- thereby providing a greater commitment to the creativity each day would bring. In fact, much of Dorothyʼs words stayed with me as I incorporated aspects of what she termed Mantle of the Expert work into the rehearsal room. I had decided that as much as I could I would use the ʻyesʼ answer to offers that would come from the cast and that this would include any offer to take over aspects of the rehearsal ritual. I considered that part of the process of them beginning to own their performance. It was important to me that a sense of play arose from the safety of a ritualised rehearsal process. Again being very accepting of how what might arise in the middle of a rehearsal might engender this. Therefore, rehearsals followed a particular format. They would begin with a 15- minute warm up exercise often in the form of a game. I would try to find exercises and games that reflected in some way what I wanted to achieve within the rehearsal. Then there would be a time of discussion (10-15 minutes), arising from the opening exercises, about aspects of the script, frame of the scene or conditions for forming their characters, after which we would put the scene up on the floor through script work, improvisation and role play (90 minutes or so). Week 1 was about the world of the characters. With the Punch and Judy characters I wanted the session to interrogate what the tradition was 12 Some notes on the Punch Play, Puppet Master Vol 2 #3 July 1948. Published by The British Puppet and Model Theatre Guild 12 Some notes on the Punch Play, Puppet Master Vol 2 #3 July 1948. Published by The British Puppet and Model Theatre Guild Gregor Cameron 198272670 95 all about- seashores and holidays, murder and mayhem. We discussed some of the comments that exist about Punch, such as from JR Cleland12 who notes in 1948 about Punch: …he is everything that is bad, immoral, subversive, sadistic and cruel, and an even worse influence upon the younger generation than the cinema. (Cleland 1948:65) I suggested that this sounds very familiar if we consider that Punch as a personification of the Carnivalesque. Punch serves admirably as the Dark Clown of the theatre and touches many of the essences of the Trickster himself. He serves as an archetype but not one that anyone would wish to emulate but rather serves as an example with which to contrast. Cleland acknowledges this as he goes on to link Punch with the ancient mystery and medieval morality plays. He senses something of the aspiration to the spirit in Punch. Through the physical and through the allegorical ancillary characters, Mr Punch tries to become more than his physical self. Clearly his presence freed from the puppet booth would be both clownish and much more violent. It was important that we talk about permissions and improvisations before we try to tie down what will be the limitations upon what was safe to place on the stage. 96 MA Thesis: The Role of Misrule in the practice of Performance With the historical story, it was all about the differences in power: how the nobility could and did get away with just about anything. How Gilles de Rais was unlikely to have been that different from the other nobles of the time although he was probably one of the most powerful, and how Joan DʼArc represented a new way of thinking. How she brought to the throne some clever ways of proving the veracity of her ʻvoicesʼ and how that still was not enough to protect her from the betrayals of manʼs politics. She also thought about her men in a way that was quite alien to Gilles. She demanded of them all the dignity of equals in the faith she placed in them and the standards she held them up to. This serves to further underline the difference in treatment that she and Gilles suffered. Bluebeardʼs Tale was introduced as a warning about the folly of trusting that all that glitters is gold. Gregor Cameron 198272670 We discussed how folk tales were a sort of short hand way of bringing an awareness of the outside world into the family and/or the village. How Bluebeard has been directly linked to the de Rais of history, how George Bernard Shaw had named de Rais in his play, Saint Joan, Bluebeard, and that perhaps Disney was a useful way to think of folk tales in the present (although very carefully considering the politics of Disney itself). I wanted that sense of a Folk Tale to come across to the audience and so the performances had to be larger than life. After this discussion, we would put the scene up onto its feet. Asking questions: what comes from the text, what opportunities there were to be found suggested by the directions and what were the prime motivators for the Gregor Cameron 198272670 97 97 characters involved. Clearly, in the first historical scene it is one of horror as Gilles rapes a child. However just before he climaxes, the spirit of Joan DʼArc possesses her and this frightens him terribly. We worked a lot on the simple physicality of the actors, where they could fit together giving a particular vision that the mediation of the camera augments. For Bluebeard the characters play out of sequence to contrast with the Puppets. We looked at the signification of power between Judith and Bluebeard at this point in their story. Bluebeard appears as this unstoppable physical presence that can throw her around like a rag doll. Posture and presence demanded a lot of time, and how such a loss of control affects the characters. As their first scene involved throwing Audrey around we also began talking about the choreography of stage combat and how we keep everyone safe. I wanted to use this scene to establish the brokenness of the bond between Judith and Bluebeard, his manic disposition and how strong consequently the bond between Clara and Judith would need to be. Figure 6. Judith, Audrey's Puppet, threatens to open the forbidden cabinet Figure 6. Judith, Audrey's Puppet, threatens to open the forbidden cabinet 98 MA Thesis: The Role of Misrule in the practice of Performance With Mr Punch, the story is in the traditional timeline sequence with the first scene being the one where Punch speaks to his wife Judy and then while looking after the baby, throws it out of the booth. He then covers his crime by killing Judy. In terms of rehearsal, this generated a conversation on tradition. We looked at some examples that included seeing a Punch and Judy man perform in a booth at the Kilbirnie Fair. We were able to talk to him for a few minutes afterwards. He was helpful, particularly in terms of not using the Swazzle, a mechanism for the mouth that produces Punchʼs voice. During rehearsals, I would work individually with Thomas in order that he not damage his voice by overdoing the falsetto that he adopted. The actors began using substitutes for the puppets right away as we had both the Mr Punch puppet that I had made over the summer and a doll about the same size for Judy. It was during this time that we also improvised around the frame of the play, which was to be a wedding- allowing us to bring in both the idea of a ritual gathering and a place where storytelling would happen. In this way, a ritual was set up for the rehearsal room, which would continue throughout the rehearsal process. Rather than detail how these rehearsals continued through this pattern, I will highlight several important moments where the vision was challenged and, through collaboration and rehearsal, changed. Each of the stories reached this stage so it is appropriate to offer these in turn. Gregor Cameron 198272670 Gregor Cameron 198272670 99 With Bluebeard, it arose out of a design decision. Early on, I wanted the actors to construct their own puppets but this became impossible due to the time constrains of the project. Thinking about the connection with Disney I thought that puppets that looked like Muppet characters might serve quite well so I started looking through the Internet to source them. After a New Zealand site let me down, I purchased them from Amazon.co.uk13. This, of course, meant that they arrived later than I might have desired. However by letting drips and drops of imagery and hints out to the actors I was able to build their anticipation quite high and they began playing the scene with their hands. By the time they did get their hands on the puppets, they leapt at the opportunity to play up the ʻDisney-nessʼ of the scene. Perhaps it was the familiarity of these types of puppets, which helped raise the level of performance. Perhaps it With Bluebeard, it arose out of a design decision. Early on, I wanted the actors to construct their own puppets but this became impossible due to the time constrains of the project. Thinking about the connection with Disney I thought that puppets that looked like Muppet characters might serve quite well so I started looking through the Internet to source them. After a New Zealand site let me down, I purchased them from Amazon.co.uk13. This, of course, meant that they arrived later than I might have desired. However by letting drips and drops of imagery and hints out to the actors I was able to build their anticipation quite high and they began playing the scene with their hands. By the time they did get their hands on the puppets, they leapt at the opportunity to play up the ʻDisney-nessʼ of the scene. Perhaps it was the familiarity of these types of puppets, which helped raise the level of performance. Perhaps it was the permissions that such a ʻmaskʼ offers that made the difference. However, we also talked about where these tales stand in a modern world. Out of this came a discussion of the article that accuses Disney of being a Nazi. By referencing Matt Rothʼs polemic article which, in seeking to define The Lion King, as Disney-fascism suggests that an illustration of the Nazi Regime can be found in the animated version of Pinocchio14. p _ _ p keywords=Doug+and+Melissa+puppets&x=0&y=0 13 http://www.amazon.co.uk/s/ref=nb_sb_noss?url=search-alias%3Daps&field- 14 http://www.ejumpcut.org/archive/onlinessays/JC40folder/LionKing.html last accessed 16/10/11 14 http://www.ejumpcut.org/archive/onlinessays/JC40folder/LionKing.html last accessed 16/10/11 15 Business in this sense being the physical and visual humour a performance can offer that does not rely upon the spoken word, in for instance the Commedia dellʼArte performance of Lazzi. Gregor Cameron 198272670 101 Gregor Cameron 198272670 In turn this lead us to the discovery of The Bloody Chamber by Angela Carter that some writers claim presents a feminist reading of the traditional story. The actors therefore were encouraged to explore this story not only as the old folk tale but also through 100 MA Thesis: The Role of Misrule in the practice of Performance this subversive retelling. This proved a wonderful example of how being prepared and doing homework can allow something innovative to exist upon the stage. For Mr Punchʼs story, I think the moment that we started really looking critically at what the script was suggesting allowed for a moment of Misrule. When they left the puppet booth and entered into the other two environments the puppet business became quite limiting. This difficulty arose because the original puppets are not very articulated, being limited to movement one hand can achieve. Business15 then must be broad and physical with none of the subtlety that a clown onstage can bring. Once they were downstairs I had a clear vision of what I wanted to reference- the Joker in the interview room from The Dark Knight (Nolan, 2008), not that we were going to be able to use the language of cuts and close ups that Christopher Nolan has to play with. However, it was a start and a clear pointer to what I wanted to get out of the scene. The challenge of this came to a head for me during the rehearsal of the Publican business in scene 4. While the re introduction of the Joey clown goes along the traditional line, the business later in the scene became complicated. In using the booth, the traditional show is limited to only having two characters on stage at any one time (most professors having only two hands of course). So with Punch and Joey and the Publican available to all be onstage this limitation wasnʼt really natural. We worked with the original scene however for some time before it was clear that it just was not working. 101 There were also problems relating to props that would need to designed and made, such as heads that thrust through bottoms of frying pans and red-hot pokers that poked into noses. 16 http://www.neilgaiman.com/works/Books/Anansi+Boys/ accessed 22/10/11 17 see appendix four 16 http://www.neilgaiman.com/works/Books/Anansi+Boys/ accessed 22/10/11 17 see appendix four p g 17 see appendix four 17 see appendix four 10 17 see appendix four 16 http://www.neilgaiman.com/works/Books/Anansi+Boys/ accessed 22/10/11 17 see appendix four Gregor Cameron 198272670 Considering this and with a wish to be balancing the other stories that were in Scene 4 I felt that the Punch story needed to be lighter and more comedic. In the end, it seemed that I would need to leave behind the director for a time and become the writer to resolve this. As it happened, I was reading Neil Gaimanʼs Anansi Boys (2005).16 I have already mentioned that Gaimanʼs work is a huge influence. In the novel, he tells a variation of a traditional Anansi story. Anansi is the spider version of the trickster stories that appear through many early cultures. In this version, Anansi cons some food from a local shopkeeper and then fools his adversary Tiger into killing his grandmother17. It seemed to me that Punch and Anansi had much in common and that perhaps I could adapt this story to fit the scene we were having trouble with. Thus at the next rehearsal I had a new scene for the actors to work with. Punch would pretend to be dead and Joey would thus con food and drink from the hapless Publican. This frame gave the clowns quite a bit of freedom to express their own ideas on what is funny and became one of my favourite scenes in the performance. For it to arise through the coincidence of my personal reading should not be thought of as an accident unless one, as I do, subscribes to the idea of the 102 MA Thesis: The Role of Misrule in the practice of Performance ʻhappy accidentʼ and the opportunity this provides. I agree with Anne Bogart when she suggests that all influences in oneʼs life contribute to the process of being an artist. As a result of a partnership with memory and the consequent journeys through the past, I feel nourished, encouraged and energized. I feel more profoundly connected to and inspired by those who came before. I feel the courage to articulate for my profession because of the shoulders upon which Iʼm standing feel sturdy. (Bogart, 2001:40) Scene 4 also provided a moment of inspiration in the rehearsing of the Gilles De Rais /Joan of Arc story. However, before that there had also been a moment of difficulty in scene three that had switched how we thought of the stage floor environment. 18 This gridwork was introduced to me by Annie Ruth while I was a student at Toi Whakaari: New Zealand Drama S h l Wh th it t f A i ʼ k l d f K ith J h t ʼ k A B tʼ I 18 This gridwork was introduced to me by Annie Ruth while I was a student at Toi Whakaari: New Zealand Drama School. Whether it arose out of Annieʼs knowledge of Keith Johnstoneʼs work or Ann Bogartʼs I am unsure. Gregor Cameron 198272670 10 18 This gridwork was introduced to me by Annie Ruth while I was a student at Toi Whakaari: New Zealand Drama School. Whether it arose out of Annieʼs knowledge of Keith Johnstoneʼs work or Ann Bogartʼs I am unsure. Gregor Cameron 198272670 103 School. Whether it arose out of Annie s knowledge of Keith Johnstone s work or Ann Bogart s I am unsure. Gregor Cameron 198272670 I did not want this story to be clownish even though we had established this as the basis of both the Punch and the Bluebeard story. This scene required the trial of Gilles de Rais to be centre stage. However there was a risk that the audience may not read this as a serious scene because of the rhythmic poetry of the words, I did not want them to be waiting for a joke that would never come, it being one of the wordiest scenes in the play. I wanted to have them listen with as little interference as possible. Earlier we had been using grid-work18 to help define the relationships between the characters. I suggested that perhaps having the court defined as a number of straight lines might stylise it so that once used to the convention the audience would listen to the nuances of the words. 103 Figure 7. In rehearsal the Inquisitor condemns Gilles to death Figure 7. In rehearsal the Inquisitor condemns Gilles to death Figure 7. In rehearsal the Inquisitor condemns Gilles to death 104 Emma dressed as the Inquisitor began what turned out to be mesmerizing approach to Des (as Gilles). For me it called up images of cats playing with prey, the hopeless captives trapped within the confines of the court. It culminated with Gilles prostrate on the stage, having begged for forgiveness, confesses his crimes and wins some boons from his condemners. He seemed from the audienceʼs point of view to be crucified upside down- a satanic image. This somewhat expressionistic approach made all the moves on stage seem deliberate and clean. It allowed the audience to hear the words without the expectation of anything physically surprising and yet still conveyed the form of the Court to shine through. This expression also helped as we found ourselves with one more character than there were actors to fill. Working with Martin, who turned out to be one of the busiest actors on stage, filling a number of 104 MA Thesis: The Role of Misrule in the practice of Performance shoes, we experimented with the idea of a Janus Mask- a face on both the front and the back of his head and through this he was able to play both of Gillesʼ servants. There is a rape in Scene 4 that mirrors the one in scene 1. Gregor Cameron 198272670 However, this rape was a far more difficult one for the actors to get their heads around, as it is quite complex in the way that, in a sense, both the characters are raped. I had a number of quite intimate conversations with Des, and Rosie was encouraged and supported by other members of the cast, especially the female cast members. I felt it important to not approach this material too quickly but with the sense that we were making sure each of us was safe in dealing with the intensity of the act. My original vision was to expose this moment as much as possible –to encourage the actors to choreograph and perform this with as much reality as the camera would allow. I hoped that placing it behind a glass would somehow make the actors safe and at the same time provide enough distance for the audience to be witnesses but not implied participants, as documentary can sometimes make us. I remember from my childhood a film shown at high school where a camera implacably focused on a nest of baby birds as a grass fire flowed through their meadow. I remember us being at once fascinated and disgusted as we realised that the film makers could have, if they had wished to, rescued the baby birds at any time and placed them in a place of safety. Instead, the camera holds upon the burning brood until the fire has swept through its gaze and continued its tidal wave way across the meadow. We found ourselves Gregor Cameron 198272670 105 caught up in the thought that we did not act. I did not want this to be the experience of our audience. I did however want the video feed to be read as a seeming documentary view, which would make the environment of naturalism that, much more affecting. A very difficult balance to find. However, as we approached the scene and talked about the context it served it was clear that Rosie was having difficulties with the subject matter. Often this would manifest as physical pain and blockages. Des became both concerned and frustrated. I think both he and I were also concerned because Rosie had just turned eighteen and we felt that possibly we were taking her to a place that could potentially prove damaging. This was something that no one intended. However, it appeared that this moment would prove to be the point upon which Then in one rehearsal we were talking about how the camera affects what it is that the audience is allowed to see. I was explaining how the camera was a way of distancing the actors from the audience and in a way that this Then in one rehearsal we were talking about how the camera affects what it is that the audience is allowed to see. I was explaining how the camera was a way of distancing the actors from the audience and in a way that this paralleled the affect that arose from the Puppet booth- after all the figures were approximately the same size. At some point the offer arrived from the actors of what would happen if we were to not allow the audience to see anything. They would still be able to hear everything. So we explored what it would be like, in effect, turning off the camera. Suddenly the audience is forced to experience 106 MA Thesis: The Role of Misrule in the practice of Performance the rape from a very different perspective. In fact in rehearsal I found myself having the hair on the back of my neck standing on end. We solved this issue with what I consider to be one of the clearest examples of how misrule provides the spark for creativity and how this can often offer the solution to a real problem that comes up in rehearsal. Gregor Cameron 198272670 In this case it involved masking the act of rape from the audiences eyes, something I will go into in more depth in the critical part of thesis Thus did rehearsals proceed, often falling into the pattern described, and sometimes breaking open to allow creative moments to appear. Later, Thus did rehearsals proceed, often falling into the pattern described, and sometimes breaking open to allow creative moments to appear. Later, rehearsals would be held to solidify our findings, to link the scenes together and to strengthen any moments that needed a deeper understanding or revision to make the best impression upon our audience- nothing very unfamiliar about this composition, which is why I choose to highlight a few key moments that affected the vision. I will talk in more detail about the production week as I talk about the design and tech used just before performance. Thoughts Gregor Cameron 198272670 107 Rehearsals are both a joy and a chore. Both these feelings colour my recall of them. Working with a group that varied in experience so much, I look back at the moments that were difficult with different eyes. One thing that stands clearly in my mind is the need to temper the requirements of the group performing the play against the differing needs of the players as individuals- bringing their own strengths and weaknesses to bear upon the text. To be able to share a vocabulary, you first have to identify the points shared in common, and then form from that beginning a shared experience which can serve to find a pace that all can share. There is a delicate balance between intellect and instinct. It becomes unbalanced if one or the other is not entertained. Generally, I found that if 15- 20 minutes spent in the head then 60- 90 minutes needed to be up on our feet. I would, perhaps, have approached the work differently if I had more experienced actors. However, the work proceeded and I am comfortable that we explored as much as we possibly could during the rehearsal period. By the time our dress rehearsals began to come together, we were able to seek and appreciate the advice of some of my mentors that attended the Tech week. Gregor Cameron 198272670 p 19 http://www.aboutfaceproductions.com.au/Site/Home.html accessed 25/10/11 20 p p 20 http://www.imdb.com/name/nm0298754/ accessed 25/10/11 p abou acep oduc o s co au S e o e accessed 5 0 20 http://www.imdb.com/name/nm0298754/ accessed 25/10/11 http://www.aboutfaceproductions.com.au/Site/Home.htm 20 http://www.imdb.com/name/nm0298754/ accessed 25/10 g 11 21 http://www.theconch.co.nz/index.html accessed 25/10/11 21 http://www.theconch.co.nz/index.html accessed 25/10/11 11 Mentors 108 I was very lucky with the mentors who gave of their time and expertise both before and over the term of the production. John Downie, my Supervisor, of course was shepherding me through this exploration and offered sage advice and useful provocation. James Davenport as technical officer of the Theatre Programme opened some doors for me and, in turn, presented some challenges. Willie Franco who assists Jim at the theatre programme generously gave of his time and advice to Elliot with the video work. I also want to acknowledge the support I have received from the other theatre academic staff. While some came to the performance and offered me feedback, others 108 MA Thesis: The Role of Misrule in the practice of Performance have been there to talk over issues as they have arisen. I have always felt that they have my back. However, some people outside of the institution have also made significant contribution to my work. During a visit to Melbourne in 2010, AboutFace Productions creatives Tim Denton and Annie Forbes19 gave me some time to talk Puppets. They helped me think about the form and function of my puppets and what differing puppets bring to a situation. They also offered direct advice on construction that proved instrumental in being able to make the Punch and Judy community of puppets. Tim and Annie also spent time with me after their production, Boxes, which ran at Capital E in Wellington. During this meeting, I was able to report back to them and get some comments on my completed performances of Saint Punch. Alex Funke20, who is a cinematographer with the miniatures and visual effects company out at Miramar, that serves Peter Jacksonʼs movies, offered some great support when I was asking about using a compositing effect on the stage. He had been doing some work at Victoria University with the honours video production course. I also worked with him on a 16mm filmmaking and cinematography course which he and Paul Wolfram facilitated. As well as offering advice, he provided the green screen paint that surrounded the actors as they travelled from castle to dungeon, police interrogation room to kitchen. I was very touched when, after attending the 109 performance, he shook my hand and simply said, “It did exactly what you said it would.” He offered this with a big smile. Mentors Perhaps the most potent mentor interaction occurred with Tom McCrory21. Perhaps the most potent mentor interaction occurred with Tom McCrory . Perhaps that is the reason that I want to report it in such detail, as it underlined, defined and turned my process upside down. Certainly he very clearly set the signposts for connecting my practice with theory better than anyone else had. Therefore I include a fairly detailed report of our meeting. Friday the thirteenth was a resonant day to begin a conversation with the dark side as a focus. Tom McCrory is a performer and tutor in movement at Toi Whakaari/ New Zealand Drama School. He attended Jacques Lecoq's school in Paris for two years working on the Bouffon, the fool as performer/provocateur. With his wife he leads The Conch a New Zealand based Pacific Theatre Company. Very generously he gave his time to speak with me at Toi Whakaari, Friday the thirteenth of August 2010. New Zealand being such a small place our conversation began by recognition of the people and places we shared in common. Jonathan Hendry, head of Acting, had told him that I was an ex-student of Toi Whakaari and so we already shared people in common. He also knew John Downie, now a lecturer at Victoria University of Wellington's Theatre and Film 110 MA Thesis: The Role of Misrule in the practice of Performance programmes, from back in 1989 at the University of Bristol- a year before I first knew and worked with him in the final year of my undergraduate degree. Then we were talking about his journey to Lecoq's school when he mentioned his wife whom he met in Europe. Something in Nina's name rang bells with me and I asked if she had worked with Rob Bennett when she was at Wellington Teachersʼ College- again a connection as we had been at college at the same time. While none of this knowledge was immediately obviously meaningful I hope it will become clear how relevant this very human process becomes within the frame of our conversation. Being human underpinned all that was said that morning. As Tom was to say it all depends on communication- that which is common, the commune, communicated. Tom spoke of his early fascination in fine art, particularly his introduction to sculpture through his grandfather. Mentors His university work began in this area but then developed into the form of theatre design which in turn lead to his awareness that the finest expression of what he wanted was to be found within his own bodies ability to perform and be- a fascination with the movement of the body. A timely workshop brought Lecoq's work to his attention and thus he found himself in Paris- an enervating competitive environment where he faced enormous success and dismal failure in the process that Lecoq has developed. He spoke of the student body being decimated after the first term and then again at the end of the first year. This Gregor Cameron 198272670 111 competition spurred those like Tom to perform intensively to survive and honed a sharp sense of finding the truth, the new, and the essence. So from this beginning we began to look at what it is to be a performer and a teacher. Tom's experience at Jacques Lecoq's school has given him a fine sense of connection with craft and tradition, how the apprentice looks to the master through to the position of journeyman and finally to a masterate of his own. We discussed this particularly with reference to the journeys of Monika Pagneux and Phillippe Gaulier, who after studying and teaching with Lecoq formed their own schools. In a sense carrying the essence into further work and discovery. I was interested to hear him talk of craft rather than art; as for me this is a key discretion in the work of a professional. Craft is the set of tools, which support the professional through the rigors of a career. Art tends to be the enthusiastʼs collection of experiments- both the success and the failures that inform the path of discovery. However taking the step beyond the norm means that the process of craft invites a level of risk that may be unsustainable. Tom commented that the traditions that are passed to the apprentice form the basis of the tools used within the craft- a way of managing such risk. From there the conversation turned to the specifics of how Tom's work related to my own. Spotting my Punch and Judy graphic novel Tom suggested seeing the cover image as a mirror. Gregor as Punch is quite a 112 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance provocation! How does the illustration relate to the inner image of Gregor? What is frightening about this revelation? What is the impulse to engage in this genre and them? And that is how much of the following discussion went- bringing up more questions than answers. However if the answers were there then the creative journey probably does not need to be taken. The impulse to explore the 'dark clown' comes from within. According to Tom, Lecoq said that we all wear three masks: What we think we are, what we share in common with others and what we really are. Lecoq's work was all involved with paring these masks down to the essential story, moving from the mask constructed by our egos in combination with our society to the nature we should be living in conjunction with and finally through to the essential sacred image, an expression of the spirit. Practice, for Lecoq, was an invitation to examine the impulse, come to terms with it and discern the difference between the ego driven destructive face of the dark impulse and the sacred call, which seeks to deliver light by defining the darkness. In short, using the energy of the fool to unpick the destruction, which often goes hand-in-hand with the constructed ego. Tom drew the analogy of the binary nature of the constructed ego- either things are good or they're bad, black or white. He suggested this forms much of the basis of much Christian practice, not all, but enough to have a profound affect upon the way most of us operate in society. The difficulty this produces as part of the way our egos are constructed is that it creates the very blocks that prevent us from experiencing simply being human. Gregor Cameron 198272670 113 This, of course, risks becoming a happy chappy new age trip so it is important to see this work within the theatrical context. Bouffon is a methodology of stripping the baggage away from the performer and stopping the three masks from interfering with the possibility of the essential story. Tom suggests this central story; the essential sacred story is one of transformation, involving the path of destruction leading to rebirth. The human, as and with nature. Bouffon, with the invitation to play, identifies those separations of ego and essence. Again Tom poses a question: Once I, as the fool/performer, touch upon the essential spirit how do I share that with others? How do I reveal the essence/spirit? Bouffon also offers 'otherness' as a connection to the divine. In effect the fool is a tool for offering transgression as a path to the sacred. It describes the way otherness as dealt with by societies other than European as perhaps offering examples. For instance the apaches dressed as women who are given a special place within the tribe or the fa'afine in Samoan society, the fools invited into the sacred places who in their disrespect move laughter into a re-appreciation of their sacred-ness. In essence it requires an attunement to the profane that offers a moment to reflect upon and brings the spectator back to the sacred. Therefore the process of destruction is always about bringing about the rebirth, creative reconstruction, responsive not reactive. This process draws a number of similarities to Bakhtin's writing 114 MA Thesis: The Role of Misrule in the practice of Performance about the carnival. It is always the intention of the carnival to use transgression as a mode of reflection upon the status quo. The fool or the clown therefore both innocent and rogue is about providing a mirror held up to the audience. “Devils may step where angels fear to tread,” says Tom. MA Thesis: The Role of Misrule in the practice of Performance about the carnival. It is always the intention of the carnival to use So it is with this new image of the sacred and the profane that I stepped more confidently on my journey. Tomʼs words had changed my perception of what I was doing but in some way had also changed me, myself. Gregor Cameron 198272670 Gregor Cameron 198272670 It was this passion and excitement that drove me through the more difficult times and laid down a pattern of what it was I wanted my audience to experience. Figure 8. The television, showing the climax of the Bluebeard story, in a kitchen. Figure 8. The television, showing the climax of the Bluebeard story, in a kitchen. Gregor Cameron 198272670 115 Gregor Cameron 198272670 Design While I had a clear sense of the three environments, that the script suggested, in the theatre, I was hoping that a designer would bring something else to the performance. Unfortunately, despite a few invitations to collaborate, I found myself doing more and more of the planning. As time went on a great team of stage management and technical crew did emerge but I felt very much stretched in coming up with the staging that we used. The play required three clear environments. Originally, I had intended them to be a punch booth, a masque space and a naturalistic stage space between them. I had some sense of placing the actors on to three levels. I had planned for the highest level to be a place of large almost operatic masques and constructs- huge to contrast with the puppets that were closest to the audience. This intention was just not practical in Old Drama House. Given this space and Downieʼs challenge I needed to rethink the design. I am quite interested in how these three stories are somewhat a shadow of each other. A process of mediation has clearly drawn one through another. This mediation is fascinating and it seemed inevitable that I capitalise on it. Therefore, my thoughts turned to making the naturalist/realist space into a reflection of both mediation and significance by using a camera. I decided that I wanted the audience to read the screen as a source of historical reality or at least some sort of recognisable claim to veracity. I also wanted to 116 MA Thesis: The Role of Misrule in the practice of Performance explore how I could bring in the outside and make the supposed space that much bigger. Thus formed the plan to use the downstairs room as a green screen studio and use the sunroom as a space that could hold a miniature set and control for the four video cameras that would allow two angles on the actors, one high and one more directly at eye level. I took advice from video developer Cameron Mackechnie at this point as to how such a set up could work but this proved too expensive. Thus working on a very small budget I relied on advice from Willie Franco to use the facilities that the theatre programme already had. Design By using a video mixer with the four cameras Elliot Lim, who had followed this project through the summer, was able to switch from one angle to the other, live editing as he went. The second major design was the Punch booth, which I decided needed to be more flexible than just the traditional one had been. As it happened, the Lighting box was about the right size and shape to be the Puppet Booth. By framing this in striped material and backing the booth with gauze I wanted to suggest tradition but also provide room for my actors to perform and ʻseeʼ their performance as well. This would also expose the illusion somewhat to the audience but not intrusively. I had been working on Punchʼs community of Puppets over the summer and they were just the right size to feature in this space. In designing the puppets I decided to not go traditional completely. In fact, I wanted the Punch puppet to reflect a more modern urban look. Thus, I dressed him in a hoodie, this being a form of dress that is rich with signification in modern stories. At times then, it can represent a morally Gregor Cameron 198272670 22 http://www.guardian.co.uk/uk/2011/aug/09/power-of-the-hoodie accessed 29/10/11 23 http://www.melissaanddoug.com/hand-puppets-animal-puppets accessed 29/10/11 Gregor Cameron 198272670 g Gregor Cameron 198272670 ttp e ssaa ddoug co a d pup Gregor Cameron 198272670 117 ambivalent figure22. Kevin Braddock in the Guardian sums up the Hoodieʼs role in the recent riots in Britain. Moreover, having made this choice I followed it through with the other Punch community puppets using a range of Baby sized clothes. When Des came on-board, I discovered he had a passion for modelling and this strength definitely deepened our collaboration together and helped putting together the show. I had purchased from the $2 shop some eight-inch toy soldiers and Barbie clones. Des was able to work on these and adapt them to our needs. He painted and subtly changed how they looked before Figure 9. The Stage: TV Screen, Curtain, Punch Booth and Grille. Figure 9. The Stage: TV Screen, Curtain, Punch Booth and Grille. 118 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance adding handles so that he and Rosie were able to play their puppet scene in the Booth. Des also made the circular mini-set that Elliot as able to combine with the green screen work. The Barbie clones became headless later on, as we needed to produce a cabinet for the Bluebeard story. With the Bluebeard story, I was having some trouble choosing an image for them. As I worked with the actors, a form became evident, so I began to search the net for suitable puppets. Very soon, I came upon a company called Melissa and Doug.23 These hand puppets call up a reference to The Muppets, a Disney property that would have a resonance for both actors and audience. Having purchased these off Amazon.co.uk I began to see a real growth in how the actors began to see their story on the stage. Des offered to make the cabinet in which Judith would find the heads of Bluebeards former wives. Finding the heads up in the booth was easier to design, and allowed Gilles and Joanʼs scene to be on the video. The Video Studio consisted of a raised floor and four flats, painted with green screen paint. Elliot placed a camera in the top corner of the room for a wide shot and another closer to the stage for close-ups. He then mirrored this with the circular miniature stage, allowing us to place photos on the walls, which would change as the action moved from one venue to another. We used photos and no furniture as this was about as far as time and resources would allow but it would have been interesting to go much further if possible. Up on the stage I built a frame for the large LCD screen that we were using and 119 made it look more like a large console Television from the 1960ʼs. Elliot would then pull the colour out of the image so that we were showing the audience a Black and White image. This was entirely deliberate as it was not my intention that they should not be aware of the conceit of the green screen, but rather see the image as a representation of how the docudrama image was produced back then. MA Thesis: The Role of Misrule in the practice of Performance I wanted the audience to read the image as representing ʻtruthʼ without forgetting that the image was entirely false. This playing with representation was entirely deliberate as one of the main objectives of the show was to encourage the audience to question the mediated images presented through the Media. Finally, the stage should connect these environments together. I wanted the stage, now the domain of the masks, to bring together the two others. All the actors on the stage would wear a mask although only the old man who bookends the play would be wearing a Commedia DellʼArte mask for real. By presenting in mask, the actors declare a place of unreality in which the fantastic and the grotesque might exist. I wanted also to signal that presence of the circus or the carnival by placing centre stage a large red curtain, which would be the major entry and exit for the action. Downstage, but off centre, a trap lead down to the video studio. I wanted to raise this so that a connection would be established that could prove that our video was still live performance. This was a difficult sell to Jim Davenport for 120 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance very good Health and Safety reasons. However, it was always my intention to somehow block the open space with a grille that would allow a view down but stop anybody from falling through. Eventually Jim agreed to this. He provided the steel grille that we used for this purpose. While this setting was sparse in some regards, it all signalled my intention for the final performances. As the play itself neared, I began to collect more Technical crewmembers. Shannon Friday, Courtney Leggett, Kate Norquay, Bop Murdoch came on-board as stage management crew lead by Rose Kynoch (MK). They assisted greatly in realising the design. Jim provided two of his scenography students, Stephen Keen and Andy Friedland to help complete the Video Studio. Andy went on to setting the lights and running the lighting board. Patricia Hetherington and Elliot Lim followed the project from the summer workshops and now ran the sound and video design. Finally, for my last illusion I had help from Dawa Devereaux, a friend who had collaborated with me when we were both students in 2006, who produced the flame circles I needed to burn Gilles and Joan in the showʼs final scene. These were placed in a pile of wood that opened up for the actors to enter. The illusion meant that as the actors stepped onto the sponge rubber pile of wood, a tube of material with a sort of flame patterning was raised to the mid-riff. Combine this with light and sound and both Gilles and Joan are burnt 121 Gregor Cameron 198272670 Gregor Cameron 198272670 121 Costumes were intended to be consistent from environment to environment, and where this was not possible for cost reasons they were to be suggestive of a commonality. Thus Gilles wears a kind of costume that suggests armour without actually being armour, Punch wears clothes similar to his puppet but not identical. Most were found costumes or cost very little to put together. It is important to note that this performance was a show and tell and far from what might have been a full production. The budget came out of my pocket, I am grateful for all the infrastructure support I received from the university, and in particular the Theatre programme. Figure 10. Looking down the Grille Figure 10. Looking down the Grille 122 Show and Tell The final week of preproduction was frenetic. While we were able to rehearse in the space in the evenings and on the weekends, it remained a teaching space so any setting of tech and stage needed to await the end of trimester. e a ee o p ep oduc o as e e c e e e e ab e o e ea se in the space in the evenings and on the weekends, it remained a teaching space so any setting of tech and stage needed to await the end of trimester. Added to this pressure were the major productions being done in 77 Fairlie Terrace, the main Theatre, which were using most of the lights and such and had to be cleared before we could access some of the resources of the Theatre Programme. Despite this, we began to construct our playing space. Andy and Stephen painted and put together the studio downstairs. Elliot began to put together the video feed. Patricia began putting together her set of sounds. On Saturday the 4th of June 2011, we began work on the upstairs five days before the first performance. Shannon, Bop, Courtney, and Kate helped with bringing my stage ideas to fruition with contributions that supported those ideas. Rose MK began taking over from me in organising cast and crew. Far faster than I imagined the stage and show began to look more and more as though it would far exceed my expectations. We were still rehearsing and it was during this process that a few select people were invited in to have a look at the work thus far. John Downie, Chris Ryan, Kerry Glamsch and Ralph Johnson all gave feedback, both to myself and to the cast that was welcome and very pertinent. Of course, some last minute glitches arose in not having enough time to test and put together with the action the technical aspects of the show. It all looked quite ambitious. Gregor Cameron 198272670 123 After a dress and a tech underlined how quick it had been to suddenly make the technical aspects of the show gel with the cast, we were off. Saint Punch ran for four nights, June 8th to 11th. The audiences were a mix of staff from Victoria University, invited friends and members of the local theatre and film community. Thoughts During the run Rose MK took on the main concern of running the show. This left me time to film the performance and to gauge what the audience was experiencing. The DVD attached to this thesis includes a recording of the final night that I believe was where everything came together about as nicely as I could ask for. In sitting in with the audience, I was able to listen as they laughed at the comedy and caught themselves laughing at events that perhaps were not quite so funny. It was a queer feeling. I spent a bit of my time trying to gauge the reactions of my fellow audience members. I realised that most of them were engaged, especially because of the broken narratives and changes of scene made by the characters. They needed to be engaged or risk missing something. This is not to say that every moment was as interesting as every other, but by and large their attention was on the performance. However it would be true to say that direct interpretation like this is a little inaccurate. 124 MA Thesis: The Role of Misrule in the practice of Performance I think that the performance stood on its own merits. I do acknowledge however that it was produced on a shoestring budget and as such suffers in its lack of complete consistency of design. Later I got the opportunity to speak to members of the audience about their experience. By and large the comments I received were positive with a number of them saying that they left the theatre quite engaged and that the play travelled with them for some time. I believe this to be as positive a response as I could want. However there was one exception who, I am told, travelled all the way back to Raumati from Wellington concerned that perhaps I should seek help and wasnʼt there a chance that I was suicidal and shouldnʼt they do something? Luckily, friends of mine, who know me all too well, and were unconcerned, had brought her along. How do I feel? Actually I am very satisfied with the show, warts and all. My intention had been to investigate the carnivalesque, to take the audience on a journey that they perhaps did not expect. However this was leavened with the knowledge that I did not want to leave any of them behind. Thoughts Thus the trick you play involves planting breadcrumbs, which will be followed through a landscape rich if they will only raise their heads. If they do then all sorts of thought could be excited, but if they donʼt there is still enough within the breadcrumbs to satiate their imaginationʼs appetite. I believe that the amount I learned from what went right has been far exceeded by the knowledge gained from what went wrong. I clearly expanded the range of my confidence and belief in my own ability – and also managed to place in front of an audience an intriguing piece of theatre. Gregor Cameron 198272670 125 125 Part Three: Critical Analysis of Misrule and its part in On rare and unforeseeable occasions, the Jester would turn the city upside-down by some prank which might be no more than an elaborate practical joke, or which might be a calculated assault on some currently cherished belief or way of life. All things considered, the name "Jester" was a highly appropriate one. There had once been men with very similar duties, operating with the same license, in the days when there were courts and kings. Letʼs start with Stanislavsky. As the role of misrule in practice, as well as performance, is what this paper is about it seems sensible to look at one of the masters of twentieth century acting/theatre systems. He offers a clear definition of how a director in the theatrical space is required to act. He suggests, as Bella Merlin (2007:180) tells us, that there are three key points that inform the directorʼs role. Firstly they need to know how to deal with the writer, something he did in his relationship with Chekov, for this relationship defines the creative moment that will move the work, the written text, outside into the light. Secondly, they must know how to work with the actors. Choosing and then provoking the actors into asking the right questions becomes the objective of the director, how to provide enough context to influence the actors own natural curiosities. Finally there is the collaboration they must negotiate with everyone else involved. This ranges from stage management to ticket 126 MA Thesis: The Role of Misrule in the practice of Performance sellers, technicians to designers. In this sense he must be able to effectively communicate the vision, and listen to the reactions to it, to bring together whatever new life is evident in the theatrical experience (Merlin, 2007:181). These relationships are a key component to producing a moment on the stage that is going to enrich the audience experience within the theatrical space. Even in his use of a fourth wall, Merlin tells us, Stanislavsky was not These relationships are a key component to producing a moment on the stage that is going to enrich the audience experience within the theatrical space. These relationships are a key component to producing a moment on the stage that is going to enrich the audience experience within the theatrical space. Part Three: Critical Analysis of Misrule and its part in Even in his use of a fourth wall, Merlin tells us, Stanislavsky was not attempting to close the audience out but instead school his actors to consider the truth on stage rather than, as the tradition had been, to pander to the audienceʼs demand for ʻentertainmentʼ. If what is placed onstage is truthful to the actor, irrespective of the system used, then the audience would be drawn into the moment of the theatre (Merlin, 2007:249-50) You should love your art because it makes it possible to talk to the spectator about the things he cares most for in life, and to make him a more useful member of society by embodying certain definite ideas on the stage in artistically creative characters. If the spectator obtains an answer to what is engaging his thoughts, he will grow fond of the theatre and will look on it as a necessity. But if all we do in the theatre is to entertain him, he will come and have a look at us and then go away. (Stanislavsky quoted in Merlin, 2007:250) Gregor Cameron 198272670 127 Richard Schechner cites to entertain and educate as a core constituent of theatre as far back as the Roman poet Horaceʼs Ars poetica (65-68 BCE). He continues to make it clear that this has been a call continuing down through the renaissance to Brecht in the twentieth century (Schechner, 2006:45). This key function, it seems to me, is allied to the carnivalesque. If the audience enters the theatrical space in the clear expectation that something is about to happen, something that they will understand and enjoy, but that will possibly open their eyes to a new worldview, then the ʻentertain and educateʼ function continues to be a potent element in the magic of the theatrical ritual. Schechner also offers a system of seven key functions of performance: Schechner also offers a system of seven key functions of performance: 1. To entertain 1. To entertain 2. To make something that is beautiful 3. To mark or change identity 4. To make or foster community 5. To heal 6. To teach, persuade, or convince 7. To deal with the sacred and / or the demonic (Schechner, 2006:46) System is a key word here, as in order to work towards an idea of creative misrule it is important to understand two key points under which misrule and carnival can occur. Anne Bogart, and then you act (2007:46) Art is an act of the spirit. It asks you to be a conduit for something larger than yourself. Part Three: Critical Analysis of Misrule and its part in There must be a frame of the status quo: the ʻreal worldʼ, a system. In the frame of the carnivalesque, it is everything outside the carnival space and, in misrulic terms, is the system within the carnival from which misrule manifests. As we see with Stanislavsky, the system is the rich bed for encouraging the manifestation of the Carnivalesque. Both of these forms require order, but the misrule of carnival is not chaos. Art is an act of the spirit. It asks you to be a conduit for something larger than yourself. than yourself. Anne Bogart, and then you act (2007:46) 128 Bakhtin and the Carnivalesque. In order to define the carnivalesque it is useful to refer to the writings of the Russian Literary Critic, Mikhail Bakhtin. Lewis Hyde (2008) recognises Bakhtin as correctly interpreting that the Renaissance, as reason and religion grappled with the minds and souls of the people, drove the carnivalesque to seek refuge in the arts. As Protestantism and its insistence on restraint took over the public sphere, the medieval carnival moved from a carnivoreʼs holiday physically enjoyed by the people before Lent into literature and art. (2008:194). According to Sue Vice (1997) Bakhtin considered the existence of the carnivalesque in his studies of the French renaissance writer, Rabelais (c.1494 – 9 April 1553). He categorizes the carnival as having three aspects: • Ritual Spectacles including comic shows and pageants, • Comic verbal compositions which feature parodies both in high and low language and, • Various genres of billingsgate (curses, oaths and popular blazons (1997:151). Bakhtin goes on to detail this list in his consideration of Russian writer Dostoevsky (1821- 1881). Carnival is further refined as inhabiting the novel: • Without footlights as there is no division between audience and performers, free and familiar, • Combinations and inversions that arise from apparent binaries (sacred/profane, high/low, wisdom/stupidity, male/female), Gregor Cameron 198272670 Gregor Cameron 198272670 Gregor Cameron 198272670 Gregor Cameron 198272670 129 • The body as governed by both high and low natures (grotesque, eliminations, parody), • Death and renewal, • Laughter pointed both at and with the participants; the laughing aspect encompassing the sense of community- the town or place which draws the players and spectators together (Vice, 1997:152). Carnival time (and space) is characterized by ʻ[m]oments of death and revival, of change and renewal [which] always lead to a festive perception of the worldʼ (Bakhtin quoted in Vice, 1997:153). It is important to note that defining things in terms of binaries is problematic and limiting, that in a dialogic world there are many other voices and positions that feed into situations. However in mythological terms they perform a useful function of definition determining limitations about a position. Thus we can use antithetical terms to perform certain positions in a given situation. Vice makes it clear that Bakhtin had some reservations about whether theatre could be dialogic, a key idea that arose from his work on the carnival. 24 Bakhtin wrote most of his works in the twenties and thirties in Soviet Russia and did not become known in the west until the sixties. Bakhtin and the Carnivalesque. However he did consider the theatre a place that could be carnivalesque in his writings, which include identifying Shakespeare as using the Carnival trope (Vice, 1997:198). It is left to others to debate whether or not Bakhtinʼs consideration of the theatre was complete or not. Ronald Knowles (1998) notes that Bakhtin believed in the ʻunfinalizabilityʼ of things- an open-ended dialogue (1998:3). Vice makes it clear that Bakhtin had some reservations about whether theatre could be dialogic, a key idea that arose from his work on the carnival. However he did consider the theatre a place that could be carnivalesque in his writings, which include identifying Shakespeare as using the Carnival trope (Vice, 1997:198). It is left to others to debate whether or not Bakhtinʼs consideration of the theatre was complete or not. Ronald Knowles (1998) notes that Bakhtin believed in the ʻunfinalizabilityʼ of things- an open-ended dialogue (1998:3). 130 Gregor Cameron 198272670 131 until the sixties. MA Thesis: The Role of Misrule in the practice of Performance Meaning, in language, and in comparison with the work of Saussure, was to be found in the space between, the dialogic. The dialogic is one of the keys to polyphony, the ʻmany-voicednessʼ which Bakhtin attributes to the novel, and unlocks the form of the heteroglossic, the 'many-differentness' (Vice, 1997:112). However, it seems reasonable to engage with the theatre as a space in which many points of view can be voiced, experienced and reacted to, at a point of live juncture- a ʻliveʼ moment of give and take between writer, performer and spectator. Robert Stam (1989) points out that Bakhtinʼs writing on the active participation and response of the reader is not at odds with the reception studies that arises out of current cultural approaches of Film and Media scholarship (1989:21). Stam also points to Bakhtin as a “liminal” thinker- “on the borders, at the junctures and points of intersection of academic disciplines as traditionally defined and institutionally regulated” (1989:17). Hence Bakhtin appears to be constantly in a state of flux and therefore it is appropriate that his thinking could and should be applied to fields that have developed considerably since he was writing in the first half of the twentieth century24. Knowles comments that Bakhtin does form a contrast between the conventions of the ʻwell made playʼ, where aesthetics, particularly those of the Greek tragedy, were, in the nineteenth century, elevated to ʻa monolith of official [high?] culture and the ʻwhole of Naturalist dramaʼ of Ibsen and his contemporaries becoming ʻnovelizedʼ (1998:9). In this instance Bakhtin does signal that the emerging theatrical traditions offer a far greater sense of many- voiced-ness than they have previously. Knowles, in his book, uses Falstaffʼs comment from Shakespeareʼs Henry IV Part 2 to illustrate the possibility: “I Meaning, in language, and in comparison with the work of Saussure, was to be found in the space between, the dialogic. The dialogic is one of the keys to polyphony, the ʻmany-voicednessʼ which Bakhtin attributes to the novel, and unlocks the form of the heteroglossic, the 'many-differentness' (Vice, unlocks the form of the heteroglossic, the 'many-differentness' (Vice, 1997:112). However, it seems reasonable to engage with the theatre as a space in which many points of view can be voiced, experienced and reacted to, at a point of live juncture- a ʻliveʼ moment of give and take between writer, performer and spectator. MA Thesis: The Role of Misrule in the practice of Performance Robert Stam (1989) points out that Bakhtinʼs writing on the active participation and response of the reader is not at odds with the reception studies that arises out of current cultural approaches of Film and Media scholarship (1989:21). Stam also points to Bakhtin as a “liminal” thinker- “on the borders, at the junctures and points of intersection of academic disciplines as traditionally defined and institutionally regulated” (1989:17). Hence Bakhtin appears to be constantly in a state of flux and therefore it is appropriate that his thinking could and should be applied to fields that have developed considerably since he was writing in the first half of the twentieth century24. Knowles comments that Bakhtin does form a contrast between the conventions of the ʻwell made playʼ, where aesthetics, particularly those of the Greek tragedy, were, in the nineteenth century, elevated to ʻa monolith of official [high?] culture and the ʻwhole of Naturalist dramaʼ of Ibsen and his contemporaries becoming ʻnovelizedʼ (1998:9). In this instance Bakhtin does signal that the emerging theatrical traditions offer a far greater sense of many- voiced-ness than they have previously. Knowles, in his book, uses Falstaffʼs comment from Shakespeareʼs Henry IV Part 2 to illustrate the possibility: “I 1997:112). However, it seems reasonable to engage with the theatre as a space in which many points of view can be voiced, experienced and reacted to, at a point of live juncture- a ʻliveʼ moment of give and take between writer, performer and spectator. Robert Stam (1989) points out that Bakhtinʼs writing on the active participation and response of the reader is not at odds with the reception studies that arises out of current cultural approaches of Film and Media scholarship (1989:21). Stam also points to Bakhtin as a “liminal” thinker- “on the borders, at the junctures and points of intersection of academic disciplines as traditionally defined and institutionally regulated” (1989:17). 131 have a whole school of tongues in this belly of mine” (Knowles, 1998:9) have a whole school of tongues in this belly of mine” (Knowles, 1998:9) have a whole school of tongues in this belly of mine (Knowles, 1998:9) suggesting that this form of dialogic has inhabited theatre even before the rise of the Naturalists. MA Thesis: The Role of Misrule in the practice of Performance Hence the spectacle of nineteenth century theatre gives way to a representational world which is not bordered by the confines of the theatre but instead suggests a wider world outside, a moment in the stream of these characters lives, illuminated with the ʻnovelʼ of the theatre. Knowles notes Bakhtinʼs recognition of the Carnival in the work of Shakespeare but his hesitation to allow theatre polyphony (1998:8). However Knowles also notes that Bakhtin also seems to ignore the work of Chekov that clearly allows for polyphony within his drama and stories (1998:10) suggesting that this form of dialogic has inhabited theatre even before the rise of the Naturalists. Hence the spectacle of nineteenth century theatre gives way to a representational world which is not bordered by the confines of the theatre but instead suggests a wider world outside, a moment in the stream of these characters lives, illuminated with the ʻnovelʼ of the theatre. Knowles notes Bakhtinʼs recognition of the Carnival in the work of Shakespeare but his hesitation to allow theatre polyphony (1998:8). However Knowles also notes that Bakhtin also seems to ignore the work of Chekov that clearly allows for polyphony within his drama and stories (1998:10) Mark Fortier (2002) cites Robert Weimann who studied the way in which Shakespeareʼs plays can be seen to operate in more than one voice. They appear to be able to represent both the official position (ruling class) and that of the people. This can happen as simply as through position on the stage- upstage for authority and downstage for an intimate and informal position to address the groundlings and lower class (2002:161). Shakespeare also becomes playful in his placing together clowns and kings, cross-dressing heroes and heroines, and lords that become friars. Thus play becomes identified as a crucial element on the carnivalesque stage. Play and laughter are key elements in the way that an audience is invited to participate, an instant feedback system if you will, in the spectacle of the theatrical space. Michael Bristol (1985) maps how misrule, burlesque, grotesquerie, and other Michael Bristol (1985) maps how misrule, burlesque, grotesquerie, and other 132 MA Thesis: The Role of Misrule in the practice of Performance inversions travel from countryside to marketplace and from there to the plays of Jonson and Shakespeare. MA Thesis: The Role of Misrule in the practice of Performance He notes that peopleʼs interaction with performance and theatre was very common across a wide if not complete array of social groups and communities. As the urban community grew so did the tradition of the development of the organized professional theatre, especially that of London (1983:638). The theatre increasingly becomes a place, a central spot where communities could meet in the expectation of seeing something festive in the spirit of the medieval markets and carnival. It becomes a defined border between the status quo and a space in which something ʻotherʼ may happen. This sense of the difference between inside and outside of the carnivalesque space fits quite well with some modern conceptions of the theatre. Bakhtin does not engage much with the theatre, as his concern is the novel. In the witness of play and laughter, in what can be presented in the theatre, society is offered with an opportunity, an invitation, but, like the proverbial horse, the provision of water does not imply the drink. The social contract that is implicit between spectator and performer allows access across these divides. At entrance and exit, performance and response to the theatrical moment, this community gathering serves to repeal one of Bakhtinʼs criticisms of the theatre as simply a spectacle ridden de-politicalized space. Marvin Carlson references Julia Kristeva in discussing who the author is in carnivalesque terms. She places the dialogic firmly in the space between the ʻauthorʼ and the spectators, with the performers acting as a conduit for the variety of voices. In a sense they are “in turn simultaneously both subject and addressee of the discourse. Gregor Cameron 198272670 133 Gregor Cameron 198272670 With this overt political agenda, and without the component of laughter, it is difficult to see how much fun a Boal production would be. The argument could be made that this very seriousness is counter- intuitive to any lasting change being made manifest. In losing the entertainment and educational aspects of the carnival Boal may well limit just how much political enlivenment he can achieve. His work on Legislative theatre however is interesting because of its direct action element- perhaps the intention to turn from allowing the audience to be affected into one which has a direct effect means that the carnival is still present but in a different form25. MA Thesis: The Role of Misrule in the practice of Performance Woolgar (2009) consider carnival as part of the celebratory theatre. They observe its liminal state: “ a borderland between two states of being” (2009:99). They discuss Augusto Boalʼs criticism of Bakhtinʼs theory. However, while Boal sees Carnival as an agency of the state oppression, his own Forum Theatre is almost a manifesto to the reflexive revolutionary aspect of the Carnival. Pickering and Woolgar invite us to consider the inverse of too much laughter, that “a population fed a constant diet of sport, celebrity or royalty is not equally impotent when it comes to changing the attitudes or policies of the governing elite” (2009:99). With this overt political agenda, and without the component of laughter, it is difficult to see how much fun a Boal production would be. The argument could be made that this very seriousness is counter- intuitive to any lasting change being made manifest. In losing the entertainment and educational aspects of the carnival Boal may well limit just how much political enlivenment he can achieve. His work on Legislative theatre however is interesting because of its direct action element- perhaps the intention to turn from allowing the audience to be affected into one which has a direct effect means that the carnival is still present but in a different form25. David Charles (2004), in commenting on Playback Theatre, offers Boalʼs Invisible theatre, along with Bread and Puppet theatre, the trestle table stages of Rome and Medieval times as example of a transient theatre full of mutability and multiplicities (2004:4). Each offered sign or mimed object displays many possible uses and readings. o pso , S e g ssay eat e o C a ge ( bout eg s at e eat e) t e o u oject <http://theforumproject.org/whatisto/what-is-theatre-of-the-oppressed/essay-theatreofchange4/>. 25 Thompson, S. Leigh. "Essay: Theatre of Change (About Legislative Theatre) \| the Forum Project". Gregor Cameron 198272670 13 25 Thompson, S. Leigh. "Essay: Theatre of Change (About Legislative Theatre) \| the Forum Projec <http://theforumproject.org/whatisto/what-is-theatre-of-the-oppressed/essay-theatreofchange4/>. Gregor Cameron 198272670 133 The carnival is also the bridge between the two split occurrences as well as the place where each of the terms is acknowledged: the author (actor + spectator) (Carlson, 2004:69). It must be noted, however, that some aspects of the carnivalesque are also apparent in ludic sports arenas. Whether this type of spectacle is able to rise above the ʻpressure releaseʼ aspect of the carnival is open to debate, take for example the annual sevens tournament played out in the Wellington Stadium (New Zealand). Here is played out a performance not only of the athletes but also in terms of the spectators and their colourful costumes which definitely have a carnival atmosphere but it would be difficult to suggest that any real critique of anything outside the stadium was being played out, a grand example of the social spectacle. David Danow (2004) places Bakhtinʼs reading of literature in an apparent binary of “two aspects, seriousness and laughter, [that] coexist and reflect each other, and are indeed whole aspects, not separate serious and comic images as in the usual modern drama” (2004:34). Thus suggested, there is a composite rather than a binary form, which Danow sees in magic realism of the Latin American literary form. In a sense laughter provides a vehicle into some aspects of the world that otherwise would be closed to scrutiny. The Carnivalesque invites both laughter and seriousness into its space in order to offer more than just an entertainment to the participants. After all, the carnival is both a place of letting off steam about, of being reflective, and possibly critical, of the status quo that exists outside. Kenneth Pickering and Mark 134 MA Thesis: The Role of Misrule in the practice of Performance Woolgar (2009) consider carnival as part of the celebratory theatre. They observe its liminal state: “ a borderland between two states of being” (2009:99). They discuss Augusto Boalʼs criticism of Bakhtinʼs theory. However, while Boal sees Carnival as an agency of the state oppression, his own Forum Theatre is almost a manifesto to the reflexive revolutionary aspect of the Carnival. Pickering and Woolgar invite us to consider the inverse of too much laughter, that “a population fed a constant diet of sport, celebrity or royalty is not equally impotent when it comes to changing the attitudes or policies of the governing elite” (2009:99). 26 Whedon, 1992 film, then 1997 – 2003 television series 13 26 Whedon, 1992 film, then 1997 – 2003 television series 13 Gregor Cameron 198272670 Improvisation connected by space and time, the here and now, is consistent with Bakhtinʼs idea of the chronotope. Playback 135 exists in the here and now, “cheerfully…. reflecting and releasing the stories, energies and emotions of its participants” (Charles, 2004:7) The chronotope defines time and space in any literary or performative text, it is the place, the duration that settles the detail of the mise-en-scène, “how fictional time, space and character are constructed in relation to one another”. Charles also acknowledges that while improvisation is free it is the ʻritualʼ or framework under which it operates that allows for this freedom. Transgression that forms in the context of the status quo, understood and then offered up to scrutiny through the improv form. The rules of governance, known, are temporarily suspended (Charles, 2004:16) Thus the frame, while not being ritual, is certainly a form which the theatre may utilise. Diane Wilson (2006) has offered a useful reading of Buffy the Vampire Slayer26 in Bakhtinian terms. She offers many examples of how various modes of the carnivalesque can offer readings of Joss Whedonʼs work. In particular Whedon is quoted as saying: If I made “Buffy the Lesbian Separatist” a series of lectures on PBS on why there should be feminism, no one would be coming to the party, and it would be boring. The idea of changing culture is important to me, and it can only be done in a popular medium -Joss Whedon. (Quoted from Nussbaum in Wilson 2006) This is telling in a number of ways. Clearly Whedon knows his audience and directly engages them. He uses humour as a mode of inversion on the horror 136 MA Thesis: The Role of Misrule in the practice of Performance trope and from this position, the political nature of what he wishes to foreground, allows for a much greater affect upon his audience. Farrell Cleary (1995) has argued for a Bakhtinian reading of James K. Baxterʼs plays. He notes that Baxter observed his own “ineradicable tendency towards vaudeville” (1995:121). Baxter wrote, in a foreword to The Band Rotunda, a warning that the characters should not be identified as “merely comic types or grotesques, though there are grotesque and comic elements in the action and dialogue” (1995:121). Cleary ends his article recalling Baxterʼs use of earth- shaking laughter in the character or Flanagan in The Day Flanagan Died. Gregor Cameron 198272670 Cleary suggests that it foregrounds Flanaganʼs freedom from the rules and that, “for all its heroʼs untidiness and irresponsibility, the play would have its audience treasure that life [before death] and escape the zombie fate of so many in Baxterʼs New Zealand (1995:131). Again Stam (1989) underlines the importance of laughter: Carnivalesque laughter can be raucous, subversive, even angry, a laughter that erases old differences and installs new, unstable ones. Carnivalesque laughter can be raucous, subversive, even angry, a laughter that erases old differences and installs new, unstable ones. Laughter is profound, communitarian, erotic, a current passing from self to self in a free and familiar atmosphere. It is the adult memory of the cascading giggles of children, who laugh not necessarily at specific localizable “jokes” but as part of a collective contagion. Carnivalesque laughter can be raucous, subversive, even angry, a laughter that erases old differences and installs new, unstable ones. Laughter is profound, communitarian, erotic, a current passing from self to self in a free and familiar atmosphere. It is the adult memory of the cascading giggles of children, who laugh not necessarily at specific localizable “jokes” but as part of a collective contagion. (Stam, 1989:120) Saint Punch brings the carnival to the stage. At the beginning the setting of a wedding serves to introduce the characters, as well as set up the frame for the telling of stories – a comfortable place for the audience. The theatre itself is set in a fairly unadventurous design with audience sitting opposite the stage They Gregor Cameron 198272670 137 perceive upon the stage the familiar trappings - the puppet booth, the ʻcircusʼ curtain and the television. After the procession of characters comes a comedic interlude establishing that the three states are not going to necessarily always be separate, as the Professor speaks with the puppet Punch. Then the first scene is played up in the puppet booth and the audience is encouraged to laugh. The television introduces the historical figures of Gilles de Rais and Joan DʼArc, which puts laughter into question as de Rais rapes the child. Then Bluebeard and Judith appear upon the stage in Mask playing out the accusation of betrayed trust. The audience must decode where in the narrative these two other stories fit, further alienating them from too much empathy with the traditional folk and historical stories. This disruption of the stories timelines is a deliberate dislocation for the audience who, up to now (scene 1), have been in a familiar theatrical frame. This intention is leavened with the knowledge that the audience must not be dislocated to the point of becoming lost- they are encouraged to decode not disengage. perceive upon the stage the familiar trappings - the puppet booth, the ʻcircusʼ curtain and the television. After the procession of characters comes a comedic interlude establishing that the three states are not going to necessarily always be separate, as the Professor speaks with the puppet Punch. Then the first scene is played up in the puppet booth and the audience is encouraged to laugh. The television introduces the historical figures of Gilles de Rais and Joan DʼArc, which puts laughter into question as de Rais rapes the child. Then Bluebeard and Judith appear upon the stage in Mask playing out the accusation of betrayed trust. The audience must decode where in the narrative these two other stories fit, further alienating them from too much empathy with the traditional folk and historical stories. This disruption of the stories timelines is a deliberate dislocation for the audience who, up to now (scene 1), have been in a familiar theatrical frame. This intention is leavened with the knowledge that the audience must not be dislocated to the point of becoming lost- they are encouraged to decode not disengage. , ( ), ( ) ( ) 28 You are There. http://www.imdb.com/title/tt0045458/ accessed 28/11/11 27 Peter Watkins, director of Culloden (1964), The War Game (1965) and Punishment Park (1971). 28 Gregor Cameron 198272670 p Gregor Cameron 198272670 p Gregor Cameron 198272670 The fourth wall is already porous as Mr Punch and Judy and the Professor directly speak to the audience, the wedding serving as the ritual and the action on stage serving as spectacle in both its horror and its comedy. This pattern of comedy skits between the more violent plays invokes the common strategy of a night at the Grand Guignol, the so-called douche écossaise or hot and cold showers (Freshwater 2007:248). Punchʼs inversion in his gentle care of Judy and the baby is unnervingly mirrored in the actions of de Rais and Bluebeard. 138 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance However we are also aware that though Punch kills the baby that there will come a renewal of their story as the tradition recognises- all things are forgiven and repeated in Punchʼs mythology. The masks of the stage serve to subsume the actor and emphasize their embodiment of their stylized characters. These characters do not represent reality as we understand it, but rather a representation of it. However, as the actors pass through the differing mediated environments, that thought is undermined, the carnival does not allow them to so easily dismiss the images before them- they laugh at Punch and Judy, can enjoy the romance of the puppets representing Judith and her sisters meeting with Bluebeard. Then the characters appear on the screen, acting more naturalistically. The screen itself defines a particular reading of the image, harking back to the sixties social realism docudramas of Peter Watkins27 or the American TV series You are There (TV Series 1953–1957)28. Suddenly the audience finds itself within the dungeon, the police interview room, the nobleʼs castle. As swiftly, the violence turns real, visceral, and discomforting. The inversion is complete and the audience should question whether what they are experiencing should be laughed at. In a sense the carnivalesque allows for a place where there is an agreed liminal space. It is a space where these characters can be re-mediated as they cross between the three performance spaces. Their basic character and the story they are telling does not change but the space can invade the context of the stories. With Punchʼs story his entry into the real (Film) environment heightens the sense of his threat. This was the intent. In placing 139 and moving the characters from place to place I intended that their stories would be thrown into question. MA Thesis: The Role of Misrule in the practice of Performance Michel Foucault, in A Preface to Transgression, notes this system of limit and taboo and suggests that without the limit there can be no transgression, no crime where there is no law (Freshwater, 2007:249). Grand Guignol, as a genre, arose from the Guignol puppet figure, which in France served as a kind of Mr Punch-like character and was emblematic of a shift from the kind of horrific image that might traditionally have stayed off-stage straying onto the stage (2005:38). Pickering also suggests a link to Soap Opera and Melodrama in Movies and TV that feature ʻgoodʼ or ʻbadʼ characters (2005:39). Thus in content it had clear ancestry with the human corpse count of a Shakespeare or a Ford but, rather than happening offstage and then reported, the characters afflictions and mutilations were performed in front of the audience. Bakhtin may have acknowledged that this entry of the grotesque body onto the stage is clearly an invocation of the carnival. This feature of the carnivalesque is key as it links back to some of the earliest traditions and rituals. The grotesque body Gregor Cameron 198272670 The Grotesque Body and the Simulacra. The grotesque character is a comic figure. It is impossible to sympathize with him, despite his agonies, because we view him from a detached perspective, and when we are not emotionally involved in his suffering, we are amused…. As with the grotesque character the entire technique of the grotesque is also essentially comic, for we always view the grotesque from a vantage point. To be certain, the subject matter of the grotesque- the raw material which creates the vision- is always potentially horrible, but the treatment of this material is comic: this explains the peculiar complexity of tone, combining both horror and the ludicrous, which characterizes the grotesque as an art form. 140 With the connotation of the Grand Guignol, the carnival allows the audience to experience an uncomfortable close up encounter with what lies behind the stories. The Grand Guignol is a place of horror and violence, often connected to the fashions of Horror and Action cinema, however in the theatre it was more likely to feature illusion, suggestion and trickery than any actual violence despite its reputation. In fact Grand Guignol is a very good example of how the carnival negotiates with the official system without. The Little Theatre in London in 1920 provoked the censorship function of the Lord Chamberlain when it began a season of Grand Guignol plays. Some plays were banned and 140 MA Thesis: The Role of Misrule in the practice of Performance some played. In response to this threat some other means of licensing needed to be found. The Old Women, was likely to be banned if the authorities knew that it would go on in London. Russell Thorndike, translator of the French Guignol play, put on the application for a license that would only be a showing to a small village. The censor felt it would do no harm there, but instead José Levy, manager of the London Grand Guignol, put it on at the Little Theatre in London as intended (Freshwater, 2007:252). This subversion of the law is interesting in that it suggests the presence of misrule in practice as well as performance. Gregor Cameron 198272670 29 Tatler, 15 September 1920 Gregor Cameron 198272670 141 is primarily concerned with the functions of the lower abdomen, elimination and procreation, shit and sex. Richard Hand and Michael Wilson trace the line between the Elizabethan and Jacobean stage through the gothic writers of Frankenstein, and Jekyll and Hyde (2007:19). They also state some of the adaptations that the Grand Guignol needed to make in its travel from Paris to London. In particular they point to the fact of its Montmartrean origins- that the sexual component of both humour and horror in the Paris version would need taming for a more conservative London audience. However it was still transgressive enough to provide a hearty dialogue in the papers of the time (2007:20). In part this explains why in most translations the location of the action remains France rather than making it local. In talking about the style of acting within the London Grand Guignol, Hand and Wilson suggest that its melodramatic core was a deliberate attempt to invite the audience into the moment- what The Times suggested might be ”realistically or romantically ʻcreepyʼ: There is a point at which horror works best when it is unconvincing, where it revels in its own artifice. If the audience knows that this is not real horror, then it is better to be able to enjoy it and surrender to its emotional intensity. There is a point at which horror works best when it is unconvincing, where it revels in its own artifice. If the audience knows that this is not real horror, then it is better to be able to enjoy it and surrender to its emotional intensity. (Hand and Wilson, 2007:43) 142 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Either by being victim or by being perpetrator the Actor becomes the grotesque body. As the Tatler called it, “a really wonderful exhibition in the art of making the audienceʼs blood curdle”29 (2007:43). This position as the grotesque allows an audience to place themselves either in opposition or in collusion with the character and the characterʼs political position. Both positions should lead to the audience reflecting upon what they experience within the theatrical space. The experience may still be one that swings from laughter to horror but that very oscillation invites reflection, and possibly change. The grotesque body also raises some questions about the use of the simulacrum. Puppets of course are the most familiar physical form of simulacra used in the theatre but the same may hold true of the masked performer and in fact of the actor themselves. In the movement of thought from Plato to postmodernism thinkers such as Nietzche, Baudrillaud and Gilles Deleuze have considered the importance of the simulacra. As they define how the simulacra is experienced by each of us they acknowledge that the simulacra, far from being a copy of a copy, plays a role “which is incommensurable with conceptual reason” (Edgar and Sedgwick, 1999:312). This quality of being without comparison means that when the audience accepts a ʻstand-inʼ in the theatre they are signing up to a social contract that includes a degree of willing disbelief. A troubling notion if the theatre piece is reliant upon the audience also being willing to empathize with the characters or situation onstage. Gregor Cameron 198272670 29 Tatler, 15 September 1920 143 Berthold Brecht is suspicious of empathy. He accepts that empathy is key to an audienceʼs enjoyment of the night out but is concerned that this may preclude them from thinking critically about the piece. He uses the actor as simulacra deliberately within his A-effect30. He says “[o]ur dramatic form is based on the spectatorʼs ability to be carried along, identify himself, feel empathy and understand.” (1978:25) Brecht is happy to use the distance between what is and what is being represented to secure an event in the theatre that challenges his audience not just to accept what is before them but invites them to be critical about what they perceive. Brecht himself co-opted the Finnish folk figure of Mr Puntila in order to use the figure to illuminate the world in which misrule governs. 30 Alienation effect for want of a better translation. 14 30 Alienation effect for want of a better translation. 14 MA Thesis: The Role of Misrule in the practice of Performance E Speidel argues that Brecht is here illustrating how harmful class division is and how much more humanistic and neatly controlled the social state might be under communism- a Marxist position (Speidel, 1970:321). In the play, Mr Puntila and his man, Matti (1940), Brecht offers a character not too far removed from that of Mr Punch, though Speidel makes it clear that Chaplinʼs Millionaire from City Lights (1931) “who, like Puntila, ʻoverflows with good humour and kindness when he is drunk, but reverts to a ʻreasonableʼ attitude, mean and businesslike, when he is sober” is a more likely ancestor (Speidel, 1970:319). Brecht is concerned with this split personality and uses it to illustrate how there is clear division between the emotional warm human and the rational Darwinistic capitalist. In 144 MA Thesis: The Role of Misrule in the practice of Performance doing this Brecht is harking back to the picaresque novel “where the main interest lies in the individual episode and not in the development of a coherent story” (Speidel, 1970:320). Here there is clear connection to the traditional structure of the Punch story. Scenes are loosely connected and it is in the episodes the scenes run that should engage the audience. Brecht was conscious however that it should not prove a populist play but be accorded the same respect as a piece of grand theatre. In this he also did not wish to emphasise the romantic, the crude or the farcical; but rather use the humour, charm and disruptive behaviour of the character to engage the audience and invite them to consider the ongoing political meaning of the piece. The audience, able to dismiss the humour as un-affecting, concerned Brecht. How does one place a clown in front of an audience and still engage them in a meaningful dialectic? His position is one of encouraging dialectic even as here he employs a clown- like figure. In the twenty-fifth paragraph of A short organum for the theatre he explains: Even the wholly anti-social can be a source of enjoyment to society so long as it is presented forcefully and on the grand scale. It then often proves to have considerable powers of understanding and other unusually valuable capacities, applied admittedly to a destructive end. MA Thesis: The Role of Misrule in the practice of Performance Even the bursting flood of a vast catastrophe can be appreciated in all its majesty by society, if society knows how to master it; then we make it our own. (Brecht, 1978:187) Gregor Cameron 198272670 145 Brecht adds an addendum to this which goes some way towards explaining how he perceives the age of reason coming to terms with the way in which the misrule of humour and surprise contribute to meaning in the theatre: The theatre of the scientific age is in a position to make dialectics into a source of enjoyment. The unexpectedness of logically progressive or zigzag development, the instability of every circumstance, the joke of contradiction and so forth: all these are ways of enjoying the liveliness of men, things and processes, and they heighten both our capacity for life and our pleasure in it. Every art contributes to the greatest art of all, the art of living. (Brecht, 1978:277) Brecht seems to be saying that not only is everything in art an artifice but that our too-close identification is a trap that disallows any form of reflexivity. 146 our too-close identification is a trap that disallows any form of reflexivity. However at the same time he is also describing a location for the theatre that has clear aspects of the carnivalesque. He works in a form that does not attempt to be reality but rather seeks to reflect and be reflected upon. He expects an active critical audience to make meaning out of dramatic texts without the confusion of empathy but with an understanding of it (Franks, 1999:192). Here Brecht enters into a kind of ʻwhat ifʼ territory. Anton Franks and Ken Jones discuss how an educationalist like Dorothy Heathcote might recognise Brechtʼs intentions. They are unconvinced about some of Heathcoteʼs understandings but acknowledge that she “suggests that the dramatic and theatrical experience is, in Brechtʼs words, like ʻvisiting another roomʼ, a room which makes the construction of an ʻas ifʼ or potential world is possible This other room operates, for Heathcote, as a place where the role However at the same time he is also describing a location for the theatre that has clear aspects of the carnivalesque. He works in a form that does not attempt to be reality but rather seeks to reflect and be reflected upon. MA Thesis: The Role of Misrule in the practice of Performance He expects an active critical audience to make meaning out of dramatic texts without the confusion of empathy but with an understanding of it (Franks, 146 146 MA Thesis: The Role of Misrule in the practice of Performance can be experienced with “the freedom to experiment without the burden of future repercussions” and in “the absence of the chance element of real life.” (Heathcote, 1991:104). Here lies a border, very likely the border of the carnivalesque. Heathcote and Brecht seem to invite their respective students and audience to collaborate in a kind of ʻlaboratoryʼ in which they remain themselves but at the same time must reflect on what manifests or is presented. Again theory and practice are beginning to reflect each other. Gavin Bolton (1985) recognises that Heathcote and Brecht rely on the “notion of ʻdistancingʼ than on verisimilitude.” He points to the Greek term Metaxis, the real and the fictional held simultaneously in mind by both performers and spectators allowing an interplay between these worlds that encourages discretion rather than immersion (Bolton 1985: 155). Franks comments on Heathcoteʼs acceptance of: …the potential for drama and theatre to distance us from everyday life, to see it afresh, to employ Verfremdungsteffekt, to reveal gestic moments, to reveal the potential mutability of both dramatized and everyday life, so that the student, the actor and the spectator can employ their powers of reasoned reflection. (Franks, 1999:190) This use of the body as simulacra, as a mediated stand-in for real-life has a number of challenges within the Carnivalesque space. It demands that the representation stand for something or risk losing meaning. In Saint Punch there are a number of simulacra, the most obvious being the puppets, and they need to be dealt with by looking at what is offered to the audience to aid decoding. However Gilles Deleuze is suspicious of representation: “It mediates Gregor Cameron 198272670 Matt Hills makes it clear that this intertextual awareness is “by definition unfinishable and non- forecloseable” (2005:40). Adolphe Haberer (2007) argues strongly for the influence of intertextuality throughout the range of cultural artefacts with particular focus on poetry. In the process he challenges Bakhtinʼs view that poetry is monologic rather than dialogic. He suggests that the intertext is exactly what makes these artefacts dialogic (2007). Thus what we read within the representation on the stage is loaded with a number of both cultural and personal resonances, some deliberate and others accidental or only recognizable through an almost accidental bringing together of these artefacts. MA Thesis: The Role of Misrule in the practice of Performance individuals but, in some sense, this is the glue of culture – that we experience enough in common to be able to read at least some commonalities in whatever is placed before us. Jen Webb and Schirato (2003) also reference the work of Bourdieu in how knowledge of a field, or fields, such as the intertextual matrix, contributes to a process of reflexivity. This leads to a process, quoted from Bourdieu and Wacquant (1992), that “disposes its subjects towards ʻthe systematic exploration of the unthought categories of thought that delimit the thinkable and predetermine the thoughtʼ” (2003:545). Matt Hills makes it clear that this intertextual awareness is “by definition unfinishable and non- forecloseable” (2005:40). Adolphe Haberer (2007) argues strongly for the influence of intertextuality throughout the range of cultural artefacts with particular focus on poetry. In the process he challenges Bakhtinʼs view that poetry is monologic rather than dialogic. He suggests that the intertext is exactly what makes these artefacts dialogic (2007). Thus what we read within the representation on the stage is loaded with a number of both cultural and personal resonances, some deliberate and others accidental or only recognizable through an almost accidental bringing together of these artefacts. This alone argues for a dialogic reading of the stage and strengthens its ties to the carnivalesque. Simulacra and the grotesque body can also have a number of readings and responses. Gregor Cameron 198272670 149 Punch, the puppet, traditionally sports a bulbous nose and a hump on his back- he is grotesque. In Saint Punch, shaped traditionally, he is not clothed in his traditional garb but wears much more contemporary costume. The reason for this is twofold. Gregor Cameron 198272670 147 everything, but mobilizes and moves nothing.” (May 2005:74). The carnival acts as a frame for representation allowing it to acknowledge its limitations and causing it to be clear about what it intends to represent within the performative text. … we read and make sense of texts in terms of their cultural contexts or frames. Contexts function as cultural frames within which to make sense of texts, frames which operate through our knowledge of, and literacy in, a variety of other contexts… All texts carry elements, or traces, of context with them. Mikhail Bakhtin, the Russian critic and theorist, coined the term dialogism to refer to the way in which texts, through their relationships with other texts and other contexts become charged with these traces. Any text resonates with the meanings of other text, and other contexts or places in which that text has been. 148 Firstly however it is important to acknowledge that writer, director, designer, actors and audience all enter the theatre space carrying with them an intertextual matrix (Hills, 2005:40). Influence lies everywhere. This matrix is the baggage accumulated by any individual moving through the rich cultural landscape that they live within. Tony Schirato and Susan Yell define intertextuality as a “process of making sense of texts in reference to their relations with other texts” (2000:53). In other words, we aggregate our experiences in a way that allows us access to all that we have produced, viewed, read or witnessed. We are aware that there is difference but treat these experiences as the basis by which we make sense of the world. Of course, what makes sense to one may not have resonance for another as 148 MA Thesis: The Role of Misrule in the practice of Performance individuals but, in some sense, this is the glue of culture – that we experience enough in common to be able to read at least some commonalities in whatever is placed before us. Jen Webb and Schirato (2003) also reference the work of Bourdieu in how knowledge of a field, or fields, such as the intertextual matrix, contributes to a process of reflexivity. This leads to a process, quoted from Bourdieu and Wacquant (1992), that “disposes its subjects towards ʻthe systematic exploration of the unthought categories of thought that delimit the thinkable and predetermine the thoughtʼ” (2003:545). 31 http://www.guardian.co.uk/politics/2005/may/13/fashion.fashionandstyle accessed 6/12/11 Gregor Cameron 198272670 As Gavin Hood says “The hoodie is the uniform of the troublemaker: its wearer may as well be emblazoned with a scarlet letter.”31 From the Grim Reaper to Robin Hood the hood stands as a marker for awareness, for moral ambiguity. As such Punchʼs representation connects him to both tradition and to a more contemporary ambivalent image. The other puppets in his narrative also wear more contemporary clothing even when their spoken language still harks back to their traditional influences. As he leaves the booth and enters the stage his mask connects him clearly to the Commedia DellʼArte tradition of clowning. Rosalind Crone (2006) chronicles Punchʼs history and makes it clear how the violence of the domestic mirrors his epochʼs ambivalence towards a growing unsettledness about the concept of marriage and domesticity (1079). She points out that Judy arose from “phrases such as ʻto make a Judy of yourselfʼ and the label ʻJudyʼ when referring to women who behaved in a disorderly manner.”(2006:1081) This was a change from her eighteenth century name, Joan. as the puppet he must also be seen as mask and as a figure on ʻfilmʼ. Thus the costume must be readily available and transferrable. Secondly he must be contemporaneous with the audience to assist them with relating to this older tradition. He wears a hoodie which has become, as Rachael Owen and Anna Sweeting imply with the title of their report for Victim Support, ʻHoodie or goodie?ʼ- an icon for ʻfalling into the wrong crowdʼ, for youth offending and for the urban culture most at risk (2007). As Gavin Hood says “The hoodie is the uniform of the troublemaker: its wearer may as well be emblazoned with a scarlet letter.”31 From the Grim Reaper to Robin Hood the hood stands as a marker for awareness, for moral ambiguity. As such Punchʼs representation connects him to both tradition and to a more contemporary ambivalent image. The other puppets in his narrative also wear more contemporary clothing even when their spoken language still harks back to their traditional influences. As Sweeting imply with the title of their report for Victim Support, ʻHoodie or goodie?ʼ- an icon for ʻfalling into the wrong crowdʼ, for youth offending and for the urban culture most at risk (2007). Gregor Cameron 198272670 One is pragmatic, this is a performance without budget and 149 as the puppet he must also be seen as mask and as a figure on ʻfilmʼ. Thus the costume must be readily available and transferrable. Secondly he must be contemporaneous with the audience to assist them with relating to this older tradition. He wears a hoodie which has become, as Rachael Owen and Anna Sweeting imply with the title of their report for Victim Support, ʻHoodie or goodie?ʼ- an icon for ʻfalling into the wrong crowdʼ, for youth offending and for the urban culture most at risk (2007). As Gavin Hood says “The hoodie is the uniform of the troublemaker: its wearer may as well be emblazoned with a scarlet letter.”31 From the Grim Reaper to Robin Hood the hood stands as a marker for awareness, for moral ambiguity. As such Punchʼs representation connects him to both tradition and to a more contemporary ambivalent image. The other puppets in his narrative also wear more contemporary clothing even when their spoken language still harks back to their traditional influences. As he leaves the booth and enters the stage his mask connects him clearly to the Commedia DellʼArte tradition of clowning. Rosalind Crone (2006) chronicles Punchʼs history and makes it clear how the violence of the domestic mirrors his epochʼs ambivalence towards a growing unsettledness about the concept of marriage and domesticity (1079). She points out that Judy arose from “phrases such as ʻto make a Judy of yourselfʼ and the label ʻJudyʼ when referring to women who behaved in a disorderly manner.”(2006:1081) This was a change from her eighteenth century name, Joan. as the puppet he must also be seen as mask and as a figure on ʻfilmʼ. Thus the costume must be readily available and transferrable. Secondly he must be contemporaneous with the audience to assist them with relating to this older tradition. He wears a hoodie which has become, as Rachael Owen and Anna Sweeting imply with the title of their report for Victim Support, ʻHoodie or goodie?ʼ- an icon for ʻfalling into the wrong crowdʼ, for youth offending and for the urban culture most at risk (2007). 32 The Dark Knight, (2008), Christopher Nolan http://www.imdb.com/title/tt0468569/ accessed 4/12/11 Gregor Cameron 198272670 g , ( ), p p Gregor Cameron 198272670 As Gavin Hood says “The hoodie is the uniform of the troublemaker: its wearer may as well be emblazoned with a scarlet letter.”31 From the Grim Reaper to Robin Hood the hood stands as a marker for awareness, for moral ambiguity. As such Punchʼs representation connects him to both tradition and to a more contemporary ambivalent image. p y g The other puppets in his narrative also wear more contemporary clothing even when their spoken language still harks back to their traditional influences. As he leaves the booth and enters the stage his mask connects him clearly to the Commedia DellʼArte tradition of clowning. Rosalind Crone (2006) chronicles Punchʼs history and makes it clear how the violence of the domestic mirrors his epochʼs ambivalence towards a growing unsettledness about the concept of marriage and domesticity (1079). She points out that Judy arose from “phrases such as ʻto make a Judy of yourselfʼ and the label ʻJudyʼ when referring to women who behaved in a disorderly manner.”(2006:1081) This was a change from her eighteenth century name, Joan. 150 MA Thesis: The Role of Misrule in the practice of Performance The grossly exaggerated violence and Punchʼs nonchalant attitude about his wifeʼs death make the scene rather humorous and ridiculous. Moreover, Punchʼs disposal of his wifeʼs body objectifies her. Thus, with his re-establishment of his mastery of the household, Punch becomes a hero, while little sympathy is left for his painful and ugly wife. Extreme violence also characterizes Punchʼs encounters with other puppets in Picciniʼs show…when Punch is finally sent to the gallows, he succeeds in tricking the hangman into placing his own neck in the noose… Gregor Cameron 198272670 The Dark Knight, (2008), Christopher Nolan h MA Thesis: The Role of Misrule in the practice of Performance The grossly exaggerated violence and Punchʼs nonchalant attitude about his wifeʼs death make the scene rather humorous and ridiculous. Moreover, Punchʼs disposal of his wifeʼs body objectifies her. Thus, with his re-establishment of his mastery of the household, Punch becomes a hero, while little sympathy is left for his painful and ugly wife. Extreme violence also characterizes Punchʼs encounters with other puppets in Picciniʼs show…when Punch is finally sent to the gallows, he succeeds in tricking the hangman into placing his own neck in the noose… (Crone, 2006:1063) His narrativeʼs characters also appear as figures in masks and the tone is one of Clown. As he and the policeman appear on the screen a different, darker moment is illuminated. Here the reference is much more the image of the Joker from the Batman canon, most particularly from the film, The Dark Knight32. This is an altogether much more disturbing image, physically much more threatening as Punch beats the Policeman to death. Similarly when Gilles de Rais and Joan DʼArc appear as themselves on the screen they carry a much heavier presence than in the other environments. De Rais is clothed in a representation of armour and Joan appears in sackcloth- there could not be more distance between them. However, between them, the power struggle is much more complex. As they leave the screen and enter the booth, instead of recognizable puppets, dolls- toy soldiers as action figures- play them. Some remodelling has been done to change the modern toy soldiers into their medieval counterparts but the image is still of two people ʻplayingʼ at war. Here the division between de Rais and Joan becomes explicit, 151 he a warrior belonging to the dominant hegemony, full of divine right and careless of the lives below him in the social order, she belonging to this lower social order, given divine leading to change things for her beloved France. As they watch the battle the division between their world views becomes wider, she intolerant of her fellow man giving in to his baser impulses (ironic given her behaviour in Scene four) and de Rais shrugging such behaviour off. This ʻplayʼ is important as it reads a particular point of view upon these characters, for the older de Rais it is play, whereas for Joan it is so much a symptom of why she acts. MA Thesis: The Role of Misrule in the practice of Performance Here played out is the division between the renaissance and the medieval. We see this even more explicitly as they move into the stage. The Mask is coupled with a more expressive set of moves upon the stage; de Rais is played almost as a chess piece about to be sacrificed. His life is in the inquisitionʼs hands, and those of his erstwhile friends and confidants. However in contrast to Joan he is given a form of mercy in being promised a quick death before burning. Thus when they return to the stage at the end of the play it is she who mirrors the giving of his ʻmercyʼ, not in the sense of forgiveness but perhaps as a way of sharing the sense of his ʻpunishmentʼ. He dies and is burnt as Joanʼs ghost screams silently and writhes in agony, terribly alive to all the pain and suffering, almost a reference to a Christ being sacrificed. The representations that the puppets, masks and actors ask us to witness, as audience, do not through their complexity and ambivalent signification allow us to fully empathise with their condition but attempt to lure us into taking a 152 MA Thesis: The Role of Misrule in the practice of Performance position or at least to consider carefully what this mediation of their narratives leave us with. In contrast Bluebeard has a much softer treatment. The performance in their native stage is deliberately large. This is melodrama, a Disney type fable. Even when they are on the television screen they are playing up the emotions and motivations of their characters. It is during their time in the Puppet Booth that their characters feel truly at home. They are glove puppets not dis-similar to ʻThe Muppetsʼ of television and cinema fame. Particularly they also hark back to the ʻMuppetsʼ beginnings on Sesame Street, an educational show for children in the United States of America (1969-present). They are perhaps therefore the most endearing of the three stories and their simulacra are charming and attractive for the most part, a welcome lightness as a foil to the other stories. However the grotesque body invades their space as well, as the cabinet is illicitly opened to reveal the six heads of Bluebeardʼs previous wives. MA Thesis: The Role of Misrule in the practice of Performance Here is an image of true horror, mediated through a mode most commonly associated with children, who are, after all, the intended audience of a tale such as this. The juxtaposition of the childish with the ghoulish is intended to deliver a wonderful moment where the audience does not know whether to laugh or cringe in fear. Such moments can be cathartic or force one into a reflexive-ness that is to be encouraged during the theatrical moment. Maria Tater tells us that Bluebeard began as adult entertainment but has fallen through “a cultural Black Hole” until it found itself behind the covers of a fairy tale collection (2004:12). She cites both Cunmar of Brittany and Gilles de Rais Gregor Cameron 198272670 153 Gregor Cameron 198272670 153 as being claimed as the progenitor of Bluebeard. She is not entirely convinced and adds that there are many comparable stories from many other cultural traditions. However she acknowledges that the common manifestation has been used as a warning to wives against “the fatal effects of curiosity and disobedience”, allying it with some of the readings that have been placed upon Punch and Judy (2004:17). Further it explains how the colour Blue became, in the eleventh and twelfth centuries, a signifier for the aristocracy leading to the nomenclature, blueblood (2004:19). However this but part of what is played out in Saint Punch. Feona Attwood (1998) compares Bluebeardʼs wife to the fate of Ada in Jane Campionʼs The Piano (1993). A useful comparison underlined by the inclusion of a community play performance of the story within the film (Chumo, 1997:173). Here the wifeʼs curiosity becomes a reading of the dangerous nature of female sexuality (1998:90) Attwood places the film as picking away at patriarchal discourse (1997:95) in a way that empowers the wife. In Campionʼs film, Peter Chumo tells us, the resonances are clearly visible as the shadow-play chopping of Bluebeard echoes the mutilation of Ada at the end of the film, the commonality of the keys to the chamber and the keys of the piano, and the play of illusion and reality of the cinema is commented on as the Maoris storm the stage to save the wife believing the image before them to be real. Finally art itself is attacked though Stewart and Adaʼs impossible distance (1997:176). Campionʼs heroines are at once both innocent and complicit in their fates. Similarly following Angela Carter (1979) Saint Punch seeks to make visible Judithʼs complicity in her fate, her want for this as being claimed as the progenitor of Bluebeard. She is not entirely convinced and adds that there are many comparable stories from many other cultural traditions. However she acknowledges that the common manifestation has been used as a warning to wives against “the fatal effects of curiosity and disobedience”, allying it with some of the readings that have been placed upon Punch and Judy (2004:17). Further it explains how the colour Blue became, in the eleventh and twelfth centuries, a signifier for the aristocracy leading to the nomenclature, blueblood (2004:19). However this but part of what is played out in Saint Punch. Gregor Cameron 198272670 Feona Attwood (1998) compares Bluebeardʼs wife to the fate of Ada in Jane Campionʼs The Piano (1993). A useful comparison underlined by the inclusion of a community play performance of the story within the film (Chumo, 1997:173). Here the wifeʼs curiosity becomes a reading of the dangerous nature of female sexuality (1998:90) Attwood places the film as picking away at patriarchal discourse (1997:95) in a way that empowers the wife. In Campionʼs film, Peter Chumo tells us, the resonances are clearly visible as the shadow-play chopping of Bluebeard echoes the mutilation of Ada at the end of the film, the commonality of the keys to the chamber and the keys of the piano, and the play of illusion and reality of the cinema is commented on as the Maoris storm the stage to save the wife believing the image before them to be real. Finally art itself is attacked though Stewart and Adaʼs impossible distance (1997:176). Campionʼs heroines are at once both innocent and complicit in their fates. Similarly following Angela Carter (1979) Saint Punch seeks to make visible Judithʼs complicity in her fate, her want for this 154 MA Thesis: The Role of Misrule in the practice of Performance glamorous man, her need to take charge of her own rights despite the possible consequences. Robin Sheets argues for Carterʼs The Bloody Chamber (a version of Bluebeard) as “writing against the interpretive tradition that emphasises the wifeʼs illicit sexual curiosity” (1991:644). Moreover she recognises its link to the story of Gilles de Rais. Of course the Marquis de Sade is also referenced in Carterʼs story. Sheets suggests, following Barthes, that as a narrator, the protagonist represents a shift in power through her control of language (1991:649). She is neither Juliette (the bad girl), or Justine (the willing victim) or Judy (the disobedient woman). Rather she is the imperfect woman: ”she has the right to act, to experience the consequences of her actions and to learn from error.”(1991:650) Her experience with the ʻgrotesque bodyʼ of the Marquis serves as a catalyst for change and self- reflection. In Kari Lokkeʼs critique of The Bloody Chamber and Max Frischʼs Bluebeard there is a strong argument for Bluebeard being grotesque simply because it is the nature of the fairy tale motif (1988:7). Gregor Cameron 198272670 She suggests, through Bakhtin, that the Medieval/Renaissance “grotesque had an emancipatory function, for the juxtaposition of opposites broke down false societal and ideological barriers to expose the truth of lifeʼs unpredictability and spontaneity and to celebrate the unity and power of that life in all its most corporeal manifestations” (1988:7). Through these two authorʼs works she illustrates how the presentation of the characters environment contribute to the way in which the grotesque is presented. With the character of Felix Schaad, the reader is invited to consider how a buttoned down but fantasy rich life can fall into pieces. Schaadʼs name, Gregor Cameron 198272670 155 significantly, she points out, “is related to both schaden, to harm or to hurt, and schade!, what a pity!, Too Bad!” which harks back to Punchʼs stock phrase (1988:9). Given how laden with meaning Bluebeard can be it is ironic that in Saint Punch it is perhaps the piece most lightly provocative. And yet as a counterpoint to the other horror and comedic forms of violence it is, perhaps, a necessary interlude in which the audience may find the pause to reflect upon what their reactions are to the rest of the work. If it is true that a work becomes grotesque by virtue of its characters and its content then Saint Punch manages to provide its audience with a clear context for reflection (should they choose to). What becomes clear is that the grotesque body motif provides a link between these three stories. The puppets, the masks and the actors on screen also are linked in their use of the simulacra as grotesque body, which both distances the audience and makes it safe for them to relate either intellectually or emotionally to differing moments of pathos and jest. The Dark Clown and the Trickster In The Road to Mars (1999), author Eric Idle (late of Monty Python's Flying Circus) offers a breakdown of how humour works using the example of the clown. He particularly cites the white face and the red nose clowns. He locates such pairings as Laurel and Hardy or Martin and Lewis. Oliver Hardy and Dean 156 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Martin are examples of the white Face Clown and are often the instigators of action- possibly action that is at odds with what society would consider acceptable, often marked by them trying either to perform or attain high status. In counterpoint Stan Laurel and Jerry Lewis are examples of the Red Nose clown, the fool whose innocence and naiveté, and position of low status, constantly complicates and defeats the red nose clownʼs efforts to subvert things. Bakhtin also acknowledges these two forms of clown- the rogue and the fool. However he adds a third category simply known as the clown- which can be seen in dramatic terms as the clown/fool, in some of Shakespeareʼs plays, who is able to comment upon high and low alike. Sue Vice suggests that Bakhtin saw their 'stylistic functions' were 'determined by [their] relationship to heteroglossia', which each one helps to introduce (Vice, 1997:69). In carnivalesque terms these clowns have a relationship to the high culture and the low, the sacred and the profane. They are often located within the low culture but through their vision, as the Fool in Shakespeare's King Lear, may be placed quite close to the centre of power. Set as commentators upon the cultures they either observe or participate in the clowns can be seen assuming very specific roles. Bakhtin, according to Vice, sees the three behaving according to these roles, thus: Gregor Cameron 198272670 The rogue parodies high languages; the fool is 'naively incomprehending' of the high languages s/he comes into contact with; the [dark] clown 'unmasks' high languages and has license to utter the otherwise unacceptable. Heteroglossia is dialogized by these human figures, as it G C 198272670 The rogue parodies high languages; the fool is 'naively incomprehending' of the high languages s/he comes into contact with; the [dark] clown 'unmasks' high languages and has license to utter the otherwise unacceptable. Heteroglossia is dialogized by these human figures, as it Gregor Cameron 198272670 157 becomes double voiced through being parodied, and revitalized by different viewpoints. (Vice, 1997:69) Thus Bakhtin sees that through incomprehension, deception and transgression/subversion the three archetypes contribute a form of organization to the heteroglossic (1997:69). The appellation 'dark' is appended to the Bakhtin description to differentiate this clown from the others. MA Thesis: The Role of Misrule in the practice of Performance This clown most closely combines the elements of carnival within their appearance and behavior. Puck in A Midsummers Night's Dream might be an essence of this misrulic clown- it is not chaotic since that would necessitate no regard for the rules- Puck is subversive of the rules but not really transgressive. The Joker out of the Batman comic mythos is perhaps a more chaotic clown except that he cannot exist but to balance the Batman, which links him irrevocably to the rules he transgresses. Both these examples are emblematic of the Dark Clown, through which the status quo might be interrogated and judged. Puck and to a lesser extent, the Joker, because he is the alter image of the Batman, challenge the way things are- one through magic and the other through acts of insanity and crime. However, as David Abrams and Brian Sutton- Smith have written the Dark Clown is not synonymous with the growing popular culture image of the psychopath or sociopath as these characters lack the flexibility that is also emblematic of the dark clown/ trickster figure. They tested both the hypothesis of the psychopath and the traditional culture hero with children aged between five to seven and a group eight to eleven, and they used as a 158 MA Thesis: The Role of Misrule in the practice of Performance popular culture hero the cartoon character Bugs Bunny33 (1977:30). Their conclusion supports the role of the trickster figure in the socialization of children, commenting particularly on the way in which the childrenʼs flexibility in defining their social roles and in their explorations of novel responses (1977:47). They then suggest that the variation in Trickster stories arises from the needs of their audiences. The cartoon character is simply one of the modern interpretations of this cultural role. In considering the role Mr. Punch has served in previous centuries it would not be too far from the role Bugs plays for his television audience. There are also examples of these clowns from the Commedia DellʼArte tradition. Arlecchino/ Harlequin is the dark clown to Brighella/ Il Capitanoʼs rogue and Pierrot/ Pantalone's fool. Of course the classic manifestation of the Commedia clowns that have entered the twentieth century are the Marx Brothers. Harpo is the rogue, Chico the fool and Groucho, of course, is the dark clown who unmasks and subverts the high and the low with considerable license. 33 http://www.imdb.com/character/ch0000574/ accessed 10/12/11 Gregor Cameron 198272670 p Gregor Cameron 198272670 http://www.imdb.com/character/ch0000574/ accessed 10/12/11 Gregor Cameron 198272670 16 p 34 http://www.imdb.com/title/tt0098904/ accessed 10/12/11 Gregor Cameron 198272670 http://www.imdb.com/title/tt0098904/ access MA Thesis: The Role of Misrule in the practice of Performance Robert Stam in Subversive Pleasures (1989) acknowledges their films as fine examples of the carnivalesque. They attack and subvert a number of controlling institutions- such as the racetrack, the opera, the law and even warring nations and their diplomats. Stam notes Bakhtin's words concerning the masks of the clowns: [which] grant the right not to understand, the right to confuse, to tease, to hyperbolize life; the right to parody others while talking, the right to not be taken literally, not “to be oneself”.... the right to rip off masks, the right to rage at others with a primeval (almost cultic) rage- and finally, the right to 159 betray to the public a personal life, down to its most private and prurient little secrets. (Stam, 1989:112-113) As hinted above, there appears to be a link between the Dark Clown and the Trickster figure that arises in Mythology. Jan Kott, in Theatre Notebook (1947- 1967), describes Harlequin like this: Harlequin has every possible gesture. He doubles and trebles himself, changes and transforms himself. He is a poor starved servant and a great conjuror. He is all intelligence, elemental, a demon of movement. He puts two flowers in his mouth and, for a dozen seconds, is a beautiful woman. He is neither a clown nor a marionette. It is he who pulls the strings of all the other characters in the comedy. He sets the mechanism of this little world in motion. But at the same time he parodies and mocks it. For he is the only one who knows all the moves. (Jan Kott quoted in Nichols, 2002: 147) (Jan Kott quoted in Nichols, 2002: 147) Nichols goes on to describe Arlecchinoʼs ability to change and transform, his one constant quality as liminal (2002:151). Furthermore she links his manifestations prior to his appearance in France in 1571 as “reminiscent of Old Comedyʼs archetypal trickster (2002:156). She traces his lineage through to his “father” Mercury (Hermes in the Greek) who is the god of deceit, disguise and trickery and most clearly the western worldʼs figure of the trickster. Hermes, Maui, Loki, Anansi, Coyote and Monkey appear all over the globe in various guises but with an abiding similarity, their liminal ability to open doors and create disruption. Klaus-Peter Koepping notes that “two forms of action and thought seem to designate the trickster across all cultural 160 Gregor Cameron 198272670 16 36 http://www.imdb.com/title/tt0056751/ accessed 10/12/11, http://www.imdb.com/title/tt0436992/ accessed 10/12/11 37 h // di i l k/ / 7/d h / / 355296/d h d d l d d i 35 Doctor Who (2005) series six in 2011 episode 11 Original Air Date—17 September 2011 37 http://www.digitalspy.co.uk/ustv/s7/doctor-who/news/a355296/doctor-who-named-most-downloaded-us-itunes- show-over-modern-family.html accessed 10/12/11 Doctor Who (2005) series six in 2011 episode 11 Original Air Date—17 September 2011 36 http://www.imdb.com/title/tt0056751/ accessed 10/12/11, http://www.imdb.com/title/tt0436992/ accessed 10/12/11 37 htt // di it l k/ t / 7/d t h / / 355296/d t h d t d l d d it 37 http://www.digitalspy.co.uk/ustv/s7/doctor-who/news/a355296/doctor-who-named-most-downloaded-us-itunes- h d f il h l d 10/12/11 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance variations, namely, his cunning form of intelligence and the grotesqueness on the body imagery used to indicate the inversion of orderʼ (1985:194). Perhaps in Mr. Punchʼs case the grotesque body serves to hide the cunning in Mr. Punch s case the grotesque body serves to hide the cunning intelligence, as he traces his origins through that of Arlecchino. Koepping cites Elisabeth Frenzel who observes that the trickster/ fool figure acts as a mediator between audience and play, “ who on the one hand stands apart from the unfolding story but on the other hand is a part with which the audience identifies most closely: one delights in the stupidity of others but is also in the fool himself, which turns means a joke on the alter ego of the listener or viewer (1985:195). This mediation is a similar one to the Trickster as culture hero who mediates between gods and men (1985:206). This view seems intrinsically Carnivalesque, through the laughter underlines “the tension between identification and distancing” (1985:197). Koepping certainly allies himself with some of Bakhtinʼs theory as he comments about how the grotesque body is somehow similar to the liminal, “the body is always in a state of becoming” (1985:200). The trickster figure can be, as Barbara Morris suggests, seen in that modern manifestation of the carnivalesque screen in the corner of the living room. The television offers an engagement with the carnival but all too often more likely to be used as a form of pressure release rather than one of fomenting change, simply the anaesthetising spectacle or gossip provider. However Morris offers George Constanza, a character out of the Seinfeld34 situational comedy, as a trickster figure appealing to adults rather than children (1999). She suggests 161 that “television comedy has immense interest and importance; it is a seasonally fluctuating barometer of public controversies, fantasies, and disillusionments regarding social norms, values and human interests (1999:47). She sees George as a hero/trickster who is among the “unattractive and disturbing outsiders or misfits in their communities, who fumble clumsily and amusingly toward some semblance of control in powerless situations” (1999:49). One of the weaknesses of Morrisʼ argument is the seeming mortality of George Constanza. The trickster figure is an outsider not only of the community but also of humanity itself- sometimes an animal totem, sometimes a god. p g py show-over-modern-family.html accessed 10/12/11 p g py show-over-modern-family.html accessed 10/12/11 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance downloaded show in the United States, beating out a number of US produced shows. The show follows the adventures of a ʻTime Lordʼ, an alien who seems to have a preternatural interest in the Planet Earth. He is able to travel about in time and space thanks to his ʻTARDISʼ (Time And Relative Distance In Space) machine. In 1963 viewers were introduced to a crotchety old man known as ʻThe Doctorʼ played by actor William Hartnell. When his ill health threatened to end the series the producers came up with the idea of the Doctor being able to regenerate into a new body. Since then ten other actors have assumed the title role. In some other manner, such as in Cinema, web animation or through charity skits, a number of other actors have also contributed to the Doctor but are not generally considered part of the canon. With a mixture of history, science fiction, fantasy, horror, science and melodrama the show has been a fixture for nearly fifty years. The Doctor operates as a trickster/ hero because he is an alien and through a number of other aspects in the make up of the show, for instance, over the years there have been times when his control of where his Tardis materialises has been quite random or disordered. He is often shown to have vast knowledge but then also ignorant of the most surprisingly small details that require others to get him out of trouble. However wherever he does appear he becomes a catalyst for some form of action or change. He is almost always travelling with a human companion who becomes essential in the action or change that follows in the Doctorʼs wake. Again often while the Doctor is capable of messianic saves from danger, he is often shown to be in need of assistance from these companions or in need of action from the people MA Thesis: The Role of Misrule in the practice of Performance Even the clown figure separates itself from the human through mask or puppet, the simulacra standing in. that “television comedy has immense interest and importance; it is a seasonally fluctuating barometer of public controversies, fantasies, and disillusionments regarding social norms, values and human interests (1999:47). She sees George as a hero/trickster who is among the “unattractive and disturbing outsiders or misfits in their communities, who fumble clumsily and amusingly toward some semblance of control in powerless situations” (1999:49). One of the weaknesses of Morrisʼ argument is the seeming mortality of George Constanza. The trickster figure is an outsider not only of the community but also of humanity itself- sometimes an animal totem, sometimes a god. Even the clown figure separates itself from the human through mask or puppet, the simulacra standing in. Every time the Doctor gets pal-ly with someone I have this overwhelming urge to notify their next-of-kin. Every time the Doctor gets pal-ly with someone I have this overwhelming urge to notify their next-of-kin. - Rory (The God Complex)35 Perhaps a better example of the trickster figure in television is the character known as the Doctor36. Doctor Who is a science fiction series that has run in various forms (television, radio plays, audioCD, comics, novels, webisodes and fan fiction) since the day after Kennedyʼs assassination in 1963. It has lately become one of the BBCʼs flagship productions, which is winning favour not only at home but also in international markets, including the significant US market. ITunes37 recently named the 2011 season as the ʻtopʼ most 162 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Whereʼs Wally, a Northern Comedian, and recently a more familiar sort of New Romantic come nerdy “Bowties are cool” type of rebellious student or disruptive youth, one can see how Britton might connect the Doctor with Harlequin, and thereby make a connection with the Trickster. In his book, Matt Hills (2010) connects the Doctor to the Bakhtinian concept of the Chronotope. The chronotope, which Hills translates as Time/Space, links time with genre discourses, of which, he suggests, may be in turn seen as a manifestation of what Bakhtin calls ʻchivalric romance…ʼa miraculous world in adventure timeʼ (2010:103). Matt Hills wishes to place the Doctor into this adventure-time, because of the ʻfantastical worldʼ that surrounds him. The Doctor is both ʻat homeʼ and cut off from his ʻhome-worldʼ but lives in this space between, the marginal- a characteristic that he shares with the Trickster. The form of the genre of the Doctorʼs adventures, Hills names as ʻintimate epicʼ (2010). Hills goes on to make a case for the Doctorʼs meandering around space/time as “impulsive wandering represent[ing] the ʻnormal course of eventsʼ rather than an intrusion of disruptive forces” (2010:105). Thus he argues that the Doctor falls into adventure that just happens along rather than seeking trouble. In this he is similar to the trickster who is often reactive rather than pro-active, wanting to change something rather than create. Hills argues that this allows Doctor Who to be multi-generic. Thus it can be science fiction or historical, fantasy or horror, dramatic or melodramatic, social critique or imaginative. Generally stories involve “the intrusion of non-human forces” in Hills opinion and he questions whether Doctor Who ever enters into a political In his book, Matt Hills (2010) connects the Doctor to the Bakhtinian concept of the Chronotope. The chronotope, which Hills translates as Time/Space, links time with genre discourses, of which, he suggests, may be in turn seen as a manifestation of what Bakhtin calls ʻchivalric romance…ʼa miraculous world in adventure timeʼ (2010:103). Matt Hills wishes to place the Doctor into this adventure-time, because of the ʻfantastical worldʼ that surrounds him. The Doctor is both ʻat homeʼ and cut off from his ʻhome-worldʼ but lives in this space between, the marginal- a characteristic that he shares with the Trickster. MA Thesis: The Role of Misrule in the practice of Performance The form of the genre of the Doctorʼs adventures, Hills names as ʻintimate epicʼ (2010). Hills goes on to make a case for the Doctorʼs meandering around space/time as “impulsive wandering represent[ing] the ʻnormal course of eventsʼ rather than an intrusion of disruptive forces” (2010:105). Thus he argues that the Doctor falls into adventure that just happens along rather than seeking trouble. In this he is similar to the trickster who is often reactive rather than pro-active, wanting to change something rather than create. Hills argues that this allows Doctor Who to be multi-generic. Thus it can be science fiction or historical, fantasy or horror, dramatic or melodramatic, social critique or imaginative. Generally stories involve “the intrusion of non-human forces” in Hills opinion and he questions whether Doctor Who ever enters into a political Gregor Cameron 198272670 Gregor Cameron 198272670 163 populating the environment he finds himself in. Piers Britton (2011) ably traces what he refers to as a Harlequinade motif through both the television series and the various other manifestations of this meta-text - in the sense that the original text is both informed, judged and added to by subsequent texts, something Henry Jenkins covers in his book, Textual Poachers (1992). Jenkins particularly looks at the fan fiction of Doctor Who as meta-text (1992:162). Britton describes the design of Gallifrey, the Doctorʼs home planet, which is: a kind of cross between the House of Lords and the most conservative of cathedrals or Oxbridge colleges, its denizens preoccupied with ritual, etiquette and precedence, and correspondingly lacking in sensitivity or imagination. Individual charactersʼ names, too- such as Goth, Spandrell, Hilred and Runcible- suggest either the medieval world or a Trollopian parody of collegiate and Episcopal institutions. Overtones of British academia and clerisy are carried through into James Achersonʼs designs for the Time Lordsʼ dress. Their daily fatigues hover in form between cassock, surplice and dalmatic, while their ceremonial robes are grotesquely bloated variants of academic regalia: gowns with voluminous sleeves, skullcaps, and monstrous collars which somehow seem to synthesize aspects of the academic hood and mortarboard, (Britton, 2011:166-167) Thrown amongst this the Doctor, who has through his various incarnations been dressed as a feisty Victorian man, a Chaplinesque clown, a sixties/seventies Dandy, a cricket player, a Tatterdemalion, a form of cosmic 164 Gregor Cameron 198272670 165 agenda (2010:104). However this is the question that Alan McKee asks (2004). He acknowledges that Doctor Who is basically an Adventure Serial: The Doctor travels to planets with various forms of social organisation. He then has to choose: either to side with rebels who aim to overthrow that social structure; or to protect that social structure from attack. Usually if a civil war is in progress, he will favour the rebels; if an interplanetary incident is threatened, he will protect the culture he finds. (McKee, 2004:202) However McKee, writing about the politics of Doctor Who, notes the Doctor deals only “with the local, and the visible and the immediate” (2004:208). One of McKeeʼs survey respondents commented: “There is something about Doctor Who, which doesnʼt have connections with particular political persuasions- of the underdog, of cleverness and inventiveness winning out over brute force…. Itʼs so woolly, the program” (2004:214). All this seems to argue for the Doctor as a Trickster figure- uninvolved but disruptive, willing to rebel but also capable of protecting the status quo, acting as a go-between or irritating force, dealing with the immediate with an ambiguous relation with the bigger picture. In this [uncanny] sense, the Doctor himself also embodies this spectre- ʻthe ghost of Christmas pastʼ, as he says in “A Christmas Carol” (25 December 2010), and also of the present and of the future- a spirit of moral and actual rebirth, resurrection and regeneration. In this [uncanny] sense, the Doctor himself also embodies this spectre- ʻthe ghost of Christmas pastʼ, as he says in “A Christmas Carol” (25 December 2010), and also of the present and of the future- a spirit of moral and actual rebirth, resurrection and regeneration. (Charles, 2011:20) (Charles, 2011:20) 166 MA Thesis: The Role of Misrule in the practice of Performance Showrunner and writer Steven Moffatʼs latest Christmas edition of Doctor Who38, The Doctor, the Widow, and the Wardrobe offers a closer look at the Doctor as Trickster. Moffat has in the past written stories that reference other texts but here, as he was last year with A Christmas Carol referencing Dickens, he is clearly honouring the works of C.S. Lewis. During World War Two, the Doctor finds himself caring for a family whose mother he owes a favour to. He appears, and names himself, as Caretaker, one who does not do but rather takes care. p 39 Which relates directly with Matt Hills writing on how the humans often use simple tech to rescue themselves and the Doctor 38 http://www.imdb.com/title/tt1998643/ accessed 21/1/12 39 http://www.imdb.com/title/tt1998643/ accessed 21/1/12 Whi h l di l i h M Hill i i h h h f i l h h l d h Gregor Cameron 198272670 167 Doctor. 38 http://www.imdb.com/title/tt1998643/ accessed 21/1/12 39 Gregor Cameron 198272670 The story leads the Doctor to follow the children through a portal; a liminal space but not a wardrobe (the Doctor identifies his TARDIS as a wardrobe when pushed). Instead this portal is through a Christmas present opened too soon, Christmas Eve instead of Day. They find themselves lost in a wintry forest with the Doctor and eventually trapped within a tower. Meanwhile their mother follows them through, her reaction to the threat of danger to her children adds a level of reality and subsequently a situation with more jeopardy- they are on an alien planet of the future about to be harvested. What makes this really interesting is that the Doctor is pretty passive in this episode, taking care but not leading. That is left to Madge, the Mother, who ultimately comes to their rescue in a gigantic transporter tripod 39, digests the life force of the forest and then leads them all home, rescuing her children, the trees and, in true Who fashion, her dead husband. Thereby making Christmas come true for her family and, by ordering the Doctor to visit Rory and Amy, the Doctorʼs own Christmas wish. We can see in this that Moffat is not only 167 referencing Lewis but also, perhaps, James Cameronʼs Aliens40 and Kevin Andersonʼs Saga of the Seven Suns41. The point is that as Caregiver the Doctor is essentially powerless, like the Trickster it is his role to offer pathways to the humans he interacts with- to inspire them and to offer them choices. He ʻcaresʼ but does not lead. He is cut off yet dependent upon both humanity and the gods, in his case the Time Lords. His space/time is liminal, constantly changing. He has no place of his own but belongs everywhere. The Marginal figure effaces spatial boundaries in several distinct ways: (1) he is a vagabond who lives beyond all bounded communities and is not confined or linked to any designated space; (2) he lives in cells, caves, ghettos, and other ʻundergroundʼ areas- like the spider inhabiting the nooks and crannies of social spaces…. He lives above or below ground but not as normal mortals on the earth- ….with places of transition, movement, and license. Temporally and spatially, he tends to confound the distinction between illusion and reality, if not deny it altogether. 40 http://www.imdb.com/title/tt0090605/ accessed 21/1/12 41 htt // dfi /i d h ? ti t t& i t &l t bl &id 61&It id 210 40 http://www.imdb.com/title/tt0090605/ accessed 21/1/12 41 http://www wordfire com/index php?option com content&view category&layout blog&id 61&Itemid 210 accessed 41 http://www.wordfire.com/index.php?option=com_content&view=category&layout=blog&id=61&Itemid=210 accesse 21/1/12 http://www.imdb.com/title/tt0090605/ accessed 21/1/12 41 http://www.wordfire.com/index.php?option=com_content&view=category&layout=blog&id=61&Itemid=210 accessed 21/1/12 40 http://www.imdb.com/title/tt0090605/ accessed 21/1/12 41 http://www.wordfire.com/index.php?option=com_content&view=category&layout=blog&id=61&Itemid=210 accessed 21/1/12 40 http://www.imdb.com/title/tt0090605/ accessed 21/1/12 41 Gregor Cameron 198272670 In fact, he casts doubt on all preconceived and expected systems of distinction between behaviors and representation thereof. (Babcock-Abrahams, 1975:155) Trickster is ”at one and the same time, creator and destroyer, giver and negator, he who dupes and who is always duped himself” (1975:161). She also links Trickster to the work of Bakhtin and Kristeva with thoughts about how “such coexistence of opposites [are] “dialogic” (1975:161). Babcock- Abrahams offers a very good list of Trickster characteristics that are appended 168 MA Thesis: The Role of Misrule in the practice of Performance to this paper as appendix three. Christopher Vecsey considers Trickster stories as being “entertainment, education, a form of humourous rebellion. They can evaluate, explain and reflect upon realities, thereby making those realities clearer and more profound to the people who tell and hear the tales” (1981:161). Vecsey points out that the story teller in the Akan tradition of Anansi tells his listeners that the story is not true even before he begins (1981:169). Thus he can tell a fanciful story and the listeners will enter into the space of the story recognising that fantastic space- and within it magic can and will happen but that the trickster is not omnipotent for: “Anansi does not teach morals when he is victorious. It is when he fails that the Akan draw ethical conclusions” (1981:173). This is similar to the tension playing through Doctor Who. The Doctors failures are more telling than his successes, his reliance on the actions of his companions and other humans he comes into contact with is what defines his heroism and his trickster ego. The same can be said of Mr Punch. His tales are always about his failure to deal with the world about him, up to the scene of the hangman. Here is played out Punchʼs resurrection, his triumph over death itself, but it still comes with the cost of the Jack Ketchʼs life. With this reversal Judy comes back from the dead (as apparently so do all the other characters Punch has killed) and the story is reset- to be played again another time. The audience witnessing this must appreciate that this means the story is not true, they must decode the moral and ethical states without leaning on Punch as an example. Regeneration and transformation also underpin the Doctorʼs adventures. Matt Hills talks of this morality behind the Doctor, Gregor Cameron 198272670 169 particularly as the Doctor plays out his dealing with some of the monsters that populate his universe. Hills quotes a line from one of the 2009 series “the Poisoned Sky”: “I have to give them a choice” (2010:134). This is the Doctorʼs dilemma. Hills makes the point when talking how the genre of horror is used in Doctor Who. He recognises that instead of reactive horror where the human race is faced with an implacable nemesis that cannot be dealt with in any reasonable manner, the Doctor faces a progressive horror, one that has a whole history behind it to explain why it is set upon this path. The Doctor has to give them a choice; “progressive horror seeks to understand monstrosity, whilst reactionary horror uses stock ideas of good and evil” (2010:133). He cites Russell Davies here who has the word evil crop up in every script: “I send them back and write, ʻCome up with a better wordʼ. Words like good and evil stop all interesting debate. Thereʼs no such thing. People do terrible things and people do great things. None of itʼs a disembodied malevolent force.” (2010:134). This moral ambiguity, complex though it is, argues for the Doctor being a Trickster rather than simply a hero. Davies again, “…youʼve got to give the Doctor an appropriate thing to fight- if itʼs just ʻevilʼ, then all you have is a war, and the Doctor isnʼt much of a warrior.” (2010:135). Hills points to Daviesʼ “Doctor Who” as particularly playing the ʻpersona ex machinaʼ as his companions act, on the Doctors provocation to save the day (2010:39). By driving a lorry, driving a forklift over the bad guy at the cost of their life or by deliberately throwing themselves in front of traffic in order to correct the timeline, it is the human action that saves the day (2010:39). And this is not 170 MA Thesis: The Role of Misrule in the practice of Performance limited to the ʻnewʼ series as companions such as Adric in the original series made similar sacrifices. In Saint Punch the carnival finds a personification in the Punch puppet, the trickster onstage. It also has some reading in both the Gilles and Bluebeard stories. Judith courts the trickster and pays the price of the curious. Gregor Cameron 198272670 Somewhere between Gilles and Joan there is a moment of change between the Medieval and the Renaissance, between divine right and righteous divine. But why is this engagement with the trickster of such consequence? Perhaps it is that while Trickster appears all over the globe in any number of guises he is also a role that can be played off the stage. If the trickster has a role within the practice of the performative text, it is in the mediation between sense and nonsense. Todd May asks the question: “What is the relation between sense and nonsense?” (2005:108). He suggests that Deleuze sees it as: “Sense is the paradoxical element that resides in the proposition but is the attribute of things. Nonsense is the paradoxical element that circulates among language and things and brings them together.” (2010:108). This seems to be an essence of what Deleuze calls the “state of becoming” which for him is the state that our lives are in, rather than the state of being, which is often cited as the state we live within. For Deleuze living is more like a constant state of difference (2005:60). Difference acknowledges that rather than being in a state of stasis, life is never-ending change. We live in the liminal just as the trickster does. However this state is resisted, there is a clear interest in maintaining the situation, particularly if we see it as beneficial to us to stay put. Deleuze finds this way of thinking troublesome. However this becomes the space of the 171 carnival. Bakhtin might have suggested that the carnival and its personification is the place where the tensions between being and becoming can be examined. Trickster is never the same twice because he is so liminal but at the same time he appears so often in the status quo, the state of being, bringing disruption and movement to those who see the status quo as a trap where some are privileged and others placed under a yoke. Iʼm no prophet. My job is making windows where there were once walls- (Michael Foucault) (Hyde, 2008:283) (Hyde, 2008:283) As Artists, there is privilege to be living in the becoming. Artists live in the community, they often reflect or engage that community both from within and without the space of the theatre. MA Thesis: The Role of Misrule in the practice of Performance However it would be a mistake to say that these artists live outside society, however on the boundaries they might choose to explore. Their art is still firmly grounded in the state of human-ness, exploring what that is, collaborating with members of the society to extend what it is to be becoming human. They find their art by extending what has come before, either by acknowledging and extending or by defining it and turning away from well-established paths. Here the trickster lives, at the crossroads, between states, directing traffic. Here we can acknowledge the benefits enjoyed by those who have gone before and anticipate what might be found as we travel beyond what has gone before. Duchamp, Hyde tells us, acknowledges and explores these shadow-lands, seeking the news but at no time releasing his culturally received knowledge. His knowledge directs the meeting with chance as, for example, he places a canvas on the floor, adds paint to some metre long threads and from a Gregor Cameron 198272670 Artists must engage with the status quo but challenge its form as a place where nothing can change. Order in its ultimate expression becomes misrulic in that it kills all that seeks to challenge it; art, science, politics and religion all have a line to the state of becoming in their various ways but become barren in the face of ultimate Order. Perhaps this is the fate of the Thousand Year Reich, the Nazis entering a stage of stasis where nobody would be able to change, where stagnation becomes the only possible escape. 172 Becoming is unfinished, liminal. It is the motivation calling to misrule. But it is not destruction and chaos. Misrule lives beside order as a horse might live in harmony with its reins. When the artist takes on the mantle of the trickster they are calling on their own disruptive imagination. Lewis Hydeʼs book (2008) links 172 MA Thesis: The Role of Misrule in the practice of Performance this disruptive imagination to the trickster. He quotes Carl Andre: “Culture is something that is done to us. Art is something we do to culture” (2008:307). The trap here is for those who do not engage with imagining something different, ”who never sense the contradictions of their cultural inheritance run the risk of becoming little more than host bodies for stale gestures, metaphors, and received ideas, all the stereotypic likes and dislikes by which cultures perpetuate themselves.” (2008:307). Hyde cites the art and attitude of Marcel Duchamp, praising his “well oiled contradiction- like his use of chance, his addiction to puns, or his turn to mechanical drawing”- as a way of avoiding being finished, as engaging becoming, especially when that becoming is undefined. Gregor Cameron 198272670 173 deliberate height lets them flutter down to etch a ʻrandomʼ image upon the canvas. He called this piece Three Standard Stoppages (2008:122). He resisted logic by choosing chance, in the process creating a ʻnew-nessʼ. However Duchamp was still an accomplished artist, he understood what it was he was resisting. His art still resonated within a system- canvas, paint, hand with only the fall determining the final result being misrulic. And here we can see a return to the beginning of this paper as a work of art arises out of a system, that without a structure there is no definition, no possibility of a ʻnewʼ arising, it needs to know what it is not. Again this seems quite carnivalesque and lies within the realm of the trickster. deliberate height lets them flutter down to etch a ʻrandomʼ image upon the canvas. He called this piece Three Standard Stoppages (2008:122). He resisted logic by choosing chance, in the process creating a ʻnew-nessʼ. However Duchamp was still an accomplished artist, he understood what it was he was resisting. His art still resonated within a system- canvas, paint, hand with only the fall determining the final result being misrulic. And here we can see a return to the beginning of this paper as a work of art arises out of a system, that without a structure there is no definition, no possibility of a ʻnewʼ arising, it needs to know what it is not. Again this seems quite carnivalesque and lies within the realm of the trickster. In the theatre art is realised by collaboration. This working together is often framed by a vision and most often through the mechanism of a script. In this it shares a form with both Film and Television. The vision is an unfinished idea of what the art might say to its receivers. It should be open-ended so that it can be improved by what is offered by the collaborators. In Saint Punch there was a moment where misrule intrinsically changed what was to be presented and lead to the work being improved. The action of scene four was most challenging for all the people involved. Gilles has broken into Joanʼs prison in 1431, just prior to her execution. She is nineteen. Joan has recanted and feels abandoned by her voices. Gregor Cameron 198272670 Gregor Cameron 198272670 Added to that she has had to fight off some attempts to rape her by English soldiers. Gillesʼ arrival sets her mind on 174 MA Thesis: The Role of Misrule in the practice of Performance another path. She will die a French Woman, her virginity taken by a countryman rather than her captors. With collaboration there is also the need to manage the group that is approaching the text. Clearly performed rape presents a certain number of problems. It was the intention of the written script that the rapes are played out in front of the audience- quite unflinchingly offering the theatre of cruelty. However that would require quite a lot from the actors who are going to be interpreting this. With this particular performance the actors experience ranged from the professional through to some who had not been on stage before. Of the two directly involved in this scene, one was an experienced professional, and the other a first year theatre student with some experience. In fact she was only just turning eighteen making her younger than the character she was portraying. Naturally this brought up a number of challenges around keeping her safe and yet still gaining from the scene the reaction the piece was looking for from the audience. Figure 11. The image in front of the rape Figure 11. The image in front of the rape Gregor Cameron 198272670 175 As written about earlier in rehearsal the group approached these scenes very carefully- making sure that there were others present- offered in support to the actor portraying Joan. The workshop performance was really blessed with her entering so generously into this ʻworldʼ. She was quite nervous about the scenes. However she displayed a real sense of courage and commitment in her willingness to engage with it as well. Still rehearsals were proceeding very slowly as she approached the material. Then in one rehearsal talk arose about how the camera affects what it is that the audience is allowed to see, how the camera has a way of distancing the actors from the audience and in a way that this paralleled the affect that arises from the Puppet booth- after all the figures are all approximately the same size. At some point the offer arrived from the actors of what would happen if we were to not allow the audience to see anything. MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance clearly the camera is still operating yet we are still, in effect, prevented from seeing the rape. This forces the audiences imagination to fill in the blank space more directly- and with less interference. Important also is the reading they can take from this card- which appears quite a lot in sixties dramas, where the threat of atomic attack means the TV goes off and people are advised to “duck and cover”. If they are sufficiently armed with cultural baggage to pick up this intertext then the awfulness of the scene behind it is underlined. As with all intertexts it does not require interpretation but offers enrichment to those who recognise it. Those who do not have prior experience are still affected by this quite rational request, set against the sounds coming from behind it- the sound of which becomes that much more awful as their imagination is engaged. It was a deliberate choice to deepen this moment in response to the ʻhappy accidentʼ that arose out of the rehearsal process. Gregor Cameron 198272670 They would still be able to hear everything. This was very intriguing since it was in opposition to what had been scripted. However, such moments are to be explored and if successful a script can be adapted, one of the benefits of having writer and director the same person. One of the problems of turning off the camera, however in effect is that while the voices could be heard there was a chance that the audience might worry that a technical hitch was happening, preventing them from seeing the scene. This could result in them being distracted from the intention experience. This problem was solved by the television card, which said PLEASE STAND BY- 176 Circling the Question of Misrule Here is the difficult phase of the argument. In setting up the system of the rehearsal room in the way that I did, was there sufficient order and chaos to produce conditions that might manifest a spontaneous creative moment? Was the offer from the actors a moment of misrule? Did the decision to say yes to this offer, change the intention of the scene or lead, through the use of the TV card, to an enrichment of the experience that the audience had. I believe so. Meaningful theater experiences do shine light in the dark places of the soul. To engage catharsis it is necessary to be sensitive to where the 177 177 dark places are to be found at any particular moment. And this demands sensitivity to context. (Bogart, 2007: 11) Anne Bogart has offered me insight into process in her writings on both acting and directing. She very neatly places what I call misrule as an opportunity that arises from preparation. She makes it clear that all of the information and work which is done before and around rehearsals informs the possibilities that will arise when you need them- solutions arising from the context of the project you are engaged with. She suggests, in fact, that practitioners should celebrate accidents and quotes Robert Altman “On the film set I create the circumstances in which these accidents can happen” (2007:48) It is not the directorʼs responsibility to produce results but, rather, to create the circumstances in which something might happen. The results come about by themselves. With one hand firmly on the specifics and one hand reaching into the unknown, you start to work. (Bogart, 2001: 124) In The Viewpoints Book she addresses the framework within which creativity can be enhanced through this step beyond. ʻViewpointsʼ offers a system by which a company may enter into a formal style of improvisation, thus opening the dialectic of the rehearsal room. Dialectic in this sense is a search for the creative moment, through collaboration, community and through reflection. Such collaboration establishes a formality that can expose the moments of inspiration that Lewis Hyde identifies as being the province of the Trickster, manifesting its presence into the room. Circling the Question of Misrule 178 MA Thesis: The Role of Misrule in the practice of Performance Similarly Bella Merlin makes it clear that while Stanislavsky developed a system it was never closed; he was constantly in a process of developing. She says, “Stanislavsky never intended his ʻsystemʼ to be gospel. If your process ainʼt broken, thereʼs no need to fix it. If the Muse descends upon you, celebrate that visitation, and donʼt overlay it with conscious intervention” (2007:12). Merlin continues to talk about other practitionersʼ definition of this moment; David Mametʼs “terrifying unforeseen”, Michael Chekovʼs “quality of ease” (darkest tragedy with lightness of touch), Jerzy Grotowskiʼs “via negative” (elimination of blocks between inner impulse and outer expression) (2007:13). She seems to be suggesting that strict adherence to any path might be constraining the creative moment and require the practitioner to consider how what they wish to accomplish might be enlivened by being open to the misrulic moment. MA Thesis: The Role of Misrule in the practice of Performance Similarly Bella Merlin makes it clear that while Stanislavsky developed a system it was never closed; he was constantly in a process of developing. She says, “Stanislavsky never intended his ʻsystemʼ to be gospel. If your process ainʼt broken, thereʼs no need to fix it. If the Muse descends upon you, celebrate that visitation, and donʼt overlay it with conscious intervention” (2007:12). Beyond a certain point, theatre cannot be controlled. That is to say, neither should actors be controlled, nor should they be wholly in control. Competent actors make things happen. Good actors let things happen. Thatʼs why theatre always has- or should have- the potential for danger, for the unpredictable. A performance should be a disciplined improvisation. Beyond a certain point, theatre cannot be controlled. That is to say, neither should actors be controlled, nor should they be wholly in control. Competent actors make things happen. Good actors let things happen. Thatʼs why theatre always has- or should have- the potential for danger, for the unpredictable. A performance should be a disciplined improvisation. Competent actors make things happen. Good actors let things happen. (Alfreds, 2007:344) Mike Alfreds makes this point at the end of his book, which is quite actor- centric and provides a very tight set of tools or system for them. He too, however, clearly understands that any performance is spiced up with a little misrule. Circling the Question of Misrule Alison Hodge in her examination of actor training strengthens this position by making it clear that a ʻsystemʼ has been rather superseded by practitioners referring to a set of first principles arising out of their own Gregor Cameron 198272670 179 particular contexts. She suggests that such first principles are transcending their origins and forming a sort of matrix of contemporary actor training (2010:xxv). She cites Copeau, Brecht, Strasberg and Mnouchkine as each having principles that demand attending to, but not constraint by. So whether it is through seeking the ʻdramatic principle in oneselfʼ, observing and ʻinterrogating the social forces at playʼ, creating out of himself by an ʻappeal to the unconscious and the subconsciousʼ or by finding a ʻcommitment to collective and communityʼ the practitioner is seeking to bring together the forces of creativity (Hodge, 2010:xxv). Chris Baldwin and Tina Bicât argue that in devised theatre the baseline of a text may not exist. In which case the company is forced to seek a new way of interacting and sharing “Everybody involved in a devising project needs not only to be conscious of their artistic brief and responsibilities, but also sensitive to the dynamics of intense collaborative group work” (Bicât, 2002:7). They argue that devised work depends heavily upon ideas and chance discoveries (Bicât, 2002:9). Within their book lie a number of strategies for managing the process of devising and improvising. particular contexts. She suggests that such first principles are transcending their origins and forming a sort of matrix of contemporary actor training (2010:xxv). She cites Copeau, Brecht, Strasberg and Mnouchkine as each having principles that demand attending to, but not constraint by. So whether it is through seeking the ʻdramatic principle in oneselfʼ, observing and ʻinterrogating the social forces at playʼ, creating out of himself by an ʻappeal to the unconscious and the subconsciousʼ or by finding a ʻcommitment to collective and communityʼ the practitioner is seeking to bring together the forces of creativity (Hodge, 2010:xxv). Chris Baldwin and Tina Bicât argue that in devised theatre the baseline of a text may not exist. In which case the company is forced to seek a new way of interacting and sharing “Everybody involved in a devising project needs not only to be conscious of their artistic brief and responsibilities, but also sensitive to the dynamics of intense collaborative group work” (Bicât, 2002:7). They argue that devised work depends heavily upon ideas and chance discoveries (Bicât, 2002:9). Within their book lie a number of strategies for managing the process of devising and improvising. Gregor Cameron 198272670 There is an important difference between improvisation as a tool of the rehearsal room and its use upon the stage. Improvisation is like a loaded gun, quite safe if used judiciously and carefully. Improvisation in and of itself can be used as performance but often this does not lead to the depth of significance and meaning that is required by storyteller and audience. As a tool it has 180 MA Thesis: The Role of Misrule in the practice of Performance enormous usefulness however. In the rehearsal room it allows a number of situations to be explored. It can provide a way of looking outside the text at the relationships between the characters. It can provide opportunity to explore the space and the movement of the text and sometimes highlight the shortcomings of the written text. It is also a tool where failure is allowed and encouraged because of the things that can be experienced and learned. It encourages that sense of spontaneity that is so important during performance and allows actors to remain fresh. It is great for building trust and community amongst the cast. However it also has a dark side. The problem with improvisation, as hinted at above, is one of order and control. Without a strong vision or system improvisation can become superficial and veer off into unknown and, largely, unusable material. Baldwin and Bicât leaven their invitation to improvise with the following caveat: The prolific imaginations of the company, if well managed and focused, can be transformed from chaos into rich, elegant and often deeply resonant productions and performances for an audience. can be transformed from chaos into rich, elegant and often deeply resonant productions and performances for an audience. (Bicât, & Baldwin 2002:9) Gregor Cameron 198272670 181 The key here is management. If a group is using a written text then it will form a focus and a source to come back to even if it is a work in progress, as Saint Punch was. All of the work in the rehearsal room was structured towards examining and mining the text for the moments onstage where the audience would be invited to interrogate their own ideas about humour, violence and storytelling. Gregor Cameron 198272670 If the original idea was the cruelty of putting the violence right out in front of them then the idea of removing it is radical to the intention but solves a difficulty and enhances the moment because it engages the audience to 181 work. This process is what I call misrule, the moment when the trickster enters the carnivalic space and offers a new solution, a choice to the creative process. We will all experience, if we have not already done so, that terrific sensation of a breakthrough. We have all sat in rehearsal for a few hours with a problem that just will not go away. Then suddenly you have a fantastic idea, which not only solves the problem but will give the audience the biggest surprise of their lives. Judith Weston, writing about directing actors in film, offers the problem of the obligation- particularly the obligation to be right. The struggle of the actor already out there on a public space, albeit stage or screen, is enormous. To make choices and have those choices appear fresh places great burden upon their shoulders. Weston suggests that the directorʼs role be one of allowing them to be wrong, in essence to seek to be free from obligation (Weston, 1996:68). Then in the moment the actor can use what comes up rather than feel they must suppress or try to ignore the moment- both of which will lead to a poorer performance. Weston also suggests that freedom allows humour. “A performance without freedom is a humorless one. Anytime in real life that people are loose and free, humor is bound to bubble up. Nothing is less entertaining than an actor who is straining to be funny” (Weston, 1996:69) Judith Weston, writing about directing actors in film, offers the problem of the obligation- particularly the obligation to be right. The struggle of the actor already out there on a public space, albeit stage or screen, is enormous. To make choices and have those choices appear fresh places great burden upon their shoulders. Weston suggests that the directorʼs role be one of allowing them to be wrong, in essence to seek to be free from obligation (Weston, 1996:68). Then in the moment the actor can use what comes up rather than feel they must suppress or try to ignore the moment- both of which will lead to a poorer performance. Gregor Cameron 198272670 Weston also suggests that freedom allows humour. “A performance without freedom is a humorless one. Anytime in real life that people are loose and free, humor is bound to bubble up. Nothing is less entertaining than an actor who is straining to be funny” (Weston, 1996:69) Of course the other side of this is that the director or dramaturge must be the managing power. In a sense they must be the ordering agent as well as the 182 MA Thesis: The Role of Misrule in the practice of Performance trickster. It is their role to take the offers given up during rehearsal or improvisation and to compose them into the finished product. This composition is the process of editing out or taking advantage of the ideas and opportunities that arises within the rehearsal period. It seems to me to be quite a carnivalic process, as the interrogation of the status quo needs this editorial function to make change or to sustain the way things are. Change need not be revolutionary unless there has been a sustained period of non-reflexivity. In fact change, as Deleuze suggests is perhaps the only natural state, ʻbecomingʼ, even if it is not a state most of us, often more comfortable with being, wish to acknowledge. This is a key point within the problem as it struggles between intentionality and the spontaneous moment of performance. Composition is the final step before the performed moment. But even as it is composed it is unfinished. It is ephemeral because the next time a piece of theatre is performed it may be subtly different. If a different group chooses to perform a text it will almost certainly be different. Composition arises from the choices made by the company or through the vision of a director or dramaturge. In Saint Punch a number of ideas were developed, and either used, or let go. The Rehearsal room became a place where the best parts were allowed air to breathe and subsequently was available to the audience but there was also an amount of material that was not going in the right direction or which were not helping the overall impact of the text and thus were left behind. These decisions were mine –both good and bad decisions and it is very gratifying to look back and be able to say that I am proud of the work. Gregor Cameron 198272670 I temper this with an acute 183 awareness of some of the things which have provided the best learning, the things that did not work- most of which were not intentional and were an important part of my learning curve. Sacred/ Profane In his book, The Moving Body, Jacques Lecoq offers the Bouffon as a form for actor training. His philosophy, arising out of the form and function of the body, suggests that investigating the world through the three Bouffon characters- mystery, the grotesque and the fantastic- can lead his students, and by implication their performances, into the realm of the carnivalesque. He expresses this by talking of the Kings foolʼs license. He suggests that this gives the Bouffon permission “to express truth in all its forms” (Lecoq, In his book, The Moving Body, Jacques Lecoq offers the Bouffon as a form for actor training. His philosophy, arising out of the form and function of the body, suggests that investigating the world through the three Bouffon characters- mystery, the grotesque and the fantastic- can lead his students, and by implication their performances, into the realm of the carnivalesque. He expresses this by talking of the Kings foolʼs license. He suggests that this gives the Bouffon permission “to express truth in all its forms” (Lecoq, 2002:118). He goes on to talk about the power of laughter, how those who are willing to laugh at even the most sacred of things were able to enter a space in which the mystery of things could be interrogated. 2002:118). He goes on to talk about the power of laughter, how those who are willing to laugh at even the most sacred of things were able to enter a space in which the mystery of things could be interrogated. Bouffons deal essentially with the social dimension of human relations, showing up their absurdities. They also deal with hierarchies of power and its reversal. (Lecoq, 2002:119) 184 Lecoqʼs project is rooted deeply within the twentieth century and thus his Bouffons are part of the conversation between the spirit and reason. His mystery gang, as he calls them, are connected to the spiritual, his fantastic gang reflect the scientific and the grotesques deal with power and hierarchy. It does not seem much of a stretch to see the carnivalesque made manifest MA Thesis: The Role of Misrule in the practice of Performance here. Tom McCrory, a pupil in the Lecoq School, points this out very clearly and indicates how each of the people entering into the process of rehearsal is similarly seeking to illuminate the sacred by exploring the profane. This is the project of the Bouffon. Sacred/ Profane Bouffons enjoy themselves, for their whole life is spent having fun imitating aspects of human life. Their great delight is to make war, fight, tear out each otherʼs guts. However, their war games never follow the logical chronology of a story that unfolds. They bring a special style of composition: they so enjoy killing one another that with each killing they want to start again. So they indulge in repeated mutual massacres just for the fun of it. (Lecoq, 2002:118) This play, so reminiscent of childhood, is at the core of what Lecoq says is the second year of training for his students. This is deliberate. Lecoq insists that this liminal state allows for experimentation to include both the failures and the successes- the failed choices to inform the development of the successes that will be composed into performance. This is such a misrulic system- it foregrounds play and allows for a great deal of creativity. In an industry where money is the motivator this would be an anathema to those seeking to reduce risk. In this space composition overwhelms play, perhaps to its own detriment. Lecoq finishes his second year by inviting his students to find their own clown. This is highly individual work but still about the space the performer enters, it is still about the dialectic between performer and spectator, one leading the other and, in turn, are themselves led. Gregor Cameron 198272670 185 In this way the dialogue whether expressed as movement and mime or through inviting the audience to watch a lifeʼs narrative played out before them, is all about seeking something beyond ourselves, even as we exist knee-deep in muck. It is about seeking the sacred through the profane, the grotesque body offering us insight by doing or saying that which we cannot. Saint Punch offered up this challenge, to engage in what it means to see violence so mediated it becomes the subject of humour. For this is what it is about, suggesting that Gilles de Raisʼ excesses are not so different from those of either Bluebeard or Mr. Punch. It is not at all plain whether this intention was able to bear fruit. The audience, while invited to reflect, may not have left the theatre with these thoughts clearly in their heads. Sacred/ Profane However it would be true to say that by being exposed to both entertainment and the profane performance of violence they were at least provided with the opportunity for reflection. This brings up a very interesting question about what is it that the artist through their art is supposed to expect of demand of their audience. If the artistic moment is dialectic, then nothing is mandated but if the art is to take a transformative position then it is expected to propose change within the space of the carnivalesque. In consideration of the role of the trickster, Lewis Hyde suggests that the artistʼs role is to linger in the liminal, the border, the joint. He suggests that the artist is cast into the space of the trickster able to see how things are but at the same time offering a glimpse across the doorway into what may be. In a sense he sees the trickster in a prophetic role, not the fortuneteller but rather as 186 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance someone who in witnessing the present offers some account of where the future might be. Clearly sitting between the sacred ʻillusion and the profane “reality” (Hyde, 2008:300). Thus the artist in seeing the real but opening up the possibility invites their spectators to escape the mundane and to fly beyond the horizon. There have been a number of comments from the audience attending Saint Punch that anecdotally suggest that while an entertaining play was experienced, it stayed with them after they left the theatre and some have spoken of their need to consider what it was that was put in front of them. It may be that at least for some people it completed what was the thematic intention; while for others they just had an intriguing night at the theatre, which may have been enjoyable or not depending on their mood. And this then raises a question about what is the artistʼs responsibility? The Trickster and the carnivalesque are all about opportunity, but they do not mandate participation. The artist offers and the dialectic allows the spectators to be responsible for their own willing activity within the liminal space. Sacred/ Profane It does not mean you can not make the leading obvious but, if the spectators participation demands some work on their part, then it is possible the reward may be greater and the change in the borders of the mundane more stable. As has been discussed before radical change is not the end intention of either the carnivalesque or the trickster, but rather that the status quo grow to encompass that which is different. That, in Deleuzeʼs sense, the becoming moves on, never satisfied just to be, seeking to defeat the static, motionless indifference of the stationary moment. The artist offers and the dialectic allows the spectators to be responsible for their own willing activity within the liminal space. It does not mean you can not make the leading obvious but, if the spectators participation demands some work on their part, then it is possible the reward may be greater and the change in the borders of the mundane more stable. As has been discussed before radical change is not the end intention of either the carnivalesque or the trickster, but rather that the status quo grow to encompass that which is different. That, in Deleuzeʼs sense, the becoming moves on, never satisfied just to be, seeking to defeat the static, motionless indifference of the stationary moment. Gregor Cameron 198272670 187 Saint Punch, then, sought to engage this idea of mediation, in particular the mediation of violence, and challenge the audienceʼs attitude to their own response to it. As Gilles picks up the girl, throws her against the table, parts her legs and thrusts, we hear the crying baby with Punch singing just prior to the baby being flung out onto the stage and as Bluebeard chucks his wife around, there is a moment where the spectators should pause and take a breath as the play begins to loosen the pins of laughter and dread in equal measure. The audience must respond to their own culpability in watching this horrendous group of violent offenders. Their position on this their own affair, it is not the artʼs responsibility to tell them how they should respond, only to try and elicit a response. Thus it is, for both the carnivalesque and the trickster, the invitation and the opportunity is there, to be taken up or not. In seeing the grotesque and the profane before them they are granted access to a sacred truth that they might discover, but it is no individuals single vision, and not within the artistʼs ability to proscribe- they have to ʻbecomeʼ on their own. 188 MA Thesis: The Role of Misrule in the practice of Performance Conclusion As Joseph Campbell has said: “Where you stumble, there you shall find your treasure.” We invite the stumbling. We hope maybe to have indicated a path but not cleared it, leaving you to work through the most thorny areas. (Bogart and Landau, 2005:xi) This thesis began with a challenge that the objects of culture, and perhaps, cultural theory, should not just make statements about what it observes but also should be seen as calls to ʻdoʼ something, to redraw the borders of ourselves and our lives. In order to do this I have explored what Bakhtin defined as the carnival, harking back to the spectacle and ritual that offered the medieval mind opportunity to take a couple of days to dream about how things might be. Bakhtinʼs theory surrounding the carnivalesque is a very attractive frame for the work that goes on in the theatre, even though he located it in the novel. Others have been left to carry on his project in how it might apply to objects and situations outside, such as the theatrical spectacle. However, we should be reminded that the carnival itself can be both constative and performative in that it can either support the status quo, or work to interrogate it and offer itʼs redrawing. Gregor Cameron 198272670 189 It is important to see the carnivalesque as a frame, a function rather than a lens or a particular reading of a work. It facilitates but does not in itself colour the product of the theatre. There are plenty of more political movements that can be drawn into the carnivalesque space that can and will encourage particular readings of works, think feminist or post-colonial lenses for example. 189 If it does become an agent it is, perhaps, through the character of the trickster. This personification as agency can and will provoke action and therefore becomes integral to the reading of the event. Saint Punch, which can be read as a critique on the mediatization of violence, is a play in which the frame of the carnival is consciously used. There are also very clear motifs of the Dark Clown in the character of Mr. Punch. He is a reflection of both Bluebeard and Gilles De Rais. In a way it is this permission to play out the politic message that interests me. MA Thesis: The Role of Misrule in the practice of Performance On the other hand there are also questions around how clear and/or successful these intentions were. However, here the words of Lewis Hyde become quite potent. Hyde suggests that the traditional culture hero, such as Hermes, Loki, Maui and Monkey do not demand from the humans, whose lives they enter, should just take up the opportunities they offer. Hyde makes the point that the trickster is not about mandating a response in the way that a system might. Rather, the trickster, a liminal figure, hangs out on the border, in the doorway, or in the joints, facilitating the travelersʼ traverse of these points. Hyde likens the role of the artist to the tricksterʼs disruptive imagination- that art itself holds no power other than what we give it. The artist offers the art but the spectator must be willing to interact with it. It is not the artists, as it is not the tricksterʼs, responsibility to mandate what reaction there is to any action Conclusion It is this function that makes theatre and performance central to how I process my world. I want a show to entertain and educate me. Cultures are most fully expressed in and made conscious of themselves in their ritual and theatrical performances. […] A performance is a dialectic of “flow”, that is, spontaneous movement in which action and awareness are one, and ʻreflexivity”, in which the central meanings, values and goals of a culture are seen “in action”, as they shape and explain behaviour. (Victor Turner, cited in Schechner, 2006:19) (Victor Turner, cited in Schechner, 2006:19) In the process of offering an exegesis a number of reflections of Schechnerʼs points appear. Firstly using a traditional story such as Bluebeard and the traditional performance that is Punch and Judy clearly offers entertainment. While much of the show is Grand Guignol inspired the point is made that by looking at the profane is simply a way of inviting the audience to consider the sacred, a beautiful thing. Saint Punch in suggesting that the main character of 190 MA Thesis: The Role of Misrule in the practice of Performance each story is, subject to mediation, essentially the same character, it seeks to mark and ring the changes in identity through history and mediation. With its beginnings, middle and end of process so clearly owing much to cast, crew, audience, tradition and mentorship a community is acknowledged- it takes a village to bring up a [child]? It is the hope of the players/ author that, through consideration of what we are still tolerant of in our ʻentertainmentʼ, there is an opportunity to heal our society of its (de)regulation of violence in all its many manifestations. That this very process of transparent mediation can offer the audience conscious understanding of what they can learn about their own behaviours. This arises out of the direct observation of both the profane and the sacred. At least that is the intention of the show. Schechnerʼs seven functions of performance also support this idea (Schechner, 2006:46). Gregor Cameron 198272670 191 that they enter into. It is a project of thinking that art invites the spectator into and what they gather there and take away with them is their own responsibility. As part of this, the thesis offers the art itself, in the form of a video recording of the show on two different nights and some ancillary extras on a DVD, along with the performance script. In part this is to provide access to the art that is central to this project but also to take a measure of where this project began over a decade ago and where it has ended up as performance. This is where the question of misrule arises. I have used this term to look at how this element entered the process of bringing Saint Punch to the stage. Undoubtedly the preparation around the context of the historical and fictional worlds of the stories in Saint Punch provided a framework, which any negotiation of meaning might be found. I have written about the second rape as a defining moment. The momentary ʻmisrulicʼ offer on the rehearsal floor was such a wonderful opportunity. Acknowledging the ʻhappy accidentʼ and accepting that what you hide from view can be more affecting than what you show, exposed meaning within the performed text that was not implicit in the written script. Therefore I would argue that misrule supported the moment I am highlighting in my play rather than leading to more difficulty. Change and ʻbecomingʼ are infused into the performed play by the attitude of both being prepared and yet open to the opportunities of the rehearsal floor. By taking an attitude of acceptance to 192 MA Thesis: The Role of Misrule in the practice of Performance what could happen, it allowed what did happen to bear significant fruit in the final performance. The carnival and the trickster are the harbingers of misrule. By harnessing their energies and opportunities the artist encounters an otherness that expands his experience of the world. It is this he offers to the world. The world in turn then decides what to do with the offer. The PLEASE STAND BY card is read by the audience informed by their previous cultural encounters, whether they be broken TV signals or precursors to the end of the sixties world. Gregor Cameron 198272670 It is not the shows responsibility to make this sign clear to them; they must do the work themselves as part of a community that endeavours together. This engagement with the trickster is an ongoing one and this work offers the Doctor as an example. By citing the Doctor and his place in the cultural literacy of those who know him an offer is made to consider the trickster as a continuing cultural hero and still a part of making our culture who it is long after its mythical formation might be aid to be long dead and buried. By using the Doctor as an exemplar it is hoped that one point is underlined- as Hyde suggests using the Hermetic Hymn as an example- that it is not the tricksters role to destroy the native system but rather to redraw its borders. That the Doctor/Trickster is travelling both “in the light taking things apart and back into the dark to stitch them back together again” (Hyde, 2008:309). In this way it is hoped that the reader will Gregor Cameron 198272670 42 Comments made during a three-weekend workshop held by Sarkies in Wellington in 2010. Author was in attendance at the Film School venue. see http://www.imdb.com/name/nm0765291/ accessed 28/2/12 G C 982 26 42 Comments made during a three-weekend workshop held by Sarkies in Wellington in 2010. Author was in attendance at the Film School venue. see http://www.imdb.com/name/nm0765291/ accessed 28/2/12 Gregor Cameron 198272670 193 engage in the frame of the sacred/profane nature of the carnival and its chief player, the trickster. There are still questions to engage with, of course. This thesis is not exhaustive on the topic of the cultural significance of the trickster or of the ongoing project of Bakhtinʼs Carnival. Neither does it judge the effectiveness of the theme of the mediation of violence that Saint Punch indicates. Instead it offers to entertain the idea of a community in which an artist points to something that they hope will have meaning to its spectators. It is offered without expectation, but in the spirit of the trickster, is a doorway towards a new way of perceiving the world. It asks a question to which there can only be an ongoing answer, just as culture can only be an ongoing definition. 194 Having said that, this thesis does engage with the object, Saint Punch. Hopefully by acknowledging the subjectivity of the artist, but also bringing to it a more formal eye, the performance, within its frame, is adequately defined and described. It is the subject of this thesis because of its conscious carnivalesque frame but also because that consciousness has enabled a particular element of the creative process to be named as misrule. If misrule is not simply an accident but actually an identifiable element that has been encountered by other practitioners, then this thesis has achieved its end. If not, then perhaps misrule belongs to the dream, a weak and idle theme. It is this 194 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance theme that gently allows systems to remain open to the creative moment- echoing Altman, Kiwi Director Rob Sarkies has admitted that if his cast and crew become too happy he is all too willing to throw a little ʻwake up callʼ their way42. By drawing these elements and frames together we seek to restore to the community itʼs ability to see beyond itself. I believe it is a need that artists acknowledge and seek in disrupting the creative imagination. And this is where I come stumbling in. By choosing to use a script I stumbled through the writing of, by stumbling through the experience of directing and by stumbling along with a group of strangers to the Laboratory presentation of this performance, I have become immersed in Joseph Campbellʼs stumble. 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Idle, Eric. The Road to Mars: A Post-Modern Novel. 1st Ed. New York: Pantheon Books, 1999. Print. Jenkins, Henry. Textual Poachers: Television Fans & Participatory Culture. Studies in Culture and Communication. New York: Routledge, 1992. Print. Knowles, Ronald Shakespeare and Carnival: After Bakhtin. Basingstoke, Hampshire: Macmillan Press; New York: St. Martin's Press, 1998. Print. Koepping, Klaus-Peter. "Absurdity and Hidden Truth: Cunning Intelligence and Grotesque Body Images as Manifestations of the Trickster." History of Religions 24.3 (1985): 191-214. Print. Lecoq, J., et al. The Moving Body: Teaching Creative Theatre. Routledge, 2002. Print. Liddo A D Alan Moore: Comics as Performance Fiction as Scalpel MA Thesis: The Role of Misrule in the practice of Performance Heathcote, D., L. Johnson, and C. O'Neill. Collected Writings on Education and Drama. Northwestern University Press, 1991. Print. Hills, Matt. How to Do Things with Cultural Theory. London: Hodder Education, 2005. Print. Hills, Matt. Triumph of a Time Lord : Regenerating Doctor Who in the Twenty-First Century. London; New York: I.B. Tauris: Distributed in the United States by Palgrave Macmillan, 2010 Hodge, Alison. Actor Training. 2nd ed. MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Schechner, Richard. Performance Studies: An Introduction. 2 Ed. New York: Routledge, 2006. Print. Schirato, T., and S. Yell. Communication and Culture: An Introduction. Sage Publications, 2000. Print. Danow, David. The Spirit of Carnival: Magical Realism and the London; New York, NY: Routledge, 2010. Print. Hyde, L. Trickster Makes This World. Canongate Books Ltd., 2008. Print. Idle, Eric. The Road to Mars: A Post-Modern Novel. 1st Ed. New York: Pantheon Books, 1999. Print. Jenkins, Henry. Textual Poachers: Television Fans & Participatory Culture. Studies in Culture and Communication. New York: Routledge, 1992. Print. Jenkins, Henry. Textual Poachers: Television Fans & Participatory Culture. Studies in Culture and Communication. New York: Routledge, 1992. Print. Knowles, Ronald Shakespeare and Carnival: After Bakhtin. Basingstoke, Hampshire: Macmillan Press; New York: St. Martin's Press, 1998. Print. Koepping, Klaus-Peter. "Absurdity and Hidden Truth: Cunning Intelligence and Grotesque Body Images as Manifestations of the Trickster." History of Religions 24.3 (1985): 191-214. Print. Koepping, Klaus-Peter. "Absurdity and Hidden Truth: Cunning Intelligence and Grotesque Body Images as Manifestations of the Trickster." History of Religions 24.3 (1985): 191-214. Print. Lecoq, J., et al. The Moving Body: Teaching Creative Theatre. Routledge, 2002. Print. Lecoq, J., et al. The Moving Body: Teaching Creative Theatre. Routledge, 2002. Print. Gregor Cameron 198272670 Liddo, A. D. Alan Moore: Comics as Performance, Fiction as Scalpel. University Press of Mississippi, 2009. Print. 199 Lokke, Kari E. "Bluebeard" and "the Bloody Chamber": The Grotesque of Self-Parody and Self-Assertion. Frontiers: A Journal of Women Studies 10.1 (1988): 7-12. Print. May, Todd. Gilles Deleuze : An Introduction. New York: Cambridge University Press, 2005. Print. McKee, Alan. "Is Doctor Who Political?" European Journal of Cultural Studies 7.2 (2004): 201-17. Print. Merlin, Bella. The Complete Stanislavsky Toolkit. London: Nick Hern Books, 2007. Morris, Barbra S. "Why Is George So Funny? Television Comedy, Trickster Heroism, and Cultural Studies." The English Journal 88.4 (1999): 47-52. Print. Nichols, Nina daVinci. "The Arlecchino and Three English Tinkers." Comparative Drama 36.1-2 (2002): 145-85. Print. Owen, Rachael and Anna Sweeting. Hoodie or Goodie? The Link between Violent Victimisation and Offending in Young People. Victim Support: Victim Support, 2007. Print. Pickering, Kenneth, and Mark Woolgar. Theatre Studies. Houndmills; New York: Palgrave Macmillan, 2009. Pickering, Kenneth. Key Concepts in Drama and Performance. Palgrave Key Concepts. New York: Palgrave Macmillan, 2005. 200 MA Thesis: The Role of Misrule in the practice of Performance Appendix One FatCircus, Workshop Number One. FatCircus, Workshop Number One. When: Saturday 29th of January 2011 from 10 to 6pm When: Saturday 29th of January 2011 from 10 to 6pm Where: 93 Kelburn Parade, the Theatre Programme of Victoria University of Wellington. In this first workshop, I want to focus an eye on the ideas of fear and frivolity, the mediation of the extreme, and the subversive element of the theatre. These elements in some way arise from my research into the Carnivalesque and Misrule. I am focusing on how misrule, rather than arising from breaking of the rules, manages to acknowledge and to interrogate the rules and traditions of the theatre and of the world beyond. Mikhail Bakhtin and Gilles Deleuze have been key thinkers in this process and I acknowledge theatre writings by Stanislavsky, LeCoq, Mike Alfreds, Ann Bogart and Judith Weston. I also think that without an element of misrule it becomes difficult to be creative. Using some exercises to ease us into the mode of misrule and then moving into a short storytelling exercise I wish to examine how these elements might inform a methodology of rehearsal and vision. This is a beginning for me with this work and as such is necessarily not as deep as if a company might spend considerable time developing a voice out of this work. It is therefore important that the people attending bring their own experiences, their own eyes and creative voice to work in the spirit of the carnival. p However, what people can bring is not as important as their willingness to talk about and put a creative exercise up on its feet. The work that comes out of this will inform the activities that will be present a fortnight later in the second workshop where the text of Saint Punch will be introduced. My intention is to bring together a number of people for the first workshop- some of whom may not be available for the performances and rehearsals of Saint Punch- however for me it would be useful to have, in addition those who are interested taking this trip with me, coming to the first of these workshops before we begin rehearsals. Saint Punch will be rehearsed for 9 weeks and performed at 93 Kelburn Parade prior to Easter. Shakespeare, W., et al. A New Variorum Edition of Shakespeare. J.B. Lippincott & co., 1955. Print. Shakespeare, W., et al. A New Variorum Edition of Shakespeare. J.B. Lippincott & co., 1955. Print. Sheets, Robin Ann. "Pornography, Fairy Tales, and Feminism: Angela Carter's "the Bloody Chamber"." Journal of the History of Sexuality 1.4 (1991): 633-57. Print. Speidel, E. "Brecht's "Puntila": A Marxist Comedy." The Modern Language Review 65.2 (1970): 319-32. Print. Stam, Robert. Subversive Pleasures: Bakhtin, Cultural Criticism, and Film. Baltimore: Johns Hopkins University Press, 1989. Tatar, M. Secrets Beyond the Door: The Story of Bluebeard and His Wives. Princeton University Press, 2004. Print. Thompson, S. Leigh. "Essay: Theatre of Change (About Legislative Theatre) \| the Forum Project". <http://theforumproject.org/whatisto/what- is-theatre-of-the-oppressed/essay-theatreofchange4/>. Theatre) \| the Forum Project". <http://theforumproject.org/whatisto/what- is-theatre-of-the-oppressed/essay-theatreofchange4/>. Gregor Cameron 198272670 is-theatre-of-the-oppressed/essay-theatreofchange4/>. Vecsey, Christopher. "The Exception Who Proves the Rules: Ananse the Akan Trickster." Journal of Religion in Africa 12.3 (1981): 161-77. Print. Vice, Sue. Introducing Bakhtin. Manchester: Manchester University Press, Distributed exclusively in the USA by St. Martin's Press, 1997. Webb, Jen, and Tony Schirato. "Bourdieu's Concept of Reflexivity as Metaliteracy."Cultural Studies 17.3/4 (2003): 539-39. Print. Vecsey, Christopher. "The Exception Who Proves the Rules: Ananse the Akan Trickster." Journal of Religion in Africa 12.3 (1981): 161-77. Print. Vice, Sue. Introducing Bakhtin. Manchester: Manchester University Press, Distributed exclusively in the USA by St. Martin's Press, 1997. G C 198272670 Webb, Jen, and Tony Schirato. "Bourdieu's Concept of Reflexivity as Metaliteracy."Cultural Studies 17.3/4 (2003): 539-39. Print. 201 Weston, Judith. Directing Actors: Creating Memorable Performances for Film and Television. Studio City, CA: M. Wiese Productions, 1996. Print. Wilson, Diane E. Buffy Vs. Bakhtin: Carnival and Dialogism in the Buffyverse. SC2: the Slayage Conference on the Whedonverses. 2006. Available online. 202 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance The commitment required for this will be one weeknight rehearsal and one weekend day rehearsal for all performers. y So if you are interested in joining us (and here I acknowledge those who with patience have already indicated their interest) or can pass this on to someone who may wish to contribute, please send me an email at Gregor@fatcircus.co.nz 203 Gregor Cameron 198272670 Gregor Cameron 198272670 Appendix Two Programme detail Appendix Two Appendix Two Programme detail Programme detail MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Finally an ode to my cast and crew, who have helped me make this one of the most pleasurable times of my life, thank you one and all. I hope you enjoy the show. Cast of Characters Thomas Pepperell: Emma Smith: Rosie Tapsell: Des Morgan: Audrey Martin: Patrick Coelho: Maria Becerro Casero: Piotr Wolodzko: Martin Quicke: Cast of Characters Thomas Pepperell: Emma Smith: Rosie Tapsell: Des Morgan: Audrey Martin: Patrick Coelho: Maria Becerro Casero: Piotr Wolodzko: Martin Quicke: Mr. Punch Judy/ Ghost/ Crocodile/ Inquisitor Joan DʼArc/ Esmerelda Gilles de Rais Judith Bluebeard Clara/Some soldiers Joey//Beadle/Policeman The Professor/ Poitou/ Prelati/ La Hire/ Publican/ Jack Ketch Emma Smith: Rosie Tapsell: Des Morgan: Audrey Martin: Patrick Coelho: Maria Becerro Casero: Piotr Wolodzko: Martin Quicke: Programme detail A story begins at the beginning, that much is obvious. A journey, a becoming. To just be, is to be trapped in a character unable to change. Our lives, however, are always caught within an act of becoming. Even in death our lives are given over to the tapestry of history, to be interpreted and used in ways we might never have thought of. Saint Punch is such to me, one story told in three versions. Becoming something else, used by others for something other than our own intentions. In this world of mediated fame and infamy it becomes all the more important that we stop and consider what these stories represent. I am caught up in Bakhtinʼs notion of Carnival. It seems to fit so nicely around the ritual of theatre and requires us to consider the mediation of our stories to exist, to become. From Bakhtin we might perceive that by entering the theatre we cross the borders into the carnival. Inside we expect to see wonders, and grotesques, to explore danger and to see the world as we know turned upside down.Thus came about the story of Gilles de Rais and Joan of Arc, of Bluebeard and Judith, and Punch & Judy. Stories dissimilar yet connected. This play has haunted me for a number of years, its voices crying to be realised. The opportunity to place this text central to my work here at Victoria was of immense value and I must thank John Downie for his constant provocation over twenty odd years which has brought me to this point. I also acknowledge the support of colleagues all over the School of English, Film, Theatre, and Media Studies. I also appreciate the help and support of my mentors-you know who you are. Of course there are also the stalwarts of the university, our general staff who make all things possible. 204 43 See Laura Makarius, "Ritual Clowns and Symbolic Behavior," Diogenes no. 69 (1970): 66, for a related list of characteristics she sees as common to both clown and trickster. Cf. Turner, "Myth and Symbol," p. 578. Barbara Babcock-Abrahams list of Trickster characteristics: In almost all cases, and to a greater or lesser degree, tricksters 1. exhibit an independence from and an ignoring of temporal and spatial boundaries; 2. tend to inhabit crossroads, open public places (especially the marketplace), doorways, and thresholds. In one way or another they are usually situated between the social cosmos and the other world or chaos; 3. are frequently involved in scatological and coprophagous episodes which may be creative, d i i l i 3. are frequently involved in scatological and coprophagous episodes which may be creative, destructive, or simply amusing; destructive, or simply amusing; destructive, or simply amusing; 4. may, similarly, in their deeds and character, partake of the attributes of Trickster- Transformer-Culture Hero; 5. frequently exhibit some mental and/or physical abnormality, especially exaggerated sexua characteristics; 6. have an enormous libido without procreative outcome; 7. have an ability to disperse and to disguise themselves and a tendency to be multiform and ambiguous, single or multiple; 8. often have a two-fold physical nature and/or a "double" and are as 8. often have a two-fold physical nature and/or a "double" and are associated with mirrors. 9. follow the "principle of motley" in dress; 10. are often indeterminant (in physical stature) and may be portrayed as both young and old, as perpetually young or perpetually aged; 11. exhibit an human/animal dualism and may appear as a human with animal characteristics or vice versa; (even in those tales where the trickster is explicitly identified as an animal, he is anthropomorphically described and referred to in personal pronouns); 12. are generally amoral and asocial - aggressive, vindictive, vain, defia 12. are generally amoral and asocial - aggressive, vindictive, vain, defiant of authority, etc.; 13. despite their endless propensity to copulate, find their most abiding form of relationship with the feminine in a mother or grandmother bond; 14. in keeping with their creative/destructive dualism, tricksters tend to be ambiguously situated between life and death, and good and evil, as is summed up in the combined black and white symbolism frequently associated with them; 15. are often ascribed to roles (i.e., other than tricky behavior) in which an individual normally has privileged freedom from some of the demands of the social code; 16. in all their behavior, tend to express a concomitant breakdown of the distinction between reality and reflection43. From Babcock-Abrahams, Barbara. Crew. Gregor Cameron: Rosanagh Kynoch: Shannon Friday, Courtney Leggett, Kate Norquay, Bop Murdoch: Stephen Keen, Andy Friedland: Welding Andy Friedland: Operator Operator Des Morgan: construction Patricia Hetherington: operation Elliot Lim: g Music Punch & Judy Man-Fraser Hines In for the Kill-Damien Brell Hell-bound Train-Victoria Police Pipe Band Acknowledgements. John Downie, Alex Funke, Paul Wolffram, Jim Davenport, William Franco, Tom McCrory, Bernard Blackburn, Damien Brell, Horst Sarubin, Kerry Glamsch, Chris Ryan, Ralph Johnson, Dawa Devereaux, ICW Productions, Shane Roberts, Roc Travers, Sean Redmond, Doug Van Belle, Matthew Wagner, Claire OʼLoughlin, Family & Friends always, The Wisebuys on Dixon shop. Various histories of Gilles De Rais and Joan of Arc. Most particularly The Saint and the Devil by Frances Winwar, 1948. The Folk tradition and tale of Bluebeard. With awareness of Angela Carter A Punch and Judy Script as it first appeared in London Labour and the London Poor by Henry Mayhew, published in 1851 The Folk tradition and tale of Bluebeard. With awareness of Angela Carter A Punch and Judy Script as it first appeared in London Labour and the London Poor by Henry Mayhew, published in 1851 Punch and Judy Script as it first appeared in London Labour and the London Poor by Henry ayhew, published in 1851 y p I appreciate that these three sources gave up major inspiration to the above work, which is somewhat truthful. However take care as any truth found herein is completely coincidental and not the intention of the author I appreciate that these three sources gave up major inspiration to the above work, which is somewhat truthful. However take care as any truth found herein is completely coincidental and not the intention of the author Gregor Cameron 198272670 205 MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance MA Thesis: The Role of Misrule in the practice of Performance Gregor Cameron 198272670 44 aren.org/prison/documents/African/14/14.pdf accessed 18/9/1 45 http://en.wikipedia.org/wiki/First_they_came... Accessed 22/1/12 Barbara Babcock-Abrahams list of Trickster characteristics: "A Tolerated Margin of Mess": The Trickster and His Tales Reconsidered." Page 159-160 43 206 44 aren.org/prison/documents/African/14/14.pdf accessed 18/9/1 45 Gregor Cameron 198272670 Appendix Four Specific Influences Here is a told original version of the Anansi story that Neil Gaiman used and I adapted for use with Mr Punch. Anansi kills his Grandmother. William Cooper, Mandeville. Specific Influences Here is a told original version of the Anansi story that Neil Gaiman used and I adapted for use with Mr Punch. Anansi kills his Grandmother. William Cooper, Mandeville. Specific Influences Here is a told original version of the Anansi story that Neil Gaiman used and I adapted for use with Mr Punch. Anansi kills his Grandmother. William Cooper, Mandeville. Anansi an' Tiger were travelling. Anansi kill him old grandmother, him put him into a little handcart was shoving him t'ru de town. After him catch to a shop jes' like out here, de shopkeeper was a very hasty-temper man; an' went in de shop an' call fe some whiskey an' give it to one of de shopkeeper carry it to his grandmother. An' said he mus' go up to de han'- cart an' call twice. An' de ol' lady did not hear. So Anansi said to de shop-keeper him mus' holla out to de ol' lady; him sleeping. So de ol' lady didn't hear, he fire de glass in de ol' lady face, an' de ol' lady fell right over. Then the shop-keeper get so frighten he cry out to Anansi, say Anansi mustn't mek no alarm in de town; he will give him a bushel of money to mek him keep quiet. So dem was going along an' borrow a quart can from Tiger an' was measuring dis money. Tiger said, "Where you get all dat money?" Anansi say, "I kill my ol' grandmother." Tiger, him went home an' kill his grandmother an' put her up in a little hand-cart an' was goin' along t'ru de town hollerin' out to all de people, "Who want a dead body to buy?" So Anansi said to Tiger he shouldn't do anyt'ing like dat; too foolish!44 "First they came…" is a famous statement attributed to pastor Martin Niemöller (1892–1984) about the inactivity of German intellectuals following the Nazi rise to power and the purging of their chosen targets, group after group. The text of the quotation is usually presented roughly as follows: First they came for the communists, y and I didn't speak out because I wasn't a communist. Appendix Four and I didn't speak out because I wasn't a communist. and I didn't speak out because I wasn't a trade unionist. y and I didn't speak out because I wasn't a Jew. Then they came for the Catholics, and I didn't speak out because I was Protestant. Martin Niemöller was a German pastor and theologian born in Lippstadt, Germany, in 1892. Niemöller was an anti-Communist and supported Hitler's rise to power at first. But when Hitler insisted on the supremacy of the state over religion, Niemöller became disillusioned. He became the leader of a group of German clergymen opposed to Hitler. He was imprisoned in Sachsenhausen concentration camp45 207 Gregor Cameron 198272670
https://openalex.org/W2624686300	https://link.springer.com/content/pdf/10.1007%2Fs00418-017-1591-3.pdf	English	null	Involvement of Atm and Trp53 in neural cell loss due to Terf2 inactivation during mouse brain development	Histochemistry and cell biology	2,017	cc-by	9,437	Histochem Cell Biol (2017) 148:489–501 DOI 10.1007/s00418-017-1591-3 ORIGINAL PAPER ORIGINAL PAPER Involvement of Atm and Trp53 in neural cell loss due to Terf2 inactivation during mouse brain development Jusik Kim1,2 · Inseo Choi1,2 · Youngsoo Lee1,2 Accepted: 8 June 2017 / Published online: 15 June 2017 © The Author(s) 2017. This article is an open access publication Accepted: 8 June 2017 / Published online: 15 June 2017 © The Author(s) 2017. This article is an open access publication Abstract Maintenance of genomic integrity is one of the critical features for proper neurodevelopment and inhibi- tion of neurological diseases. The signals from both ATM and ATR to TP53 are well-known mechanisms to remove neural cells with DNA damage during neurogenesis. Here we examined the involvement of Atm and Atr in genomic instability due to Terf2 inactivation during mouse brain development. Selective inactivation of Terf2 in neural pro- genitors induced apoptosis, resulting in a complete loss of the brain structure. This neural loss was rescued partially in both Atm and Trp53 deficiency, but not in an Atr-defi- cient background in the mouse. Atm inactivation resulted in incomplete brain structures, whereas p53 deficiency led to the formation of multinucleated giant neural cells and the disruption of the brain structure. These giant neural cells disappeared in Lig4 deficiency. These data demonstrate ATM and TP53 are important for the maintenance of tel- omere homeostasis and the surveillance of telomere dys- function during neurogenesis. Abbreviations DSBs DNA double-strand breaks DDR DNA damage response P Postnatal day E Embryonic day VZ Ventricular zone CC Corpus callosum * Youngsoo Lee ysoolee@ajou.ac.kr 1 Genomic Instability Research Center, School of Medicine, Ajou University, Suwon 16499, Republic of Korea 2 Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon 16499, Republic of Korea Introduction Genomic instability resulting from DNA damage induced by either endogenous or exogenous insults could lead to defective neurodevelopment and neurological diseases (Lee et al. 2016). Ataxia telangiectasia mutated (ATM) is one of the early responders to DNA damage, particularly to DNA double-strand breaks (DSBs). ATM mutations cause Ataxia Telangiectasia (A-T) characterized by ataxia due to loss of Purkinje and granule cells in the cerebellum (McKinnon 2012, 2013). Similarly, Ataxia-telangiectasia and RAD3- related (ATR) recognizes single-stranded DNA resulting from replication stress. Seckel syndrome 1 (SCKL1) due to hypomorphic mutations in the ATR gene is characterized by microcephaly and mental retardation (Nam and Cortez 2011; McKinnon 2013). Once activated by DNA damage, ATM and ATR phosphorylate several overlapping sub- strates including tumor protein p53 (TP53/Trp53 in mice) to regulate DNA damage repair, apoptosis, and cell cycle arrest (Lee et al. 2001, 2012b; Lovejoy and Cortez 2009). Although it is well known that ATM and ATR are required to maintain genomic integrity, the precise roles of ATM and ATR during brain development are not fully under- stood, especially related to neuropathology such as ataxia, neurodegeneration, and microcephaly observed in human patients. Keywords Atm · DNA damage · Apoptosis · Brain development Electronic supplementary material The online version of this article (doi:10.1007/s00418-017-1591-3) contains supplementary material, which is available to authorized users. 2 Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon 16499, Republic of Korea 1 3 Histochem Cell Biol (2017) 148:489–501 490 proper breeding scheme. Conditional knockout animals could not be used for breeding. Terf2LoxP/+;Nestin-Cre or Emx1-Cre animals did not show any discernable defects or shortened life span, and they were fertile. So these animals were included in control groups. Mutant alleles of Atm, AtrLoxP, Trp53LoxP, Lig4LoxP, and Pot1aLoxP genes and the polymerase chain reaction (PCR) conditions for genotyping were as previously described (Lee et al. 2001, 2012b, 2014; Shull et al. 2009). All animals were maintained in a mixed strain of C57BL/6 X 129 genetic background. Since we could not find any gender differ- ence of DDR in the brain before (Lee et al. 2001, 2012a, 2014), we analyzed the experimental materials regardless of gender for the current study. Also Nestin-Cre expres- sion was maintained in females for breeding to minimize any ectopic Cre recombinase activity outside of the nerv- ous system. Animals Floxed Terf2 animals (Karlseder et al. 1999; Celli and de Lange 2005) were purchased from the Jackson Labo- ratory (JAX #006568). Germline deletion of the Terf2 gene caused embryonic lethality at mid-gestation in the mouse (Celli and de Lange 2005). In order to restrict inactivation of the Terf2 gene in the nervous system dur- ing development, Terf2LoxP/+ animals were interbred with Nestin-Cre animals (JAX #003771) or Emx1-Cre animals (JAX #005628). Cre recombinase expression driven by the Nestin promoter is active around embryonic day (E) 11 throughout the neural progenitors, whereas the Emx1 promoter is activated only in the dorsal telencephalon and the hippocampal progenitors around E10 during mouse embryogenesis (Gorski et al. 2002; Graus-Porta et al. 2001; Tronche et al. 1999). Terf2LoxP/LoxP;Nestin-Cre and Terf2LoxP/LoxP;Emx1-Cre mice were obtained through a Materials and methods All of animal materials for experiments were courte- ously provided by Dr. Peter McKinnon (St. Jude Children’s Research Hospital, USA). The presence of a vaginal plug was indicated as E.5 and the day of birth as postnatal day (P) 0. All animals were housed in an AAALAC accred- ited facility and were maintained in accordance with the National Institutes of Health ‘Guide for the Care and Use of Laboratory Animals’. All procedures for animal use were approved by the Institutional Animal Care and Use Committee. Introduction Genotypes of genetically engineered animals were determined by a routine PCR method using the fol- lowing primers: Terf2 (Forward: 5′ ccaaccagggatacaca- gtga, Reverse: 5′ atccgtagttcctcttgtgtctg), Pot1a (Forward: 5′ ctcgaattccatctcctcccagtactctctcag, Reverse: 5 ggaactgg- tacgtatcagtgtgtgtgg), Atm wildtype ( WT) allele (Forward: 5′ gcctgtatcttctatgtgcaccgtcttcgc, Reverse: 5′ ggtgcggtgtg- gatgggactggagg), Atm targeted allele (Forward: 5′ gtgat- gacctgagacaagatgctgtc, Reverse: 5′ gggaagacaatagcag- gcatgc), Atr (Forward: 5 tacattttagtcatagttgcataacac, Reverse: 5 cttctaatcttcctccagaattgtaaaagg), Lig4 (Forward: 5′ atcgctcttgtcccagtacacctgc, Reverse: 5 gtgcattaaatggagt- gctgtgc), Trp53 (Forward: 5′ cacaaaaacaggttaaacccag, Reverse: 5′ agcacataggaggcagagac). Telomere dysfunction is one of the endogenous sources to induce DNA damage response (DDR), since the unpro- tected telomere ends could be recognized as DNA strand breaks (de Lange 2005). To protect this DNA region, the telomere ends are coated with the Shelterin complex, which is composed of several proteins including telomeric repeat binding factor 2 (TERF2) and protection of telomeres 1 (POT1), to prevent telomere attrition and inappropriate DDR induction (de Lange 2005; Palm and de Lange 2008). TERF2 homodimers bind to double-stranded regions of tel- omeres and inactivation of TERF2 triggers ATM-dependent DDR signals, whereas POT1 protects the 3′ single-stranded overhang and its inactivation initiates ATR-dependent DDR signals at the cellular level (Karlseder et al. 1999; Zhang et al. 2006, 2007; Denchi and de Lange 2007; Sfeir and de Lange 2012). The exposed telomere ends as a result of Terf2 inactivation were processed by DNA ligase IV (Lig4) which is the ligase for the canonical Non-homologous end- joining repair (NHEJ) pathway for DSBs (Celli and de Lange 2005; Lee et al. 2016; Smogorzewska et al. 2002). The involvement of ATM and ATR in DDR induced by tel- omere dysfunction was mutually exclusive in this context. However, selective deletion of the Pot1a gene during mouse brain development resulted in Atm-dependent neu- rological phenotypes including cerebellar defects, suggest- ing that telomere dysfunction might induce DDR in a tis- sue-specific manner during neurogenesis (Lee et al. 2014). For the current study, we asked whether telomere dysfunc- tion due to Terf2 inactivation also induces neuro-specific DDR associated with Atm or Atr signaling pathway during neurogenesis. PCR products for Terf2 genotyping were amplified for 35 cycles of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 45 s. PCR products for WT and floxed alleles (LoxP) of the Terf2 gene were 233 and 300 bp, respectively. Histology The scale bar is 1.2 mm Fi 1 T f2 i ti ti d i i lt d i blit ti d (T f2 Li 4 T 53)Emx1-Cre) Al T 53 d fi i d and (Terf2;Lig4;Trp53)Emx1-Cre). Also Trp53 deficiency rescued embryonic lethality as well as partially the missing brain structure in Terf2Nes-Cre animals. Lower panel the hippocampus, one of the important brain structures for memory, in the conditional knock- out brains was not formed. Either Atm or Trp53 deficiency was not sufficient enough to restore the hippocampal structure. However, the (Terf2;Lig4;Trp53)Emx1-Cre brain displayed a partial recovery of the hippocampus. In the parasagittal section, the matched brain part containing the hippocampus in the Emx-1 Cre background was com- pletely gone, since the Emx1-Cre was expressed in this particular brain structure during neurogenesis. The dotted red lines demarcate some brain areas affected by Terf2 inactivation, such as the cerebral cortex and hippocampus. CC corpus callosum, ce cerebellum, Cpu caudate putamen, ctx cerebral cortex, th thalamus, hipp hippocampus (CA1/2/3 Cornu Ammonis areas 1/2/3, DG dentate gyrus), tm tectum. The scale bar is 1.2 mm Fig. 1 Terf2 inactivation during neurogenesis resulted in obliteration of the brain structure. a The view from the top of Terf2 conditional knockout brains at 3 weeks of age. The skull was partially removed to show the brain structure. The eyeballs were intact. The control brain was fully developed, yet the Terf2Emx1-Cre brain had the cerebral corti- cal part missing in which the Emx1 promoter-driven Cre recombinase was expressed, so the colliculus (col) was fully exposed. Red arrows indicate the part lost in the Terf2Emx1-Cre brain. These missing brain parts were partially restored in both Atm and Trp53-null backgrounds. ce cerebellum, col colliculus, ctx cerebral cortex, ob olfactory bulb. Ctrl control, the ruler in mm. b Nissl staining of the mature brains. The sagittal (upper panel) and parasagittal (lower panel) views of 3-week-old brains in different genetic backgrounds. Upper panel the brain areas expressing Cre recombinase controlled by the Emx1 promotor were completely missing (Terf2Emx1-Cre), but the partial recovery of the brain areas was observed in Atm-, Trp53- and Lig4/ Trp53-null backgrounds (Terf2Emx1-Cre;Atm−/−, (Terf2;Trp53)Emx1-Cre medical GmbH) cryostat for pathological analysis. Hema- toxylin and Eosin (H/E), and Nissl staining were carried out in a routine procedure. Histology Histopathological procedures were performed as previously described (Lee et al. 2012a, 2014). In brief, both embryos and brains were collected at indicated time points after the fixation step using 4% phosphate-buffered paraformalde- hyde. Cryosections of embryos or brains at 10 μm were collected using an HM500M (Microm) or an MEV (SLEE 3 1 491 Histochem Cell Biol (2017) 148:489–501 Fig. 1 Terf2 inactivation during neurogenesis resulted in obliteration of the brain structure. a The view from the top of Terf2 conditional knockout brains at 3 weeks of age. The skull was partially removed to show the brain structure. The eyeballs were intact. The control brain was fully developed, yet the Terf2Emx1-Cre brain had the cerebral corti- cal part missing in which the Emx1 promoter-driven Cre recombinase was expressed, so the colliculus (col) was fully exposed. Red arrows indicate the part lost in the Terf2Emx1-Cre brain. These missing brain parts were partially restored in both Atm and Trp53-null backgrounds. ce cerebellum, col colliculus, ctx cerebral cortex, ob olfactory bulb. Ctrl control, the ruler in mm. b Nissl staining of the mature brains. The sagittal (upper panel) and parasagittal (lower panel) views of 3-week-old brains in different genetic backgrounds. Upper panel the brain areas expressing Cre recombinase controlled by the Emx1 promotor were completely missing (Terf2Emx1-Cre), but the partial recovery of the brain areas was observed in Atm-, Trp53- and Lig4/ Trp53-null backgrounds (Terf2Emx1-Cre;Atm−/−, (Terf2;Trp53)Emx1-Cre and (Terf2;Lig4;Trp53)Emx1-Cre). Also Trp53 deficiency rescued embryonic lethality as well as partially the missing brain structure in Terf2Nes-Cre animals. Lower panel the hippocampus, one of the important brain structures for memory, in the conditional knock- out brains was not formed. Either Atm or Trp53 deficiency was not sufficient enough to restore the hippocampal structure. However, the (Terf2;Lig4;Trp53)Emx1-Cre brain displayed a partial recovery of the hippocampus. In the parasagittal section, the matched brain part containing the hippocampus in the Emx-1 Cre background was com- pletely gone, since the Emx1-Cre was expressed in this particular brain structure during neurogenesis. The dotted red lines demarcate some brain areas affected by Terf2 inactivation, such as the cerebral cortex and hippocampus. CC corpus callosum, ce cerebellum, Cpu caudate putamen, ctx cerebral cortex, th thalamus, hipp hippocampus (CA1/2/3 Cornu Ammonis areas 1/2/3, DG dentate gyrus), tm tectum. Telomere in situ using telomere‑PNA probe To detect telomere in situ, tissue sections were dehydrated in alcohol series, denatured at 80 °C for 4 min, followed by incubation for 4 h in the dark at room temperature with CY3-CCCTAACCCTAACCCTAA peptide nucleic acid (PNA) telomere probe (0.7 μg/ml, Panagene) in 70% formamide, 10 mM Tris–HCl pH 7.5, 5% MgCl2 buffer (82 mM Na2HPO4, 9 mM citric acid, 25 mM MgCl2), and 1% blocking reagent (Roche). Then, sections were washed with Solution I (70% formamide, 10 mM Tris–HCl pH 7.2, 0.1% BSA) and Solution II (0.05 M Tris–HCl pH 7.2, 0.15 M NaCl, 0.05% Tween 20) several times. After wash- ing, slides were mounted with DAPI-containing aqueous mounting medium (Vector). Images were captured and ana- lyzed as described in the “Histology” method section. Histology Right panel apoptosis detected by TUNEL (green color) shows increased apoptosis in the Emx1-Cre expressing area of the Terf2 conditional knockout brains (Terf2Emx1-Cre and (Terf2;Atr)Emx1-Cre), but not in the GE. Atm and Trp53 deficiency (but not Atr deficiency) attenuated apoptosis resulting from Terf2 inactivation in the devel- oping brain. d Apoptosis measured by TUNEL at E15.5. Terf2- inactivated brain parts were gone completely in the Terf2Emx1-Cre and (Terf2;Atr)Emx1-Cre embryos by E15.5 (No CTX formed). Even though the missing part in the developing brain was partially restored, the Terf2Emx1-Cre;Atm−/− forebrain (cortex: CTX) showed apoptosis restricted to the ventricular zone (VZ), and there was no significant sign of programmed cell death observed in the (Terf2;Trp53)Emx1-Cre and (Terf2;Lig4;Trp53)Emx1-Cre developing brains. However, the restored brain area in a Trp53-null background contained irregular cel- lularity (white asterisk), which was not observed in the Terf2Emx1-Cre; Atm−/− and (Terf2;Lig4;Trp53)Emx1-Cre embryonic brains. Since Atr or Lig4 inactivation during brain development also induces neuronal cell death, the level of programmed cell death found in the AtrEmx1-Cre and Lig4Emx1-Cre embryonic brains was as expected. GE ganglionic eminence, VZ ventricular zone. The scale bars in b and c left panels are 1.2 mm, and the scale bars in the rest of the photomicrographs are 200 μm ◂ Histology The missing brain parts were restored in both Atm and Trp53 null backgrounds (Terf2Nes-Cre;Atm−/− N C ◂ Fig. 2 Massive apoptosis occurred in the Terf2-deficient develop- ing brain. a TUNEL analysis to detect neural apoptosis. The embry- onic forebrain (cortex) and colliculus area at embryonic day (E) 13.5 with TUNEL staining. The controls (Terf2ctrl, AtrNes-Cre, Atm−/− and Trp53Nes-Cre) showed only the basal levels of TUNEL positiv- ity (green staining), and there was no difference among the control groups. However, massive apoptosis was observed in the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre developing brains, while a dramatic reduction of programmed cell death was evident in both Terf2Nes-Cre;Atm−/− and (Terf2;Trp53)Nes-Cre embryonic cortex and colliculus. Green fluores- cent apoptotic debris were falling off inside of the ventricle (V) in the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre embryonic brains. Ctrl control. b Tuj1 (Neuron-specific class III beta-tubulin, marker for postmitotic neurons) positive immuno-staining. Left panel the heads of embryos (at E15.5) with Tuj1 immunoreactivity (purple color) at low magni- fication show that the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre brains were completely gone by E15.5 (red arrowheads), yet the brain structure was partially rescued in both Atm and Trp53 deficiency (red arrows). The ventricular zone was not positive for Tuj1 immunoreactivity, so methyl green staining (counterstaining) was visible in this zone. Right panel high magnified views of the forebrains (cortex) in experimen- tal groups. The Terf2Nes-Cre and (Terf2;Atr)Nes-Cre embryos did not have any brain structure which was positive for Tuj1 immuostaining (pur- ple color). Two pictures of Terf2Nes-Cre and (Terf2;Atr)Nes-Cre heads show only a part of the skull and scalp. The missing brain parts were restored in both Atm and Trp53 null backgrounds (Terf2Nes-Cre;Atm−/− and (Terf2;Trp53)Nes-Cre). Methyl green was used for counterstaining so that the ventricular zone could be identifiable. V ventricle. c H/E and TUNEL staining of the Emx1-Cre expressing brains at E13.5. Left panel Hematoxylin/Eosin (H/E) staining of the embryonic brains shows that the Cre expressing area (red line) was gone in Terf2Emx1-Cre and (Terf2;Atr)Emx1-Cre embryos, but the affected brain area was par- tially restored in both Atm−/− and Trp53Emx1-Cre backgrounds. The con- trol animals including Terf2ctrl, AtrEmx1-Cre, Atm−/− and Trp53Emx1-Cre brains did not show any morphological defects. The ganglionic eminence (GE) was intact in the (Terf2)Emx1-Cre embryonic brains, since Emx1-Cre is not expressed in the GE. The red lines demarcate the Emx1-Cre expressing area. GE ganglionic eminence, V ventri- cle. Histology Immunoreactivity was visualized by either colorimetric detection using the VIP substrate kit (Vector Labs) reactive with biotinylated secondary antibod- ies or fluorometric detection using FITC/CY3 conjugated secondary antibodies (Jackson Immunologicals) after incu- bation with primary antibodies. Counterstaining was done with 0.1% methyl green in 0.1 M sodium acetate buffer for colorimetric staining followed by mounting with DPX (Sigma) or DAPI/PI counterstaining mounting medium (Vector Laboratories) for fluorometric staining. Antibodies used for this study were Calbindin (mouse, 1:2000, Sigma-Aldrich), Ctip2 (rat, 1:100, Abcam), Cux1 (rabbit, 1:100, Santa Cruz), Foxp2 (rabbit, 1:500, Abcam), GFAP (mouse, 1:500, Sigma-Aldrich), γ-H2AX (rabbit, 1 3 Histochem Cell Biol (2017) 148:489–501 492 492 Histochem Cell Biol (2017) 148:489–501 1:100, Cell signaling), γ-tubulin (mouse, 1:1000, Sigma- Aldrich), Histone H3phosphoS10 (rabbit, 1:2000, Cell signaling), MBP (myelin basic protein, rabbit, 1:200, Abcam), Myelin-PLP (rabbit, 1:200, Abcam), NeuN (mouse, 1:500, Millipore), Neuronal Class III β tubulin (clone TUJ1, mouse, 1:1000, Covance Research), Tbr1 (rabbit, 1:500, Abcam). Depending on primary antibodies, the citric acid-based antigen retrieval method was applied to enhance immunoreactive signals. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay using Apoptag (Chemicon) was applied to measure apoptosis. Multiple histological slides were examined and imaged using an Axio Imager A1 microscope (Zeiss) or B600TiFL (Optika). Microscopic 1:100, Cell signaling), γ-tubulin (mouse, 1:1000, Sigma- Aldrich), Histone H3phosphoS10 (rabbit, 1:2000, Cell signaling), MBP (myelin basic protein, rabbit, 1:200, Abcam), Myelin-PLP (rabbit, 1:200, Abcam), NeuN (mouse, 1:500, Millipore), Neuronal Class III β tubulin (clone TUJ1, mouse, 1:1000, Covance Research), Tbr1 (rabbit, 1:500, Abcam). Depending on primary antibodies, the citric acid-based antigen retrieval method was applied to enhance immunoreactive signals. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay using Apoptag (Chemicon) was applied to measure apoptosis. Multiple histological slides were examined and imaged using an Axio Imager A1 microscope (Zeiss) or B600TiFL (Optika). Microscopic the citric acid-based antigen retrieval method was applied to enhance immunoreactive signals. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay using Apoptag (Chemicon) was applied to measure apoptosis. Multiple histological slides were examined and imaged using an Axio Imager A1 microscope (Zeiss) or B600TiFL (Optika). Microscopic 1 3 3 Histochem Cell Biol (2017) 148:489–501 493 Fig. 2 Massive apoptosis occurred in the Terf2-deficient develop- ing brain. a TUNEL analysis to detect neural apoptosis. The embry- onic forebrain (cortex) and colliculus area at embryonic day (E) 13.5 with TUNEL staining. Histology The controls (Terf2ctrl, AtrNes-Cre, Atm−/− and Trp53Nes-Cre) showed only the basal levels of TUNEL positiv- ity (green staining), and there was no difference among the control groups. However, massive apoptosis was observed in the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre developing brains, while a dramatic reduction of programmed cell death was evident in both Terf2Nes-Cre;Atm−/− and (Terf2;Trp53)Nes-Cre embryonic cortex and colliculus. Green fluores- cent apoptotic debris were falling off inside of the ventricle (V) in the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre embryonic brains. Ctrl control. b Tuj1 (Neuron-specific class III beta-tubulin, marker for postmitotic neurons) positive immuno-staining. Left panel the heads of embryos (at E15.5) with Tuj1 immunoreactivity (purple color) at low magni- fication show that the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre brains were completely gone by E15.5 (red arrowheads), yet the brain structure was partially rescued in both Atm and Trp53 deficiency (red arrows). The ventricular zone was not positive for Tuj1 immunoreactivity, so methyl green staining (counterstaining) was visible in this zone. Right panel high magnified views of the forebrains (cortex) in experimen- tal groups. The Terf2Nes-Cre and (Terf2;Atr)Nes-Cre embryos did not have any brain structure which was positive for Tuj1 immuostaining (pur- ple color). Two pictures of Terf2Nes-Cre and (Terf2;Atr)Nes-Cre heads show only a part of the skull and scalp. The missing brain parts were restored in both Atm and Trp53 null backgrounds (Terf2Nes-Cre;Atm−/− and (Terf2;Trp53)Nes-Cre). Methyl green was used for counterstaining so that the ventricular zone could be identifiable. V ventricle. c H/E and TUNEL staining of the Emx1-Cre expressing brains at E13.5. Left panel Hematoxylin/Eosin (H/E) staining of the embryonic brains shows that the Cre expressing area (red line) was gone in Terf2Emx1-Cre and (Terf2;Atr)Emx1-Cre embryos, but the affected brain area was par- tially restored in both Atm−/− and Trp53Emx1-Cre backgrounds. The con- trol animals including Terf2ctrl, AtrEmx1-Cre, Atm−/− and Trp53Emx1-Cre brains did not show any morphological defects. The ganglionic eminence (GE) was intact in the (Terf2)Emx1-Cre embryonic brains, since Emx1-Cre is not expressed in the GE. The red lines demarcate the Emx1-Cre expressing area. GE ganglionic eminence, V ventri- cle. Right panel apoptosis detected by TUNEL (green color) shows increased apoptosis in the Emx1-Cre expressing area of the Terf2 conditional knockout brains (Terf2Emx1-Cre and (Terf2;Atr)Emx1-Cre), but not in the GE. Atm and Trp53 deficiency (but not Atr deficiency) attenuated apoptosis resulting from Terf2 inactivation in the devel- oping brain. d Apoptosis measured by TUNEL at E15.5. Histology Terf2- inactivated brain parts were gone completely in the Terf2Emx1-Cre and (Terf2;Atr)Emx1-Cre embryos by E15.5 (No CTX formed). Even though the missing part in the developing brain was partially restored, the Terf2Emx1-Cre;Atm−/− forebrain (cortex: CTX) showed apoptosis restricted to the ventricular zone (VZ), and there was no significant sign of programmed cell death observed in the (Terf2;Trp53)Emx1-Cre and (Terf2;Lig4;Trp53)Emx1-Cre developing brains. However, the restored brain area in a Trp53-null background contained irregular cel- lularity (white asterisk), which was not observed in the Terf2Emx1-Cre; Atm−/− and (Terf2;Lig4;Trp53)Emx1-Cre embryonic brains. Since Atr or Lig4 inactivation during brain development also induces neuronal cell death, the level of programmed cell death found in the AtrEmx1-Cre and Lig4Emx1-Cre embryonic brains was as expected. GE ganglionic eminence, VZ ventricular zone. The scale bars in b and c left panels are 1.2 mm, and the scale bars in the rest of the photomicrographs are 200 μm ◂ images were captured and processed using Photoshop (v.CS6.5, Adobe). Fig. 2 Massive apoptosis occurred in the Terf2-deficient develop- ing brain. a TUNEL analysis to detect neural apoptosis. The embry- onic forebrain (cortex) and colliculus area at embryonic day (E) 13.5 with TUNEL staining. The controls (Terf2ctrl, AtrNes-Cre, Atm−/− and Trp53Nes-Cre) showed only the basal levels of TUNEL positiv- ity (green staining), and there was no difference among the control groups. However, massive apoptosis was observed in the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre developing brains, while a dramatic reduction of programmed cell death was evident in both Terf2Nes-Cre;Atm−/− and (Terf2;Trp53)Nes-Cre embryonic cortex and colliculus. Green fluores- cent apoptotic debris were falling off inside of the ventricle (V) in the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre embryonic brains. Ctrl control. b Tuj1 (Neuron-specific class III beta-tubulin, marker for postmitotic neurons) positive immuno-staining. Left panel the heads of embryos (at E15.5) with Tuj1 immunoreactivity (purple color) at low magni- fication show that the Terf2Nes-Cre and (Terf2;Atr)Nes-Cre brains were completely gone by E15.5 (red arrowheads), yet the brain structure was partially rescued in both Atm and Trp53 deficiency (red arrows). The ventricular zone was not positive for Tuj1 immunoreactivity, so methyl green staining (counterstaining) was visible in this zone. Right panel high magnified views of the forebrains (cortex) in experimen- tal groups. The Terf2Nes-Cre and (Terf2;Atr)Nes-Cre embryos did not have any brain structure which was positive for Tuj1 immuostaining (pur- ple color). Two pictures of Terf2Nes-Cre and (Terf2;Atr)Nes-Cre heads show only a part of the skull and scalp. Terf2 was essential for brain development It has been reported that germline deletion of the Terf2 gene caused embryonic lethality at mid-gestation in the mouse (Celli and de Lange 2005). So, we restricted Terf2 inactivation to the nervous system by cross-breeding of the Terf2 floxed animal model with either Nestin-Cre (Terf2LoxP/LoxP;Nestin-Cre, hereafter Terf2Nes-Cre) or Emx1- Cre (Terf2LoxP/LoxP;Emx1-Cre, hereafter Terf2Emx1-Cre) animal lines during development. Although the Terf2 gene was targeted only in the nerv- ous system during mouse development, all of Terf2Nes- Cre mice were born dead without a proper brain structure (data not shown). The overall body size of Terf2Nes-Cre animals was comparable to that of controls at birth (data not shown). On the other hand, Terf2Emx1-Cre animals were born at normal Mendelian ratio. These mutant ani- mals became runted and succumbed around 1 month after birth. The Terf2Emx1-Cre brains showed complete loss of the brain region in which Emx1-Cre is expressed (Fig. 1a, b). Terf2LoxP/+;Nestin-Cre or Emx1-Cre animals did not show any discernable neurological defects, so these genotypes of animals alone with WT animals were considered as control groups. 1 3 1 3 Histochem Cell Biol (2017) 148:489–501 494 erf2 inactivation induced massive neural apoptosis uring development no proper brain structure found by E15.5 in a Nestin background (Fig. 2b). Similarly, Terf2Emx1-Cre emb had only the vestige of the forebrain with residual a Terf2 inactivation induced massive neural apoptosis during development Loss of the brain regions in which Cre recombinase was expressed at birth led us to examine the embryonic brains during neurogenesis. The Terf2Nes-Cre embryos at E13.5 showed massive apoptosis measured by TUNEL through- out the developing brain (Fig. 2a). As a result, there was no proper brain structure found by E15.5 in a Nestin-Cre background (Fig. 2b). Similarly, Terf2Emx1-Cre embryos had only the vestige of the forebrain with residual apop- tosis at E13.5 (Fig. 2c), and a complete disappearance of the Emx1-Cre expressing areas by E15.5 (Fig. 2d), while the ganglionic eminence in which Emx1-Cre is not expressed was intact during neurogenesis. These data suggest an essential requirement of TERF2 and the Terf2 inactivation induced massive neural apoptosis during development Loss of the brain regions in which Cre recombinase was expressed at birth led us to examine the embryonic brains during neurogenesis. The Terf2Nes-Cre embryos at E13.5 showed massive apoptosis measured by TUNEL through- no proper brain structure found by E15.5 in a Nestin- background (Fig. 2b). Terf2 was essential for brain development Similarly, Terf2Emx1-Cre embr had only the vestige of the forebrain with residual ap tosis at E13.5 (Fig. 2c), and a complete disappeara of the Emx1-Cre expressing areas by E15.5 (Fig. 2 while the ganglionic eminence in which Emx1-Cre not expressed was intact during neurogenesis. Th no proper brain structure found by E15.5 in a Nestin-Cre background (Fig. 2b). Similarly, Terf2Emx1-Cre embryos had only the vestige of the forebrain with residual apop- tosis at E13.5 (Fig. 2c), and a complete disappearance of the Emx1-Cre expressing areas by E15.5 (Fig. 2d), while the ganglionic eminence in which Emx1-Cre is not expressed was intact during neurogenesis. These data suggest an essential requirement of TERF2 and the Terf2 inactivation induced massive neural apoptosis during development Loss of the brain regions in which Cre recombinase was expressed at birth led us to examine the embryonic brains during neurogenesis. The Terf2Nes-Cre embryos at E13.5 showed massive apoptosis measured by TUNEL through- out the developing brain (Fig. 2a). As a result, there was 3 3 495 Histochem Cell Biol (2017) 148:489–501 Fig. 3 Terf2 inactivation disrupted the brain structure. a Develop- mental defects in the (Terf2;Trp53)Nes-Cre cerebellum. Nissl staining indicates that the (Terf2;Trp53)Nes-Cre cerebellum was much smaller (red arrowhead) and the size of cells inside was bigger and not organ- ized. The Purkinje cell layer (yellow arrowhead) detected by Cal- bindin immunostaining displayed the complete misalignment of the layer and could not be found as a single cell layer as shown in the control brain. Glial fibrillary acidic protein (GFAP) immunoreactivity shows the disruption of the Bergmann glial network (yellow arrow). The populations of neurons (NeuN immuno-positive) and oligoden- drocytes (MBP and Myelin-PLP immuno-positive) were strikingly reduced, and the structure was disorganized (red arrows). These cerebellar defects lead to severe ataxia in the (Terf2;Trp53)Nes-Cre animals (see the accompanied video clip). Mo the molecular layer in the cerebellum, Gr the granule cell layer in the cerebellum, Ctrl control. Scale bars are as indicated in the figures. b Disruption of the six-layered cortical structure. The layered cortical structure in the 3-week-old brain was examined with four different markers; the upper layers—Cux1; the lower layers—Tbr1, Ctip2, Foxp2. The res- cued part of the Terf2-null cortex in either Atm or Trp53 deficiency ((Terf2;Trp53)Nes-Cre, Terf2Emx1-Cre;Atm−/− and (Terf2;Trp53)Emx1-Cre) did not form the upper layers (Cux1 immuno-positive), and the layers IV, V, VI were located to the upper part of the cortex. The multiple defects were found in the Terf2‑null brains importance of telomere stability for cell survival during neurogenesis. Although neuron-specific class III β-tubulin (Tuj1), which is a marker for postmitotic neurons, showed a normal distri- bution in the developing brains of the Terf2/Atm and Terf2/ p53 double null embryos (Fig. 2b), the restored portion in the mature brains exhibited several faulty features. Trp53 inactivation led to better restoration of the missing parts in the Terf2-null brain than Atm deficiency did. However, the restoration was still incomplete (Fig. 1b). The hippocampus was not restored in the Terf2/Atm and Terf2/Trp53 double null brains (Fig. 1b). Terf2 was essential for brain development In contrast, the (Terf2;Lig4;Trp53)Emx1-Cre cortex displayed all six layers including the upper layers (Cux1 immuno-positive). The size of immunoposi- tive cells to the layer markers in the Terf2/Trp53 dual null cortex was much bigger and less organized than that of controls. Roman numeric signs indicate the cortical layers. The white dotted lines indicate the surface of the cerebral cortex. The scale bar is 100 μm ◂ Noticeably, (Terf2;Trp53)Nes-Cre animals displayed severe ataxia, hence could live only up to the time of weaning (supplementary video clip). This severe ataxic phenotype resulted from the malformation of the cerebellum which was very small and did not have any organized structure of the cerebellum, particularly a complete disruption of the Purkinje cell layer (Fig. 3a). Yet the mutant cerebellum contained all of the cellular components including Purkinje cells (Calbindin immuno-positive), Bergmann glia (glial fibrillary acidic protein (GFAP) immuno-positive), and oligodendrocytes (myelin basic protein (MBP) and myelin proteolipid protein (Myelin-PLP) immuno-positive), except granule cells (NeuN immuno-positive) (Fig. 3a). Tumo- rigenicity in the nervous system of the (Terf2;Trp53)Nes-Cre animals could not be evaluated because of a short life span. Although both Atm and Trp53 inactivation could reduce apoptosis dramatically in the Terf2-null brains (Fig. 2), there was a difference between these two genetic back- grounds. Neural cell death in both Terf2Nes-Cre and Ter- f2Emx1-Cre embryos had disappeared by E15.5 in a Trp53- null background, whereas Atm inactivation could not stall most of the apoptosis in the ventricular zone (VZ) where neural progenitor cells are located in (Fig. 2d). Atm and Trp53, but not Atr, inactivation rescued neural apoptosis resulting from Terf2 deficiency Previously, it has been demonstrated that DNA damage trig- gered by Terf2 inactivation in vitro induced Atm-dependent Atr-independent DDR (Karlseder et al. 1999; Celli and de Lange 2005; Zhang et al. 2006, 2007; Denchi and de Lange 2007). So, next we tested the in vivo involvement of Atm and Atr, as well as Trp53, a common downstream substrate, in lethality and massive neural apoptosis resulting from Terf2 inactivation during neurogenesis. Floxed Terf2 animals were cross-bred with Atr (conditional inactivation: AtrNes-Cre and AtrEmx1-Cre), Atm (germline inactivation: Atm−/−) or Trp53 (conditional inactivation; Trp53Nes-Cre and Trp53Emx1-Cre) animals. Similar to the in vitro situation (Denchi and de Lange 2007; Karlseder et al. 1999), Atr inactivation did not have any influence on the phenotypes observed in both Terf2Nes-Cre and Terf2Emx1-Cre animals (Fig. 2). Terf2Emx1-Cre; Atm−/− and (Terf2;Trp53)Emx1-Cre animals also did not show any discernible improvement in gross phenotypes includ- ing smaller body size and shorter life span observed in the Terf2Emx1-Cre animals (data not shown). g Next, we analyzed the six-layered cerebral cortical struc- ture created in an inside-out manner during development (Greig et al. 2013). The Foxp2, Ctip2, and Tbr1 immuno- positive neurons, which are generated during an early stage of cortical development and localized in the lower layers of the cerebral cortex in normal development, were found in the upper cortical part of the (Terf2;Trp53)Nes-Cre and (Terf2;Trp53)Emx1-Cre brains (Fig. 3b). Similarly, the Ter- f2Emx1-Cre;Atm−/− cortex showed mis-localization of Ctip2 and Tbr1 immuno-positive neurons. Furthermore, there were no proper upper layers formed (layers I, II, and III), which were Cux1 immuno-positive, in both the Terf2/Atm and Terf2/Trp53 double null cortices, suggesting that the Terf2-null developing brain could not generate cortical neu- rons at the later stage during development in both Atm- and Trp53-deficient backgrounds (Fig. 3b). In a Nestin-Cre background, Atm inactivation could not rescue prenatal lethality resulting from Terf2 inactiva- tion, while (Terf2;Trp53)Nes-Cre animals were born alive. 1 3 3 Histochem Cell Biol (2017) 148:489–501 496 496 Histochem Cell Biol (2017) 148:489–501 496 Histochem Cell Biol (2017) 148:489 501 Fig. 4 Defective glia network was formed in the mutant brain. a Increased immunoreactivity of GFAP in the Terf2 mutant brain. The Terf2-null brains in both Atm- and Trp53-deficient backgrounds showed the high level of GFAP immuno-staining compared to that of the control brain, which is a common feature found in the brain of genomic instability animal models. Atm and Trp53, but not Atr, inactivation rescued neural apoptosis resulting from Terf2 deficiency The corpus callosum, which is a glia-rich structure, was not defined in the Terf2-null brain ((Terf2;Trp53)Nes-Cre, Terf2Emx1-Cre;Atm−/− and (Terf2;Trp53)Emx1-Cre). The (Terf2;Lig4;Trp53)Emx1-Cre brain showed partial recovery of the corpus callosum. The red dotted lines demarcate the normal corpus callosum in the control brain and the partially restored cor- pus callosum in the (Terf2;Lig4;Trp53)Emx1-Cre brain. The size of GFAP immuno-positive cells in the Terf2/Trp53 double null brains, not in both Terf2/Atm double and Terf2/Lig4/Trp53 triple deficient brains, was bigger than that of controls (red arrows). The scale bar Fig. 4 Defective glia network was formed in the mutant brain. a Increased immunoreactivity of GFAP in the Terf2 mutant brain. The Terf2-null brains in both Atm- and Trp53-deficient backgrounds showed the high level of GFAP immuno-staining compared to that of the control brain, which is a common feature found in the brain of genomic instability animal models. The corpus callosum, which is a glia-rich structure, was not defined in the Terf2-null brain ((Terf2;Trp53)Nes-Cre, Terf2Emx1-Cre;Atm−/− and (Terf2;Trp53)Emx1-Cre). E 1 C Fig. 4 Defective glia network was formed in the mutant brain. a Increased immunoreactivity of GFAP in the Terf2 mutant brain. The Terf2-null brains in both Atm- and Trp53-deficient backgrounds showed the high level of GFAP immuno-staining compared to that of the control brain, which is a common feature found in the brain of genomic instability animal models. The corpus callosum, which is a glia-rich structure, was not defined in the Terf2-null brain ((Terf2;Trp53)Nes-Cre, Terf2Emx1-Cre;Atm−/− and (Terf2;Trp53)Emx1-Cre). The (Terf2;Lig4;Trp53)Emx1-Cre brain showed partial recovery of the corpus callosum. The red dotted lines demarcate the normal corpus callosum in the control brain and the partially restored cor- pus callosum in the (Terf2;Lig4;Trp53)Emx1-Cre brain. The size of GFAP immuno-positive cells in the Terf2/Trp53 double null brains, not in both Terf2/Atm double and Terf2/Lig4/Trp53 triple deficient brains, was bigger than that of controls (red arrows). The scale bar Fig. 4 Defective glia network was formed in the mutant brain. a Increased immunoreactivity of GFAP in the Terf2 mutant brain. The Terf2-null brains in both Atm- and Trp53-deficient backgrounds showed the high level of GFAP immuno-staining compared to that of the control brain, which is a common feature found in the brain of genomic instability animal models. The corpus callosum, which is a glia-rich structure, was not defined in the Terf2-null brain ((Terf2;Trp53)Nes-Cre, Terf2Emx1-Cre;Atm−/− and (Terf2;Trp53)Emx1-Cre). The (Terf2;Lig4;Trp53)Emx1-Cre brain showed partial recovery of the corpus callosum. Atm and Trp53, but not Atr, inactivation rescued neural apoptosis resulting from Terf2 deficiency The red dotted lines demarcate the normal corpus callosum in the control brain and the partially restored cor- pus callosum in the (Terf2;Lig4;Trp53)Emx1-Cre brain. The size of GFAP immuno-positive cells in the Terf2/Trp53 double null brains, not in both Terf2/Atm double and Terf2/Lig4/Trp53 triple deficient brains, was bigger than that of controls (red arrows). The scale bar is 200 μm. b Defective oligodendrocyte network in the Terf2 mutant brain. Oligodendrocytes were detected by two different markers: MBP (myelin basic protein) and Myelin-PLP (proteolipid protein). The Terf2ctrl cerebral cortex displayed a rich network of oligoden- drocytes, particularly in the corpus callosum. However, the Terf2 conditional knockout brains in all different genetic backgrounds showed a dramatic reduction of immunopositivity to MBP/Myelin- PLP, and agenesis of the corpus callosum (red arrows), in contrast to the increased astrocyte population in the Terf2-null brains (A panel above). And the genetic combination with Atm or Trp53 deficiency could not improve this defect. However, Lig4 inactivation resulted in partial recovery of the corpus callosum in the (Terf2;Trp53)Emx1-Cre brain. The corpus callosum shown in the lower panel was the poste- rior part of the corpus callosum. The scale bars are 100 μm 1 Trp53 inactivation resulted in multinucleated giant neural cells in the Terf2‑null brain g Neuropathological analysis revealed that the neu- rological abnormalities were moderately corrected in the (Terf2;Lig4;Trp53)Emx1-Cre brain. During embry- ogenesis, the reduction of neural cell death in the (Terf2;Lig4;Trp53)Emx1-Cre embryos was the same as the (Terf2;Trp53)Emx1-Cre embryos, most likely due to the effect of Trp53 inactivation (Fig. 2d). However, prolifer- ating cells at metaphase in the (Terf2;Lig4;Trp53)Emx1-Cre VZ were similar to those with two centrosomes found in control embryos (Fig. 5b). Consequently, the most obvious correction was disappearance of multinucleated giant cells in the triple conditional null mature brain (Figs. 4a, 5a). In addition, the upper layers of the cerebral cortex, which are Cux1 immuno-positive, were formed in the triple null brain (Fig. 3b). However, this recovery by Lig4 inactiva- tion was not complete, such as partial reconstruction of the hippocampus and CC compared to the control groups (Figs. 1b, 4). Furthermore, we noticed the existence of giant neural cells only in the Terf2/Trp53 double null brains, not in the Terf2/ Atm double null brains (Figs. 3, 4a, 5a). Each giant cell contained irregular shaped multiple nuclei connected to each other by thin strings (Fig. 5a). Also, the considerable amounts of DNA damage visu- alized by phosphorylated H2AX (γ-H2AX) foci forma- tion, which is a good marker for DNA strand breaks, were detected in all of the Terf2-null brains, but not in the control brain (Fig. 5a). In addition, telomere in situ visualization using a telomere-PNA probe showed its conjugated CY3 signals in the Terf2-null brains (Fig. 5a). These telomere- PNA signals were not noticeable in the control brain, sug- gesting that telomeres in Terf2-null neural cells were read- ily accessible by the telomere-PNA probe since they were not well protected. Next we examined whether multinucleated giant neural cells were formed during neurogenesis. Certainly, γ-H2AX foci formation in the Terf2-null brains, particularly in the VZ of the developing brains, was dramatically increased in both Atm- and Trp53-deficient backgrounds (Fig. 5b). However, the chromosome mass at metaphase visualized by phosphorylated H3 immunoreactivity (H3pS10) showed a significant increase only in the Terf2/Trp53 double null neu- ral progenitors (Fig. 5b). This giant chromosomal mass was not observed in an Atm-null background. It is possible that this giant chromosomal aggregation might partially result from incomplete cell division, since multiple centrosomes detected by γ-tubulin immunoreactivity in a single cell were observed only in the Terf2/Trp53 double null neural cells at metaphase (Fig. 5b). DNA ligase IV plays a role in neural abnormalities due to Terf2 deficiency during neurogenesis Previously it was reported that Lig4 plays a role in abnormal fusions of telomere ends in Terf2-deficient cells (Celli and de Lange 2005; Smogorzewska et al. 2002). So we tested whether Lig4 inactivation during neurogenesis could alleviate neural abnormalities observed in the (Terf2/Trp53)Emx1-Cre brain. As described before (Lee et al. 2012a), the (Lig4;Trp53)Emx1-Cre brain did not show any neural phenotypes related to telomere dysfunction. The triple conditional knockout animals (here- after (Terf2;Lig4;Trp53)Emx1-Cre) were born alive, but the (Terf2;Lig4;Trp53)Emx1-Cre animals died around 1 month of age similar to the (Terf2;Trp53)Emx1-Cre animals. 3 3 Histochem Cell Biol (2017) 148:489–501 497 embryonic brains (Fig. 5b); consequently, there were no multinucleated giant neural cells found in the Pot1aNes-Cre mature brains (Lee et al. 2014). Increased GFAP immunoreactivity was one of common features found in the brains of genomic instability animal models (Shull et al. 2009; Lee et al. 2012a, b). So we tested whether Terf2-deficient brains also exhibit increased GFAP immunoreactivity as a sign of genomic instability. Indeed the Terf2-null cortex showed increased GFAP immuno- positivity in both Atm- and p53-null backgrounds (Fig. 4a). Interestingly, the size of GFAP immuno-positive cells in Terf2/Trp53 double null brains was relatively big (Fig. 4a). On the contrary, the dramatic reduction of oligodendrocyte population which was visualized by MBP and Myelin-PLP immunoreactivity was found in the Terf2/Atm and Terf2/ Trp53 double null cortices (Fig. 4b). However, all of the Terf2-null cortices were in the absence of the corpus cal- losum (CC) structure, which functions as a communication path between two cerebral hemispheres (Fig. 4a, b). Trp53 inactivation resulted in multinucleated giant neural cells in the Terf2‑null brain Even though DNA damage detected by γ-H2AX foci was evident, neural progenitor cells with giant chromosomal mass were not observed in the Pot1aNes-Cre Telomere homeostasis is essential for brain development in the mouse A big conglomeration of chromosomes detected by phosphorylated H3 (H3pS10) immunoreactivity (green staining) was observed only in the Terf2/Trp53 double null metaphase cells ((Terf2;Trp53)Nes-Cre and (Terf2;Trp53)Emx1-Cre), not in Terf2/Atm dou- ble and Terf2/Lig4/Trp53 triple null metaphase cells. Centrosomes were visualized by γ-tubulin immunoreactivity (yellow arrowheads). Only Terf2/Trp53 double null metaphase cells in both Nestin and Emx-1 Cre backgrounds had multiple centrosomes (red staining) in a single-metaphase cell. Terf2Nes-Cre;Atm−/−, Terf2Emx1-Cre;Atm−/− and (Terf2;Lig4;Trp53)Emx1-Cre neural progenitor cells at metaphase had two centrosomes the same as the control embryonic brain (yel- low arrowheads). Pot1a deficiency (Pot1aNes-Cre) did not show any similar defects such as a big aggregation of chromosomes and mul- tiple centrosomes. The scale bars are as indicated in the figure. c A schematic illustration of signaling pathways induced by either DNA damage or telomere dysfunction to trigger apoptosis or cell cycle arrest during brain development. Telomere dysfunction resulting from either TERF2 or POT1a inactivation induces neural apoptosis which is ATM-Trp53 dependent during neurogenesis. Terf2 inactivation directly activates ATM-dependent signaling, while Pot1a inactivation induces ATM activation via ATR signaling during neurogenesis ◂ resting cells, most likely contributes to this difference in cellular viability as suggested before (Martinez et al. 2014). Interestingly, there was no similarity of the neural phe- notypes between the Terf2Nes-Cre and Pot1aNes-Cre animals (Lee et al. 2014), even though germline deletion of the Terf2 or Pot1a gene in the mouse resulted in embryonic lethality (Celli and de Lange 2005; Hockemeyer et al. 2006; Wu et al. 2006), Pot1a inactivation in neural progeni- tor cells did not induce massive neural apoptosis during embryogenesis, and the neurological defects resulting from Pot1a inactivation were restricted mainly to the cerebellum (Lee et al. 2014). Furthermore, Pot1aNes-Cre;Trp53−/− ani- mals did not show any sign of ataxia. On the contrary, Terf2 inactivation in the neural progenitor cells induced more global effects throughout the central nervous system. Also it appeared that there was a differentiation defect of late- born neurons originated from Terf2-null neural progenitor cells which underwent more cell divisions in a Trp53-defi- cient background. As illustrated in Fig. 5c, it is possible that the Shelterin complex with all components including POT1 presents only at the end of telomeres where the 3′ single-stranded GC rich overhang is exposed (de Lange 2005; Gramat- ges and Bertuch 2013; Palm and de Lange 2008). Telomere homeostasis is essential for brain development in the mouse For this reason, the impact resulting from Terf2 inactivation in the neural progenitor cells was more severe than that of Pot1a inactivation in the murine nervous system. Telomere homeostasis is essential for brain development in the mouse The role of TERF2 and POT1 as components of the Shel- terin complex is to protect the telomere ends so that genomic integrity is maintained (Celli and de Lange 2005; Denchi and de Lange 2007; Karlseder et al. 1999). Pre- viously the Terf2 gene of the mouse was conditionally targeted only in the liver and skin (Bojovic et al. 2013; Lazzerini Denchi et al. 2006; Martinez et al. 2014). Terf2 inactivation in the basal layer of epidermis using a K14- Cre animal line did not cause embryonic lethality (Bojovic et al. 2013), while Terf2 inactivation in epidermal stem cells 1 3 1 Histochem Cell Biol (2017) 148:489–501 498 1 3 499 Histochem Cell Biol (2017) 148:489–501 Fig. 5 The multinucleated giant neural cells were found only in the Terf2/Trp53 double null brain. a Giant neural cells in the Terf2/ Trp53 double null brains and a sign of DNA damage in the Terf2 null brain. The neural cells including neurons in the brain, particu- larly the cerebral cortex (CTX), were examined by Nissl staining and NeuN immunoreactivity. Giant neural cells were found only in the (Terf2;Trp53)Nes-Cre and (Terf2;Trp53)Emx1-Cre cortices. Giant neural cells had multiple nuclei in a single cell visualized by DAPI staining in the Terf2/Trp53 double deficient cortex. This kind of giant neural cells were not observed in the Terf2Emx1-Cre;Atm−/− and (Terf2;Lig4;Trp53)Emx1-Cre cortices. DNA damage or breaks could be visualized by immunostaining of phosphorylated H2AX (γ-H2AX) as foci formation in the nucleus. γ-H2AX foci were abundant in the giant nuclei of the Terf2/Trp53 double null cortices. Since Lig4 deficiency itself results in accumulation of γ-H2AX foci, the (Terf2;Lig4;Trp53)Emx1-Cre cortex showed strong foci formation in the nucleus. Telomeres were visualized by hybridization with Telomere- PNA probe conjugated with CY3 (red). Only the Terf2-null cortices in all different genetic backgrounds showed the high level of PNA probe positivity indicating that the PNA-telomere probe was hybrid- ized better with telomeres in the Terf2-null brains. The blue staining was DAPI staining for γ-H2AX immunostaining and PNA-telomere in situ detection. The scale bars are 100 μm except γ-H2AX immu- nostaining. b The high level of γ-H2AX foci and big chromosomes in the Terf2-null embryonic cortex during embryogenesis. DNA damage visualized by γ-H2AX foci was evident in the Terf2-null embryonic fore brains in different genetic backgrounds as well as in the Pot1aNes-Cre embryonic brain, particularly in the ventricular zone (VZ) of the cortex. References Bojovic B, Ho HY, Wu J, Crowe DL (2013) Stem cell expansion during carcinogenesis in stem cell-depleted conditional telom- eric repeat factor 2 null mutant mice. Oncogene 32(43):5156– 5166. doi:10.1038/onc.2012.555 Celli GB, de Lange T (2005) DNA processing is not required for ATM-mediated telomere damage response after TRF2 dele- tion. Nat Cell Biol 7(7):712–718. doi:10.1038/ncb1275 Davoli T, de Lange T (2012) Telomere-driven tetraploidiza- tion occurs in human cells undergoing crisis and promotes transformation of mouse cells. Cancer Cell 21(6):765–776. doi:10.1016/j.ccr.2012.03.044 Davoli T, Denchi EL, de Lange T (2010) Persistent telomere dam- age induces bypass of mitosis and tetraploidy. 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We thank the Hartwell Center for biotech support, the cytogenetics core for chromosome analysis and the Animal Resource Center for animal husbandry. ATM and Trp53 are key signaling mediators in telomere dysfunction due to Terf2 inactivation during brain development in the mouse One of the interesting observations was the entity of mult- inucleated giant neural cells only in the Terf2/Trp53 con- ditional knockout mature brain, possibly resulting from endoreduplication without proper cell division as sug- gested in other Terf2 conditional knockout animal models (Lazzerini Denchi et al. 2006; Martinez et al. 2014; Ullah et al. 2009). It had been demonstrated that endoredupli- cation and mitotic failure in a Trp53-dependent manner during telomere crisis lead to polyploid cell accumula- tion (Davoli and de Lange 2012; Pampalona et al. 2012; Davoli et al. 2010). This defect of multinucleated giant cells was resolved by Lig4 inactivation, suggesting that the ligation function of LIG4 plays a role in this particu- lar neural defect due to telomere dysfunction resulting from Terf2 inactivation during brain development. using a K5-Cre animal line led to partial embryonic lethal- ity and impaired skin development (Martinez et al. 2014). Similarly, here we demonstrated that a selective inactiva- tion of the Terf2 gene in the neural progenitor cells resulted in embryonic fatality and loss of brain structures, provid- ing another example that the protective role of TERF2 is important to maintain cellular and organismal viability. This result is clearly different from the animal model for selective inactivation of the Terf2 gene in the liver that showed normal liver function and regeneration through endoreduplication without cell division (Lazzerini Denchi et al. 2006). The status of cells, such as proliferating vs. Trp53 inactivation suppressed neural apoptosis trig- gered by Terf2 inactivation in the entire developing nerv- ous system including the VZ in which big metaphase cells could be formed, whereas Atm deficiency could not inhibit programmed cell death in the VZ of the Terf2-null embry- onic brain. Therefore, multinucleated giant neural cells 1 Histochem Cell Biol (2017) 148:489–501 500 were not observed in the Terf2/Atm knockout brain. Also this result suggests that Trp53 activation to induce neu- ral apoptosis in the Terf2-null VZ was Atm-independent. This observation was consistent with the previous reports demonstrating that ATM is involved in neural apoptosis via Trp53 activation in the postmitotic zone, not in the VZ (Lee et al. 2000, 2001; Orii et al. 2006). 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https://openalex.org/W3110775596	https://www.nature.com/articles/s41599-020-00675-9.pdf	English	null	Correction: Satisfaction of scientists during the COVID-19 pandemic lockdown	Humanities & social sciences communications	2,020	cc-by	299	OPEN Correction: Satisfaction of scientists during the COVID-19 pandemic lockdown Isabel J. Raabe , Alexander Ehlert , David Johann & Heiko Rauhut Isabel J. Raabe , Alexander Ehlert , David Johann & Heiko Rauhut Correction to: Humanities and Social Sciences Communications https://doi.org/10.1057/s41599-020-00618-4, published online 4 November 2020. © The Author(s) 2020 Table 2 in the original paper has been corrected to ensure that the columns in the top section are aligned appropriately with those in the lower section. Table 2 in the original paper has been corrected to ensure that the columns in the top section are aligned appropriately with those in the lower section. In addition, the following sentence in the legend to Fig. 1 has been corrected as follows to improve clarity: Original: For illustration purposes, this distribution was rescaled to rescaled to cover only a fraction of the ﬁgure. ed to: For illustration purposes, this distribution was rescaled to cover only a fraction of the ﬁgure. 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/. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2020) 7:168 \| https://doi.org/10.1057/s41599-020-00675-9 1
https://openalex.org/W4210927690	https://link.springer.com/content/pdf/10.1007/s00784-022-04400-5.pdf	English	null	The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo	Clinical oral investigations	2,022	cc-by	9,818	The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo Mira Günther1 · Lamprini Karygianni2 · Aikaterini Argyropoulou3 · Annette Carola Anderson Alexios Leandros Skaltsounis3 · Annette Wittmer4 · Kirstin Vach5 · Ali Al‑Ahmad1 Received: 22 December 2021 / Accepted: 29 January 2022 © The Author(s) 2022 / Published online: 9 February 2022 https://doi.org/10.1007/s00784-022-04400-5 Clinical Oral Investigations (2022) 26:4369–4380 https://doi.org/10.1007/s00784-022-04400-5 Clinical Oral Investigations (2022) 26:4369–4380 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Objective In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clini- cal ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms. i Materials and methods Initial in situ biofilm samples (2 h) from six healthy volunteers were treated ex vivo with R. officinalis extract at concentrations of 20 mg/ml and 30 mg/ml. The number of viable bacterial cells was determined by counting the colony-forming units. All surviving bacteria were isolated in pure cultures and identified using MALDI-TOF and biochemical testing procedures. Additionally, live/dead staining in combination with epifluorescence microscopy was used for visualizing the antimicrobial effects in the initial biofilms.fiii fi Results The number of colony-forming units in the R. officinalis–treated biofilms was significantly lower than in the untreated controls (p < 0.001). The reduction range of log10 was 1.64–2.78 and 2.41–3.23 for aerobic and anaerobic bacteria, respec- tively. Regarding the bacterial composition, large intra- and interindividual variability were observed. Except for Campylo- bacter spp., the average amount of all bacterial taxa was lower after treatment with R. officinalis than in the untreated biofilms. A total of 49 different species were detected in the untreated biofilms, while only 11 bacterial species were detected in the R. officinalis–treated biofilms. Live/dead staining confirmed that the R. officinalis–treated biofilms had significantly lower numbers of surviving bacteria than the untreated biofilms.fiii i Conclusions The treatment with R. officinalis extract has a significant potential to eliminate microbial oral initial biofilms. Clinical relevance The results of this study encourage the use of R. officinalis extracts in biofilm control and thus in the treatment of caries and periodontitis as a herbal adjuvant to synthetic substances. Introduction In dentistry, antimicrobial substances are used to prevent and treat bacteria-associated oral infections by reduc- ing the oral biofilm [1]. New substances are increasingly sought due to growing primary and secondary resistances against conventional synthetically produced antimicro- bial and antiseptic substances, e.g., chlorhexidine (CHX). Several studies show that, especially in the oral cavity, the prevalence of antibiotic-resistant bacterial strains is on the rise [2–5]. For this reason, the focus is shifting back towards plant-based alternatives, as plants and their extracts have been used as medicines for centuries. More than 300,000 plant extracts have been described to date [6, 7] and while many of these have a potential pharmacologi- cal application, their antimicrobial effects have only been studied in a few cases.ii Rosemary (Rosmarinus officinalis) extracts have been studied as potential therapeutic agents against various dis- eases [25, 26]. Several studies showed that R. officinalis exhibits hepatoprotective, anti-hyperglycemic, antifungal, antitumor, and anti-ulcerogenic effects [27, 28]. In addition, R. officinalis is reported to have antioxidant and antimicro- bial activities [27, 29]. These properties are thought to be due to the high content of phytochemicals such as carnosic acid, rosmarinic acid, or chlorogenic acid [27]. In organic rosemary extracts, diterpene derivatives, camphor, urolic acid, carnosic acid, rosmanol, and rosmarinic acid are con- sidered the main phenolic compounds [28, 30]. The different phenolic compositions are responsible for the antimicrobial properties of rosemary extracts [27]. In the aqueous R. offici- nalis extract, carnosol, rosmanol, carnosic acid, methyl car- nosate, and various flavonoids dominate [31]. Biofilm formation, defined as an organizational form of oral microorganisms and salivary components that irreversibly attach to oral surfaces and are embedded in a matrix of extracellular polymeric substances (EPS), leads to a change in the properties of microorganisms compared to those in the planktonic form [8–11]. Thus, biofilms show significantly higher resistance to antimi- crobial substances (100 to 1,000-fold) [2, 12, 13]. Oral biofilms are closely associated with diseases of the teeth and periodontium [14]. Although the genesis of inflamma- tory periodontal and carious diseases is multifactorial, the presence of an oral biofilm is a crucial etiological factor. Supragingival biofilms attached to tooth surfaces can lead to caries, whereas subgingival biofilms can lead to peri- odontal disease and peri-implantitis. Due to their high polyphenol content and its associated strong antioxidant effect, some extracts obtained from R. officinalis are already used in food preservation [28]. Abstract Keywords Initial biofilm · In situ · Alternative treatment · Rosmarinus officinalis extract · Antimicrobial effect · Live/dead staining · Culture technique * Ali Al‑Ahmad ali.al-ahmad@uniklinik-freiburg.de Mira Günther mira.guenther@web.de Lamprini Karygianni lamprini.karygianni@zzm.uzh.ch Annette Carola Anderson annette.anderson@uniklinik-freiburg.de Elmar Hellwig elmar.hellwig@uniklinik-freiburg.de 1 Department of Operative Dentistry and Periodontology, 2 Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland 3 Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece 4 Institute of Medical Microbiology and Hygiene, Faculty of Medicine, University of Freiburg, Freiburg, Germany 5 Institute for Medical Biometry and Statistics, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany Vol.:(0123456789) 1 3 * Ali Al‑Ahmad ali.al-ahmad@uniklinik-freiburg.de Mira Günther mira.guenther@web.de Lamprini Karygianni lamprini.karygianni@zzm.uzh.ch Annette Carola Anderson annette.anderson@uniklinik-freiburg.de Elmar Hellwig elmar.hellwig@uniklinik-freiburg.de 1 Department of Operative Dentistry and Periodontology, Medical Center, Faculty of Medicine, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany 2 Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland 3 Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece 4 Institute of Medical Microbiology and Hygiene, Faculty of Medicine, University of Freiburg, Freiburg, Germany 5 Institute for Medical Biometry and Statistics, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany 2 Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland 2 Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland 3 Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece 4 Institute of Medical Microbiology and Hygiene, Faculty of Medicine, University of Freiburg, Freiburg, Germany 5 Institute for Medical Biometry and Statistics, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany 3 Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece Lamprini Karygianni lamprini.karygianni@zzm.uzh.ch 4 Institute of Medical Microbiology and Hygiene, Faculty of Medicine, University of Freiburg, Freiburg, Germany Annette Carola Anderson annette.anderson@uniklinik-freiburg.de Elmar Hellwig elmar.hellwig@uniklinik-freiburg.de 1 Department of Operative Dentistry and Periodontology, Medical Center, Faculty of Medicine, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany Hugstetter Str. 55, 79106 Freiburg, Germany (0121 3456789) 3 Clinical Oral Investigations (2022) 26:4369–4380 4370 spectrum of activity against microorganisms and a low level of resistance [6]. Abstract Natural substances such as coffee extracts [18, 19], tea extracts [20], cranberry extracts [21], cranberry juice concentrate [22], or Manuka honey [23] have been shown to reduce the total bacterial count of adherent microorganisms. They all exhibit anti-adhesive and antimicrobial activity based on different mechanisms. For example, a decrease in bacterial membrane hydropho- bicity or reduced enzyme activity of fructosyl- and glyco- syltransferase are proposed as explanations for the strong anti-adhesive properties and the pronounced inhibition of biofilm by cranberry extract [15]. Tea extracts, on the other hand, are thought to lead to lysis of the bacterial cell membrane, resulting in the death of the bacterium [24].fi Introduction In pre- vious studies, it was shown that a methanolic crude extract of R. officinalis can inhibit the growth of Streptococcus mutans [29]. In addition, the antimicrobial activity of aque- ous and methanolic R. officinalis extracts against Streptococ- cus sobrinus and Streptococcus sanguinis has been reported. These bacterial species account for a large proportion of cariogenic biofilms. If targeted elimination or inactivation of these species was achieved, this could represent a funda- mental step in the prevention of carious lesions.fi p p For the prevention and treatment of oral diseases, diverse antimicrobial substances are used that either lead to a reduction of oral biofilm or which selectively inhibit bacterial taxa associated with specific oral diseases. Sub- stances that inhibit the growth and proliferation of micro- organisms or prevent their secondary adhesion mecha- nisms such as coaggregation and coadhesion are also applied. Given the growing inefficacy of synthetically produced agents, plant-derived compounds have gained interest in the development of therapies aiming to control oral biofilms [15]. Many plants have an intricate defense system to protect them from fungal and bacterial attacks. Various antimicrobial metabolites such as phytoanticipins, which provide a chemical barrier against microbial attack, and phytoalexins, which are secondary metabolites with antimicrobial activity, can be produced as part of the plant defense system [16, 17]. Representatives of phytoalexins include flavonoids, glycosteroids, terpenoids, or polyphe- nols [6]. In addition, plants have the ability to produce endogenous antimicrobial peptides that have a broad Thus, it can be assumed that R. officinalis extract sup- presses the growth of cariogenic streptococci, which would recommend its application for caries prevention [29]. To date, however, little is known about its use in dentistry [27]. It is unknown whether R. officinalis extract is effective against other oral bacterial taxa, for example, periodontal pathogens such as Porphyromonas gingivalis and Prevotella intermedia. Its potential as a natural antigingivitis or anti- periodontitis agent also needs further investigation. Therefore, the aim of the present study was to investigate the effect of R. officinalis extract on the initial oral biofilm, 1 3 3 4371 Clinical Oral Investigations (2022) 26:4369–4380 which is the onset of biofilm formation in the oral cavity. We investigated the antimicrobial activity of R. officinalis extract and determined its influence on the survival and diversity of the bacterial species within an initial oral bio- film. Preparation of the extract Powdered extract of R. officinalis (100 mg) was dissolved in 1000 µl dimethyl sulfoxide (DMSO) resulting in a concen- tration of 100 mg/ml. Complete dissolution of the extract was achieved by alternately heating in a water bath to 36 °C and vortexing for up to 20 min. This solution was used as a stock solution and was stored in a cool, dark place for a maximum of 48 h. On the day of the experiment, dilutions were made with phosphate-buffered saline (PBS) to obtain extract concentrations of 20 mg/ml and 30 mg/ml. A 0.2% CHX solution served as a negative control and a DMSO/ PBS (1/10) solution and two 0.9% NaCl solutions as posi- tive controls. The preparation of the extraction and the analysis were performed as previously described [32]. The investigated plant extract of Rosmarinus officinalis L. (Lamiaceae) was isolated from superficial plant parts collected from public ground in the Attica region of Greece (Lat.: 37°58′07.98″ N, Long.: 23°47′11.34″ E, elevation: 253 m). The Univer- sity of Athens has permission to collect small amounts of plant parts for research purposes if they are not from endan- gered or protected species. A sample of the collected plant parts was deposited at the herbarium in the Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrias University of Athens under number: R. officinalis-KL 163. To isolate the extract, the collected plant parts were ground into a fine powder with homogeneous particle size (SCIS, Allenwest-Eac ltd) followed by a 15-min ultrasound-assisted extraction (Elma S 100H) with 100% methanol as the extraction solvent. Extrac- tion took place for 15 min at room temperature and a ratio of 1:10 of plant per solvent. Finally, the extract was separated from the solvent by evaporation at 40 °C under reduced pres- sure (Buchi Rotavapor R-200). The prepared dry extract was provided by the Faculty of Pharmacy, National and Kapodis- trias University of Athens, for the study of its antimicrobial activity against initial oral biofilms. Introduction For this purpose, initial oral biofilms were cultivated in situ on bovine enamel slabs and treated ex vivo with R. officinalis extract solution of different concentrations and control solutions. As a null hypothesis, it was assumed that the R. officinalis extract showed no significant antimicrobial and antibiofilm effect on initial oral biofilms. distance from the lower edge: 8 mm, distance from the left and right edges: 20 mm, distance between the different tracks: 10.4 mm. For fully automated development of the HPTLC plates, the ADC2 automated development chamber (CAMAG) was used under the following settings: chamber satura- tion: 20 min, preconditioning of the plate: 10 min at 33% relative humidity (MgCl2), drying of the plate: 5 min. The mobile phases used were dichloromethane, methanol, water (70:30:4; v/v/v) and ethyl acetate, methanol, formic acid, and water (50:10:7:1; v/v/v/v). Subsequent measurements and documentation were performed with the CAMAG® TLC Visualizer 2, taking images at 254 nm and 366 nm. Protocol for the treatment of the initial biofilm with the R. officinalis extract Each of the six volunteers wore the splint system with six BES twice for 120 min each in two different experiments. In total, 12 BES were treated with each concentration of R. officinalis extract. A total of 24 biofilm-covered BES were treated with 0.9% NaCl solution as a negative control, and 12 BES were treated with DMSO/PBS solution as a second neg- ative control. After wearing, to ensure that all non-adherent bacteria were removed, the splints and BES were rinsed with 0.9% NaCl solution for 30 s. The BES were then removed from the splint system using sterile dental forceps without touching the surface covered with initial biofilm. One of the six biofilm-covered BES was placed in the sample extract of R. officinalis at a concentration of 20 mg/ml and one at a concentration of 30 mg/ml. One served as a positive control and was treated with 0.2% CHX. Two BES were treated with 0.9% NaCl and one with DMSO/PBS solution. These three served as negative controls. The BES covered with the initial oral biofilm were incubated for 10 min in the differ- ent treatment solutions consisting of R. officinalis extract as well as the controls. The test concentrations were determined in a pilot test, which showed that it is sufficient to evalu- ate the extract at concentrations of 20 mg/ml and 30 mg/ ml, respectively. The BES were then rinsed again with 0.9% NaCl solution to clear them of extract or control solution residues. Subsequently, the BES were submitted to further microbiological and molecular biological test procedures. As proof of sterility, the disinfected BES were placed in an NaCl solution (0.9%) in the ultrasonic bath for 3 min. The NaCl-treated biofilms were plated onto yeast-cysteine blood agar (HCB) plates and Columbia blood agar (CBA) plates, incubated accordingly and evaluated. The BES were removed (26 h before further use), placed in a sterile Petri dish for 2 h to allow the ethanol to evaporate, and then placed in 0.9% NaCl solution for 24 h to hydrate [33, 35]. An individual intraoral acrylic splint system for the upper jaw was fabricated for each volunteer. It contained six retention wells on the inner surface of the vestibular acrylic components. The retentions were located in the approxi- mal area between the first premolar and the second molar (Fig. 1). Selection of the study participants A total of six volunteers aged between 21 and 51 years were selected to participate in the study. All participants were in good general health. The following exclusion criteria were defined: (1) cardiovascular diseases, (2) blood coagulation disorders or blood diseases, (3) intake of medication, (4) allergy to the materials used for the splints, (5) pregnancy, (6) use of antibiotics or mouth rinses in the last 6 months prior to the start of the study, (7) suffering from xerostomia (dry mouth). The volunteers did not regularly consume alco- hol or drugs and were nonsmokers. A healthy adult complete dentition or adult dentition with adequate prosthetic and/or conservative care was required to ensure adequate stability of the splint system and undisturbed bacterial attachment. All participants were free of active carious lesions or peri- odontal disease. The study participants were neither allowed to eat anything nor to perform oral hygiene measures 2 h prior and during initial biofilm formation. Before the start of the study, all participants gave their written informed con- sent. The study was approved by the Ethics Committee of the Albert-Ludwigs-University Freiburg (No. 502/13). To analyze the composition of the extract, high-per- formance thin-layer chromatography (HPTLC) was per- formed at the Faculty of Pharmacy, National and Kapo- distrias University of Athens, using a CAMAG® system. To prepare the sample, 10 mg of extract was added to 1 ml of methanol and this solution was subsequently applied to a 20 × 10 cm TLC plate (silica gel 60 F254, Merck) with the aid of an automatic sampler (ATS4, CAMAG). The process was controlled using VisionCats 2.3 software (CAMAG). The following settings were selected: applica- tion volume: 8 µl, number of lanes: 6, band length: 8 mm, 1 1 3 4372 Clinical Oral Investigations (2022) 26:4369–4380 Protocol for the treatment of the initial biofilm with the R. officinalis extract Before each experiment, the splint systems were disinfected with isopropyl alcohol and then rinsed with 0.9% NaCl solution. The BES were fixed in the wells using the addition-cured Silicone Panasil® (Kettenbach GmbH & Fig. 1 Individual upper jaw acrylic splint system. The BES (bovine enamel slabs) were attached at positions 1 to 6 of the splint with their upward-facing surfaces exposed to the buccal tooth surfaces. All other surfaces were covered with silicon Obtaining initial oral biofilm from intraoral splint systems Co. KG, Eschenburg, Germany) as previously described [33–36]. They were positioned so that only the enamel sur- face was in contact with the oral cavity. To avoid unwanted contact between the BES and the vestibular tooth surfaces, the gingiva, or the alveolar mucosa and to allow saliva flow, the vestibular area of the splint was bent 1 mm outward. All six volunteers carried the splint twice for 120 min. Co. KG, Eschenburg, Germany) as previously described [33–36]. They were positioned so that only the enamel sur- face was in contact with the oral cavity. To avoid unwanted contact between the BES and the vestibular tooth surfaces, the gingiva, or the alveolar mucosa and to allow saliva flow, the vestibular area of the splint was bent 1 mm outward. All six volunteers carried the splint twice for 120 min. Bovine enamel slabs (BES) were used to generate an ini- tial biofilm in vivo. For this purpose, bovine mandibular incisors were extracted from freshly slaughtered 2-year-old BSE (bovine spongiform encephalopathy)-free cattle at the Freiburg abattoir (Freiburg, Germany). Cylindrical enamel slabs (diameter of 5 mm) were punched from the labial sur- faces of the teeth and reduced to a height of approximately 1.5 mm. The enamel side was ground to a flat surface using a wet disc grinder (Knuth-Rotor 3, Struers GmbH, Ballerup, Denmark) with 220 and 500 grit sandpaper and then fin- ished and polished with 1200, 2400, and 4000 grit, as was described previously [33–35]. The specimens were ground so that no dentin was exposed on the surface. The BES had a thickness of around 1.0 mm and a diameter of 5 mm and thus an enamel surface area of 19.635 mm2. The BES were disinfected in 70% ethanol for at least 48 h. Statistical analysis The effects of treatment with R. officinalis extract were ana- lyzed in a detailed evaluation for all six volunteers. In a descriptive analysis, the median, mean, and standard devia- tion were calculated. A graphical representation was per- formed using boxplots. Mixed linear models with random intercepts for each subject were fitted to compare the treat- ment groups. Here, because of the multiple measurements for each subject, the subject was considered a cluster. A correction was made for multiple testing using the Scheffe method. The significance level was set to p = 0.05. All statis- tical analyses were performed using the statistical software STATA 14.1. Quantification of the adherent oral microorganisms in the initial biofilms Afterward, the number of colony-forming units (CFU) was determined. The quantification was performed using an opti- cal system (WTW BZG 40, Weilheim, Germany). Quantification of the adherent oral microorganisms in the initial biofilms The six treated BES were each added into Eppendorf tubes containing 1 ml 0.9% NaCl. They were treated for 3 min at 70% in an ultrasonic bath and subsequently vortexed for 60 s to enable the removal of the adherent microorganisms from the BES surfaces. A dilution series with 0.9% NaCl was then prepared for each sample up to a dilution of 1:103 and 1:104. The bacterial species were subsequently cultured and identified as previously described [37]. A 100-µl ali- quot of each sample and dilution was plated onto one HCB plate and one CBA plate using a sterile glass spatula. The HCB plates served to cultivate and isolate anaerobic bacteria and were incubated in an anaerobic pot with a gas generator Fig. 1 Individual upper jaw acrylic splint system. The BES (bovine enamel slabs) were attached at positions 1 to 6 of the splint with their upward-facing surfaces exposed to the buccal tooth surfaces. All other surfaces were covered with silicon 1 3 3 4373 Clinical Oral Investigations (2022) 26:4369–4380 that the enamel side containing the initial biofilm was facing downward. Immediately thereafter, the Axio Observer.Z1 epifluorescence microscope (Carl Zeiss Microscopy, Jena, Germany) with a 63 × oil immersion objective was used for analysis. The Filter Set 38 HE was used to visualize the SYTO-9 staining (live bacteria), and the Filter Set 43 HE from Zeiss (Carl Zeiss Microscopy, Jena, Germany) was used for PI (dead bacteria). Images were recorded using the Zeiss AxioCam ICc5 CCD 5-megapixel camera (Carl Zeiss Microscopy, Jena, Germany). Ten images with 968 × 728 pixels were taken per sample. The scale per pixel was 0.144 µm × 0.144 µm, resulting in an image size of approxi- mately 140 µm × 105 µm. The area of one image corre- sponds to 0.0147 mm2 of the sample surface. The individual images were taken using an ApoTome.2 system (Carl Zeiss Microscopy, Jena, Germany) which prevents stray light in the focal plane from being perceived as an interfering signal by detecting the magnification and pushing an appropriate grating into the beam path. Thus, stray fluorescence signals caused by unfocused light are reduced. Some exemplary images are shown in Fig. 2. (GENbox, Biomerieux) to achieve an anaerobic environment at 37 °C for 7–10 days. The CoBl plates served to cultivate and isolate aerobic and facultative anaerobic bacteria and were incubated in an atmosphere with 5% CO2 for 3–5 days. Live/dead staining and epifluorescence microscopy for visualization and quantification of initial biofilms The fluorescent SYTO® 9 stain and propidium iodide (PI) assay (LIVE/DEAD® BacLight™ Bacterial Viability Kit, L7007, Life Technologies GmbH, Darmstadt, Germany) was applied to determine bacterial viability and to differ- entiate between live and dead bacteria within the biofilm samples as previously described [39]. Both stains, SYTO-9 and PI, were removed from the freezer (− 22 °C), thawed at room temperature, and mixed in a 1:1 ratio. Each BES was individually stained and microscopically examined. Staining solution (1 µl) was added to a well plate containing 500 µl NaCl, and the BES was placed inside and incubated for 10 min in a dark chamber at room temperature. After briefly washing it in 0.9% NaCl solution, it was placed on a special chamber slide (µ-Slide 8 Well, ibidi GmbH) that had a drop of 15 µl 0.9% NaCl on it. It was important to ensure that the dye-treated enamel slide was positioned so Identification of the adherent oral microorganisms within the initial biofilms In order to differentiate the surviving microorganisms, the different colony types were determined based on their color and shape, odor, and hemolysis behavior, and their respec- tive numbers were determined. Isolates were sub-cultivated to obtain pure cultures. For identification of these pure cul- tures, a MALDI-TOF analysis was performed in a MALDI BiotyperMicroflex LT (Maldi Biotyper, Bruker Daltonik GmbH, Bremen, Germany) as previously described [38]. The integrated Biotyper 3.0 software recorded mass spectra according to the manufacturer’s instructions. In addition, the software equates the recorded spectra with a reference database incorporating 3,740 reference spectra (319 gen- era and 1,946 species), whereby the results are listed as log score values. These values are used to indicate the validity of the results and correlate with the probability of correct species identification. Values of ≥ 2.0 were assumed to indi- cate species identification, and values ≥ 1.7 were assumed to indicate genus identification. For values below 1.7, no sig- nificant similarity with a spectrum deposited in the database could be detected. In cases where ambiguous results were obtained, the procedure was repeated. Effects of R. officinalis extract on the viable counts of oral microorganisms in initial biofilms Figure 3 shows the efficacy of R. officinalis extract on the log counts of adherent oral microorganisms in the initial biofilms, as well as the untreated positive (CHX), negative (NaCl), and neutral (DMSO) controls. The R. officinalis extract displayed a pronounced antimicrobial effect on the total bacterial counts of initial microbial biofilms in situ. The untreated controls revealed mean log10 CFU/cm2 values of 4.20 ± 0.44 for initially adherent oral aerobic microor- ganisms and mean log10 CFU/cm2 values of 4.05 ± 0.73 for initially adherent oral anaerobic microorganisms; the neutral 1 3 1 3 controls (DMSO) revealed mean log10 CFU/cm2 values of 4.10 ± 0.79 for aerobic microorganisms and 4.16 ± 0.68 for showed no bacterial growth, resulting in a 100% reduc- tion. Incubation of initial biofilms with R. officinalis extract Fig. 2 Epifluorescence micros- copy images demonstrating the effect of R. officinalis on initial bacterial biofilm (2 h) examined by live/dead staining followed by fluorescence microscopy. Differentiation between live (green) and dead (red) micro- organisms. Example images of a DMSO-treated biofilms, b R. officinalis (20 mg/ml)–treated biofilms, c CHX-treated bio- films, d NaCl-treated biofilms (negative control) Fig. 3 A, B Boxplots depicting colony forming unit (CFU) counts, showing the antimicrobial efficacy of R. officinalis against aerobic (A) and anaerobic (B) oral microorganisms after initial adhesion (2 h). The following treatment groups are shown: CHX-treated posi- tive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ ml and 30 mg/ml. The CFUs are presented on a log10 scale per square centimeter and the p-values are shown in the diagram 4374 Clinical Oral Investigations (2022) 26:4369–4380 4374 Clinical Oral Investigations (2022) 26:4369–4380 Fig. 2 Epifluorescence micros- copy images demonstrating the effect of R. officinalis on initial bacterial biofilm (2 h) examined by live/dead staining followed by fluorescence microscopy. Differentiation between live (green) and dead (red) micro- organisms. Example images of a DMSO-treated biofilms, b R. officinalis (20 mg/ml)–treated biofilms, c CHX-treated bio- films, d NaCl-treated biofilms (negative control) Fig. 3 A, B Boxplots depicting colony forming unit (CFU) counts, showing the antimicrobial efficacy of R. officinalis against aerobic (A) and anaerobic (B) oral microorganisms after initial adhesion (2 h). The following treatment groups are shown: CHX-treated posi- Fig. 3 A, B Boxplots depicting colony forming unit (CFU) counts, showing the antimicrobial efficacy of R. Effects of R. officinalis extract on the viable counts of oral microorganisms in initial biofilms Thus, a sig- nificantly lower number of different species is present in the samples treated with R. officinalis extract (p < 0.05). Treatment with CHX killed all bacteria, thereby result- ing in the elimination of the original bacterial community. biofilms (20 mg/ml and 30 mg/ml). In summary, a total of 49 different species could be detected in samples from six study participants with a clear dominance (30.92%) of 10 streptococcal species (S. oralis, S. mitis, S. sanguinis, S. parasanguinis, S. gordonii, S. salivarius, S. vestibularis, S. anginosus, S. infantis, S. intermedius). As shown in Fig. 5, in both untreated and neutral control biofilms, a total of 46 different bacterial species were identified, 38 of which were only isolated from the untreated biofilms. In the R. officinalis extract–treated samples, the number of differ- ent surviving bacterial species was reduced from 46 to 11 over six individuals. Three of these bacterial species could not be detected in the controls (NaCl or DMSO). The bacteria detected in the R. officinalis extract–treated biofilms included species of the genera Streptococcus, Rothia, Haemophilus, and Campylobacter. Thus, a sig- nificantly lower number of different species is present in the samples treated with R. officinalis extract (p < 0.05). Treatment with CHX killed all bacteria, thereby result- ing in the elimination of the original bacterial community. 20 mg/ml with a mean decrease of 2.92 log steps to a mean value of 1.28 ± 1.24 log10 CFU/cm2 (p < 0.001 both) for aerobic bacteria and a mean decrease of 3.23 log levels to a mean value of 0.82 ± 1.32 log10 CFU/cm2 (p < 0.001 both) for anaerobic bacteria. At a concentration of 30 mg/ml R. officinalis extract, a mean decrease of 3.49 log levels to a mean value of 0.71 ± 1.16 log10 CFU/cm2 (p < 0.001 both) for aerobic bacteria and a mean decrease of 3.64 log levels to a mean value of 0.41 ± 1.01 (p < 0.001 both) for anaerobic bacteria could be detected. Effects of R. officinalis extract on the viable counts of oral microorganisms in initial biofilms officinalis against aerobic (A) and anaerobic (B) oral microorganisms after initial adhesion (2 h). The following treatment groups are shown: CHX-treated posi- tive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ ml and 30 mg/ml. The CFUs are presented on a log10 scale per square centimeter and the p-values are shown in the diagram tive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ ml and 30 mg/ml. The CFUs are presented on a log10 scale per square centimeter and the p-values are shown in the diagram Fig. 3 A, B Boxplots depicting colony forming unit (CFU) counts, showing the antimicrobial efficacy of R. officinalis against aerobic (A) and anaerobic (B) oral microorganisms after initial adhesion (2 h). The following treatment groups are shown: CHX-treated posi- tive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ ml and 30 mg/ml. The CFUs are presented on a log10 scale per square centimeter and the p-values are shown in the diagram controls (DMSO) revealed mean log10 CFU/cm2 values of 4.10 ± 0.79 for aerobic microorganisms and 4.16 ± 0.68 for anaerobic microorganisms. The biofilms treated with CHX showed no bacterial growth, resulting in a 100% reduc- tion. Incubation of initial biofilms with R. officinalis extract showed a significant reduction both in the concentration of 1 3 3 4375 Clinical Oral Investigations (2022) 26:4369–4380 biofilms (20 mg/ml and 30 mg/ml). In summary, a total of 49 different species could be detected in samples from six study participants with a clear dominance (30.92%) of 10 streptococcal species (S. oralis, S. mitis, S. sanguinis, S. parasanguinis, S. gordonii, S. salivarius, S. vestibularis, S. anginosus, S. infantis, S. intermedius). As shown in Fig. 5, in both untreated and neutral control biofilms, a total of 46 different bacterial species were identified, 38 of which were only isolated from the untreated biofilms. In the R. officinalis extract–treated samples, the number of differ- ent surviving bacterial species was reduced from 46 to 11 over six individuals. Three of these bacterial species could not be detected in the controls (NaCl or DMSO). The bacteria detected in the R. officinalis extract–treated biofilms included species of the genera Streptococcus, Rothia, Haemophilus, and Campylobacter. The shift of bacterial composition in the initial microbial biofilm after incubation with R. officinalis The different bacterial species were determined for the cultivable bacteria of the untreated biofilms and were compared to the composition of the R. officinalis extract–treated samples. Figure 4 shows the composition of the initial biofilms of the untreated biofilms, the neutral controls (DMSO), and of the R. officinalis extract–treated Fig. 4 Heatmap demonstrating the absolute distribution (in log10/ml) of different bacterial groups/species among six study probands after a 2-h treatment with R. officinalis extract. An untreated negative con- trol (NaCl 0.9%), a positive control (CHX 0.2%), and a control with DMSO (10%) were also used, as was the R. officinalis extract (20 mg/ ml and 30 mg/ml) with a 10-min exposure time. The participant num- bers for each treatment group are shown in columns and the variables (bacterial groups, species) in rows. The colors as depicted on the color scale bars on the right vary to indicate the change in data values for the different samples (low: 0–35, moderate: 35–70, high: 70–100) 1 Fig. 4 Heatmap demonstrating the absolute distribution (in log10/ml) of different bacterial groups/species among six study probands after a 2-h treatment with R. officinalis extract. An untreated negative con- trol (NaCl 0.9%), a positive control (CHX 0.2%), and a control with DMSO (10%) were also used, as was the R. officinalis extract (20 mg/ ml and 30 mg/ml) with a 10-min exposure time. The participant num- bers for each treatment group are shown in columns and the variables (bacterial groups, species) in rows. The colors as depicted on the color scale bars on the right vary to indicate the change in data values for the different samples (low: 0–35, moderate: 35–70, high: 70–100) Fig. 4 Heatmap demonstrating the absolute distribution (in log10/ml) of different bacterial groups/species among six study probands after a 2-h treatment with R. officinalis extract. An untreated negative con- trol (NaCl 0.9%), a positive control (CHX 0.2%), and a control with DMSO (10%) were also used, as was the R. officinalis extract (20 mg/ ml and 30 mg/ml) with a 10-min exposure time. The participant num- bers for each treatment group are shown in columns and the variables (bacterial groups, species) in rows. The colors as depicted on the color scale bars on the right vary to indicate the change in data values for the different samples (low: 0–35, moderate: 35–70, high: 70–100) 1 3 Clinical Oral Investigations (2022) 26:4369–4380 4376 Fig. 7 Boxplots showing the percentages of live bacteria on the BES surface after two-hour (2 h) oral exposure followed by treatment with R. officinalis extract and control solutions. The following treat- ment groups are shown: CHX-treated positive control, DMSO-treated neutral control, NaCl-treated negative control, R. Discussion Given the continuous global increase in antimicrobial resist- ance, the present study aimed to introduce a natural, plant- based antimicrobial agent for the prevention of oral diseases. For this purpose, the effect of R. officinalis extract on initial oral biofilms in situ was investigated. To the best of our knowledge, this study is the first to address the antimicro- bial activity of R. officinalis extract against bacteria directly gained from the oral cavity in the form of in situ generated initial oral biofilm. Preventing the initial microbial adhesion is the most important step to avoid the severe consequences of mature biofilm formation, in which microorganisms are highly protected against the effects of different antimicrobi- als such as disinfectants and antibiotics [2, 13, 16, 40].i Figure 6 shows the average amount of detected bacterial species among the six probands. Fig. 7 Boxplots showing the percentages of live bacteria on the BES surface after two-hour (2 h) oral exposure followed by treatment with R. officinalis extract and control solutions. The following treat- ment groups are shown: CHX-treated positive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ml and 30 mg/ml. The graph shows the bacterial counts per square centimeter as percentages and the p-values are shown in the diagram ml and to 2.09% at an extract concentration of 30 mg/ml (p < 0.001). After treatment with 0.2% CHX, the vitality rate of viable bacteria in the initial biofilms was also significantly decreased to a mean percentage of 1.95% (p < 0.001). There was no significant difference between the positive control CHX and the extract-treated samples at either concentration (20 mg/ml and 30 mg/ml) (p = 0.758/p = 1.000). Fig. 6 Average number of different bacterial species detected in ini- tial oral biofilm treated with R. officinalis extract. The following treat- ment groups are shown: CHX-treated positive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ml and 30 mg/ml officinalis extract at two different concentrations of 20 mg/ml and 30 mg/ml. The graph shows the bacterial counts per square centimeter as percentages and the p-values are shown in the diagram Fig. 5 Diagram showing the distribution of bacterial species among six study probands. A total of 49 different species were detected: 41 different species in the untreated biofilms, 31 species in the DMSO controls, and only 11 different species in the R. officinalis extract- treated biofilms Fig. 5 Diagram showing the distribution of bacterial species among six study probands. A total of 49 different species were detected: 41 different species in the untreated biofilms, 31 species in the DMSO controls, and only 11 different species in the R. officinalis extract- treated biofilms Fig. 6 Average number of different bacterial species detected in ini- tial oral biofilm treated with R. officinalis extract. The following treat- ment groups are shown: CHX-treated positive control, DMSO-treated neutral control, NaCl-treated negative control, R. officinalis extract at two different concentrations of 20 mg/ml and 30 mg/ml Live/dead assay reveals the high bactericidal activity of R. officinalis extract against initial bacterial adhesion officinalis) is a widely used plant, it is easy to grow, and inexpensive. In addition, studies have proven that R. officinalis extract is nontoxic and antimuta- genic at the concentrations used [25, 50]. For example, the use of a 10-min mouth rinse with R. officinalis extract to minimize initial bacterial adhesion would be conceivable in dentistry. The fluorescence microscopy experiments after live/dead staining confirmed the microbiological results that R. officinalis extract has a significant disintegrating effect on initial biofilms. In principle, the finding that the number of green-stained live bacteria accounts for a high proportion in untreated initial biofilms is consistent with other studies [51, 52]. However, concerning the question of the percent- ages of live and dead bacteria present after live/dead stain- ing, previous studies sometimes derived results that differed from those of the present work. For example, in a study by Tawakoli et al. [52], only 42 to 66% of vital bacteria were found in the initial biofilm after 120 min of intraoral expo- sure. The divergent results of the present work could be due to the fact that these are in situ studies in which individual differences are common due to a wide variety of factors. In addition, it could be due to the intraoral positions of the enamel slabs, which were positioned in different places in the various studies Furthermore it must be considered that The antimicrobial effect of R. officinalis extract was not only demonstrated by the reduction of the total bacterial counts and the reduced number of viable bacteria, but also by the fact that the total number of different detected bac- terial species was significantly reduced after R. officinalis extract treatment. In the untreated biofilms, a total of 49 bacterial species out of 16 bacterial groups were detected. In the extract samples, there was a total of only 11 spe- cies out of five bacterial groups (mitis group streptococci, salivarius group streptococci, Rothia spp., HACEK group, Campylobacter spp.). In all samples (excluding R. offici- nalis [30 mg/ml] and CHX-treated biofilms), non-mutans streptococci were the predominant taxa (DMSO: 52.44%, NaCl: 41.58%, R. officinalis 20 mg/ml: 56.41%), which is confirmed by previous studies [47, 53]. However, the absolute bacterial counts were significantly reduced by R. officinalis extract treatment. For a long time, mainly mutans streptococci were considered to be the relevant species in caries formation. Live/dead assay reveals the high bactericidal activity of R. officinalis extract against initial bacterial adhesion The live/dead staining results are shown in Fig. 7. There was a mean percentage of 89.89% and 84.25% viable bacteria in the untreated initial biofilms of the negative (NaCl) and neutral controls (DMSO), respectively. The treatment of ini- tial biofilms with R. officinalis extract significantly reduced these values to 5.05% at an extract concentration of 20 mg/ In the present study, a highly significant antimicrobial effect of R. officinalis extract was demonstrated on both aerobic and anaerobic bacteria of initial oral biofilms. The 1 3 Clinical Oral Investigations (2022) 26:4369–4380 4377 fluorescence results after live/dead staining could depend on the temporary environment within the oral cavity of each volunteer. number of CFUs after initial biofilm treatment with R. offici- nalis extract was significantly lower than the CFUs of the untreated biofilms. Nevertheless, the CFUs were comparable to the CFUs yielded by the CHX-treated biofilms. Regarding the antibacterial properties of R. officinalis extract, other studies based on the minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) also came to the same conclusion that R. officinalis extract has a bactericidal effect. For example, Sienkiewicz et al. [41] showed inhibition of E. coli growth with an R. officinalis essential oil. Various studies also confirmed the efficacy of R. officinalis against S. aureus [26, 42], Bacillus cereus [43], Clostridium perfringens [44], and various Salmonella spe- cies [45, 46]. In a study by Hickl et al. [32], the effect of sev- eral plant extracts including R. officinalis was tested on oral bacteria for the first time using in vitro models. The authors concluded that rosemary extract led to a significant reduc- tion in the growth of all tested bacteria [32]. All of these studies used specific bacterial strains, whereas in the present study we chose an experimental setup that reflects the actual complexity and diversity of in situ oral biofilms in the oral cavity. By collecting in situ biofilms on enamel slabs, which are positioned intraorally on individual splint systems, the actual environmental parameters in the oral cavity (e.g., oxy- gen availability, pH, nutrient supply) can be integrated into the experimental model to better reflect the oral conditions and investigate the effect on a more diverse oral microbial spectrum of biofilms. This model has been used in previous studies and was proven to be sufficient for ex vivo studies [47–49]. Rosemary (R. Live/dead assay reveals the high bactericidal activity of R. officinalis extract against initial bacterial adhesion However, recent studies assume that non- mutans streptococci may also have a significant impact on caries initiation [47, 54], as the increase in acidogenic non- mutans streptococci is considered to precede the attachment of mutans streptococci and other cariogenic germs. Accord- ing to this hypothesis, reducing non-mutans streptococci in the initial biofilm could help prevent the development of carious lesions. The presence of Actinomyces spp. was reduced by 100% by the R. officinalis extract treatment. Similar to non-mutans streptococci, Actinomyces spp. are able to produce acids that increase the risk of demineralization of dental enamel [47, 54]. In particular, large amounts of non-mutans strep- tococci and Actinomyces spp. have been detected in white spot lesions [55]. Again, the present results indicate that R. officinalis extract could be used for caries prevention by reducing the concentration of Actinomyces spp. in the oral biofilm. Interestingly, mutans streptococci were not detected in any of the study participants’ biofilms. Due to various virulence factors, S. mutans is considered to play a particu- lar role in the development of caries [56]. The fact that no mutans streptococci were detected in the present study may be attributed to the fact that the microbial composition was examined after only 2 h of initial microbial colonization. Previous studies also showed that the proportion of mutans streptococci in initial biofilms was very low, at 2% or even less [53]. All results showed large intraindividual and interindi- vidual differences, which is in line with previous studies investigating initial oral biofilms [20, 33, 51]. The composi- tion of a biofilm is highly dependent on local or individual factors [57] such as diet, salivary flow rate, and salivary pH. 1 4378 Clinical Oral Investigations (2022) 26:4369–4380 treatment of caries, periodontitis, and peri-implantitis should be evaluated in further clinical studies. These factors may differ from subject to subject, and could also vary within the oral cavity, for example, depending on the position of the enamel slabs. Therefore, it was ensured that the enamel slabs were always placed in the same posi- tion. Here, the R. officinalis extract samples were opposite the control samples in the splint system. Acknowledgements The authors thank Bettina Spitzmüller for her excellent technical assistance. References 1. Pitten FA, Splieth C, Kramer A (2000) Prophylactic and thera- peutic application of antimicrobial agents in the oral cavity. Pharmazie 55:635–639ii Live/dead assay reveals the high bactericidal activity of R. officinalis extract against initial bacterial adhesion Author contribution MG collected the samples, performed the experi- ments, analyzed the results, and drafted the manuscript; LK concep- tualized and supervised the study, prepared the figures, and edited the manuscript; AA provided reagents and resources; ACA critically revised the manuscript; EH critically revised the manuscript; ALS provided reagents and resources; AW performed and analyzed experi- ments; KV performed the statistical analysis; AAA conceptualized and supervised the study and edited the manuscript. All authors approved the final article. The present study has some limitations which should be considered in future investigations of this topic. One limita- tion of the present study is that only one treatment time over 10 min was tested. This long treatment time was intended to simulate the intensive use of R. officinalis ingredients in tea preparation. However, the inclusion of shorter rinse times such as 1 min in future studies would allow for a compari- son with other mouth rinses, which are usually only used for 1 min in the oral cavity. Another limitation is the small number of probands involved in the study, as the number of microbial species isolated using the culture technique would have been increased if biofilm samples from a larger number of volunteers would have been studied. In addition to its activity against initial oral biofilm, the effects of R. officinalis extract against the oral biofilm formed over longer periods is an important aspect that should also be included in follow up-studies. Furthermore, testing the minimal inhibi- tory concentrations of the extract against a diverse array of oral bacterial species would complement the data of the pre- sent study in addition to the aspects highlighted here.i Funding Open Access funding enabled and organized by Projekt DEAL. This study was supported in part by the German Research Foundation (DFG, AL 1179/4–1). Conflict of interest The authors declare no competing interests. Conflict of interest The authors declare no competing interests. Further clinical studies on biofilms grown over a longer period are needed to investigate whether there is also a com- parable antimicrobial effect of R. officinalis extract in such cases. In this study, the investigations were performed on 2-h-old initial biofilms. With the aid of a similar experi- mental set-up, in particular, through the use of intraoral splint systems, a mature biofilm of 2–3 weeks could be obtained for further investigations with R. officinalis extract. A determination of whether there is efficacy against other oral species, e.g., periodontal pathogens such as Porphy- romonas gingivalis and Prevotella intermedia, and whether R. officinalis extract can be used as a natural substance in the prevention of periodontitis, requires further investigation in clinical studies including appropriate patient collectives. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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. 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Sci Rep 8:14202. https://doi.org/10.1038/ s41598-018-32544-6 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 48. Al-Ahmad A, Wiedmann-Al-Ahmad M, Faust J et al (2010) Biofilm formation and composition on different implant materi- als in vivo. J Biomed Mater Res B Appl Biomater 95:101–109. https://doi.org/10.1002/jbm.b.31688 1 3 1 3 3
https://openalex.org/W2162048896	https://pure.uva.nl/ws/files/2194082/153413_Mineralocorticoid_receptors.pdf	English	null	Mineralocorticoid Receptors Guide Spatial and Stimulus-Response Learning in Mice	PloS one	2,014	cc-by	11,136	Link to publication Citation for published version (APA): Arp, J. M., ter Horst, J. P., Kanatsou, S., Fernández, G., Joëls, M., Krugers, H. J., & Oitzl, M. S. (2014). Mineralocorticoid receptors guide spatial and stimulus-response learning in mice. PLoS ONE, 9(1), e86236. https://doi.org/10.1371/journal.pone.0086236 Citation for published version (APA): Arp, J. M., ter Horst, J. P., Kanatsou, S., Fernández, G., Joëls, M., Krugers, H. J., & Oitzl, M. S. (2014). Mineralocorticoid receptors guide spatial and stimulus-response learning in mice. PLoS ONE, 9(1), e86236. https://doi.org/10.1371/journal.pone.0086236 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Citation for published version (APA): Arp, J. M., ter Horst, J. P., Kanatsou, S., Fernández, G., Joëls, M., Krugers, H. J., & Oitzl, M. S. (2014). Mineralocorticoid receptors guide spatial and stimulus-response learning in mice. PLoS ONE, 9(1), e86236. https://doi.org/10.1371/journal.pone.0086236 Introduction the vast majority of male C57Bl/6j mice use a spatial strategy to navigate on a CHB [17]; to acquire a stimulus-response task, male mice have to overcome their natural tendency to use the spatial strategy [22]. Brief exposure to stress causes a shift from spatial towards stimulus-response strategies, an effect which is mediated via MRs [17]. Corticosteroid hormones are secreted from the adrenals in an ultradian and circadian pattern, as well as in response to stressful experiences [1,2]. Corticosteroid hormones regulate brain func- tion via activation of high affinity mineralocorticoid receptors (MRs) and lower affinity glucocorticoid receptors (GRs) which are both expressed in brain areas which are crucial for learning and memory such as hippocampus, amygdala, prefrontal cortex and striatum [3]. Via activation of MRs and GRs, corticosteroid hormones promote behavioral adaptation to stressful experiences [1,3–5]. MRs mediate initial behavioral responses to novel situations, and are required for adequate spatial and fear learning and memory processes [6–10]. Activation of GRs is crucial for consolidation of spatial and emotional information [6,11–13]. In these studies, the CHB task was designed to allow animals to make a choice. However, it remains to be established to what extent activation of MRs determines stimulus-response and spatial strategies to solve navigational tasks per se, i.e. when animals do not have to make a choice. In the present study we therefore studied the role of MRs in spatial (hippocampus-dependent) and stimulus-response (striatum- dependent) learning separately; i.e., we used, separately, the spatial and stimulus-response learning versions of the CHB task [22]. Moreover, to gain better insight into the role of brain MRs, we did not only examine the consequences of MR deficiency [7] but also investigated the other end of the spectrum, i.e. MR overexpression [8]. We hypothesized that MR overexpression will lead to improved performance in both tasks and might ease the shift from the spatial to the acquisition of the stimulus-response task, while the opposite is expected in the MR-deficient animals. We initially focused on female MR mutants, in view of the recently Recently we demonstrated that corticosteroids are particularly important for the choice of strategies to solve navigational tasks. Different strategies such as spatial (hippocampus-dependent) or stimulus-response (striatum-dependent) strategies can be used to solve navigational tasks [14–16]. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparati Funding: This study was supported by grant NWO-NIBC433-09-251 to MA, GF, MJ, HJK, MSO, NWO-DN-95-420 to MSO, the European Science Foundation (07- EuroSTRESS-FP-005) to JPtH. 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: h.krugers@uva.nl Competing Interests: The authors have declared that no competing interests exist. * E-mail: h.krugers@uva.nl * E-mail: h.krugers@uva.nl Abstract Adrenal corticosteroid hormones act via mineralocorticoid (MR) and glucocorticoid receptors (GR) in the brain, influencing learning and memory. MRs have been implicated in the initial behavioral response in novel situations, which includes behavioral strategies in learning tasks. Different strategies can be used to solve navigational tasks, for example hippocampus-dependent spatial or striatum-dependent stimulus-response strategies. Previous studies suggested that MRs are involved in spatial learning and induce a shift between learning strategies when animals are allowed a choice between both strategies. In the present study, we further explored the role of MRs in spatial and stimulus-response learning in two separate circular holeboard tasks using female mice with forebrain-specific MR deficiency and MR overexpression and their wildtype control littermates. In addition, we studied sex-specific effects using male and female MR-deficient mice. First, we found that MR-deficient compared to control littermates and MR-overexpressing mice display altered exploratory and searching behavior indicative of impaired acquisition of novel information. Second, female (but not male) MR-deficient mice were impaired in the spatial task, while MR-overexpressing female mice showed improved performance in the spatial task. Third, MR-deficient mice were also impaired in the stimulus-response task compared to controls and (in the case of females) MR-overexpressing mice. We conclude that MRs are important for coordinating the processing of information relevant for spatial as well as stimulus-response learning. Citation: Arp JM, ter Horst JP, Kanatsou S, Ferna´ndez G, Joe¨ls M, et al. (2014) Mineralocorticoid Receptors Guide Spatial and Stimulus-Response Learning in Mice. PLoS ONE 9(1): e86236. doi:10.1371/journal.pone.0086236 Editor: Mathias V. Schmidt, Max Planck Institute of Psychiatry, Germany Received November 6, 2013; Accepted December 10, 2013; Published January 21, 2014 Received November 6, 2013; Accepted December 10, 2013; Published January 21, 2014 Copyright: 2014 Arp 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. Copyright: 2014 Arp et al. This is an open-access article distributed under the terms of the Creative Commons Attrib use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grant NWO-NIBC433-09-251 to MA, GF, MJ, HJK, MSO, NWO-DN-95-420 to MSO, the Europe EuroSTRESS-FP-005) to JPtH. Disclaimer/Complaints regulations Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:24 Oct 2024 J. Marit Arp1, Judith P. ter Horst1, Sofia Kanatsou1, Guille´n Ferna´ndez3, Marian Joe¨ls2, Harm J. Krugers1, Melly S. Oitzl1 1 Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands, 3 Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands January 2014 \| Volume 9 \| Issue 1 \| e86236 Timeline Male and female forebrain MR-deficient (MRCaMKCre; [7]) mice and their control littermates (MR flox/flox)(n = 12 per group; males approximately 3 months and females approximately 4 months old) were bred in the animal facility of Leiden University. The MRCaMKCre mice were obtained by breeding MRflox/flox with MRflox/wtCaMKCre mice from the German Cancer Research Center, Heidelberg, Germany. The conditional MR allele was generated in embryonic stem cells of 129Ola mice and CaMKCre transgene was injected in FVB/N mice [24]. The MR flox allele and the Cre transgene were backcrossed into C57Bl/6J mice. For a detailed description of the design and breeding of the MRCaMKCre mice see [7]. In an additional experiment, female forebrain-specific MR-overexpressing transgenic (MR-Tg; [8]) mice and their control littermates were used (n = 19220 per group; approximately 4 months old) that were bred in the animal facility of the University of Amsterdam. The MR-Tg mice were obtained from the Centre for Cardiovascular Science, Edinburgh, UK. A haemagglutinin (HA) epitope tag was inserted into the N- terminus of the full-length human MR cDNA. Transgenic mice were generated by injection of a CaMKIIa-HA-MR construct in C57Bl/6J CBA embryos (Babraham Institute, Cambridge, UK). For a detailed description of the design and breeding of the MR- Tg mice see [8]. After arrival, the mice were allowed to acclimatize to the animal facility for three weeks. Male and female MR- deficient mice were derived from 7 litters (2–6 mice per litter); 5 out of 7 litters contributed both MRCaMKCre and controls (MR flox/flox). MR-overexpressing female mice were derived from 5 litters (3–4 mice per litter); we used MR-Tg mice and control littermates from all litters. The experimental design of the experiment is schematically shown in Figure 1. We tested both male and female MRCaMKCre mice, female MR-Tg mice and the control littermates of each group in two versions of the CHB task: a spatial task and a stimulus-response task. First, mice were given a free exploration trial (FET). One week later the spatial tasks started. Mice received six training trials (inter trial-interval 15 min), where only extra- maze spatial cues were available to locate the exit hole. One day after the spatial training, each mouse performed one spatial memory test trial with all holes closed. Timeline The stimulus-response task started one week after this spatial memory test and consisted of two subsequent days of each six training trials (again 15 min inter trial- interval), where an intra-maze stimulus (the bottle) marked the exit hole. Free Exploration Trial For the free exploration trial (FET) the mice were allowed to explore the CHB for 5 min. All holes were closed. At the end of the 5 min, the exit hole was opened and the animals were guided there by the experimenter. This exploration trial allowed to analyze exploratory behavior and general activity of the mice. One week before the behavioral testing started, mice were moved to the experimental room (temperature: 20uC; humidity: 55% 615), under a 12:12 hour light/dark cycle, lights on at 07:30 h) and single housed in Macrolon cages with sawdust bedding and with food and water ad libitum. Testing was carried out between 08:30 and 12:30 h. The experiments were approved by the committee on Animal Health and Care from Leiden University, The Netherlands, in accordance with the EC Council Directive of September 2010 (2010/63/EU). The Spatial Task One week after the FET, mice were given six successive training trials with a maximum of 120 s per trial. The location of the exit hole was always fixed relative to the distal extra-maze cues in the room. There were no proximal cues present, so the exit could only be found by using the extra-maze cues. This task was used to assess spatial learning. Twenty-four hours later, we tested long-term spatial memory. Stimulus-response Task In the stimulus-response (S-R) task, the position of the exit hole was marked by a bottle and varied from trial to trial in the same sequence for all mice. The position of the exit was never at the same location or a location adjacent to it within a six trial session. Furthermore, the exit hole location of the spatial task was not used as an exit hole position during the stimulus-response task. There were two subsequent days of six trials each. A trial lasted 120 s. The distal extra-maze cues were present, but only the proximal intra-maze cue (a transparent bottle filled with water; 0.5 L; 22 cm high; 5 cm diameter), located next to the exit hole, marked the Tunnel Training One week before the behavioral experiments started the mice were weighed and trained to climb through a tunnel on every second day (three times in total). This familiarized mice with the task requirements. Apparatus The mouse was placed on the CHB for two minutes with all holes closed. The behavior and movement pattern of the mouse allowed to analyze search strategy and spatial memory. The circular hole board (CHB) is a revolvable grey round plate (Plexiglas; 110 cm in diameter; situated 1 m above the floor) with twelve holes at equal distances from each other, located 10 cm from the rim of the board. Holes are 5 cm in diameter and can be closed by a lid at a depth of 5 cm. Whether a hole is open or closed can only be detected if the mouse puts its head over the edge of the hole. An S-shaped tunnel (5 cm in diameter; 15 cm long) leads from the open exit hole to the home cage of the animal. Multiple distant cues in the room allow spatial orientation on the board [17,18,22]. Mineralocorticoid Receptors in Learning Strategies Mineralocorticoid Receptors in Learning Strategies board and exit through the open tunnel. If the mouse did not find the exit hole within 120 s, it was gently guided to the exit using a grid. The board was cleaned after each trial with 1% acetic acid solution to dissipate odor cues and rotated until another hole was at the position of the exit. The home cage was placed under the board at the position of the exit hole such that the mouse could not see the cage from the board. reported clear phenotype of female (but not male) MR-deficient mice in fear conditioning and the dual-solution CHB task [18,19,23]. Since we observed significant behavioral effects of MR deficiency in females, we next also examined the effects of MR deficiency in male mutants. January 2014 \| Volume 9 \| Issue 1 \| e86236 Introduction We designed a version of the circular hole board (CHB) task such that both spatial and stimulus- response strategies could be used to locate the exit hole (dual- solution task; [17–21]). When exposed to such a dual-solution task, 1 PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e86236 Mineralocorticoid Receptors in Learning Strategies Spatial Learning All groups acquired the task, as reflected by decreasing exit latencies over the trials (latency of first visit to the exit hole, MR- Tg females and their controls: F(5,185) = 6.338, p,0.0001; Figure 2A; MRCaMKCre females and their controls: F(5,110) = 10.736, p,0.0001; Figure 2B; MRCaMKCre males and their controls: F(5,110) = 10.011, p,0.0001; Figure 2C). Estrous Cycle The stage of the estrous cycle was determined in female mice by vaginal cytology after each behavioral task. Using a plastic loop (inoculation loops 1 ml, Mediscan, Greiner Bio-one), a vaginal smear was obtained. The loop was dipped in water and then inserted into the vagina and gently rubbed against the vaginal wall. Cells were smeared on a glass slide in a drop of water. After air drying, the cells were stained with Giemsa (Sigma) for 10 minutes. The stage of the cycle was determined based on the presence or absence of nucleated epithelial, cornified epithelial and leukocyte cells. Proestrus: many cells with a nucleus and some epithelial cells; estrus: many epithelial cells and some cells with a nucleus; metestrus: some epithelial cells and many macrophages; diestrus: many macrophages and some cells with a nucleus. We did not encounter the metestrus stage. ( , ) , p ; g ) MRCaMKCre and MR-Tg female mice. MRCaMKCre females showed significantly impaired performance compared to their controls (exit latency: F(1,22) = 6.587, p = 0.019), while MR-Tg females out-performed the controls (F(1,37) = 4.893, p = 0.033). MRCaMKCre females stayed longer in the center (F(1,22) = 4.986, p = 0.036) and had a lower velocity (F(1,22) = 7.335, p = 0.013) than their controls (Table 1) and than the MR-Tg mice (F(1,29) = 23.072, p,0.0001; Table 1). To figure out whether the impaired performance of the MRCaMKCre compared to control females was caused by the longer time spent in the center, the exit latency was corrected for the latency to leave the center. When corrected, the MRCaMKCre females still needed significantly more time to find the exit hole compared to their controls (F(1,22) = 4.941, p = 0.037; data not shown). Conversely, the MR-Tg females had a higher velocity during the trials than their controls (F(1,37) = 7.917, p = 0.008; Table 1). Other behavioral parameters were comparable between MR-Tg females and their controls. MRCaMKCre female mice took longer to locate the exit than the MR-Tg females (F(1,29) = 16.090, p,0.0001), while controls of both groups had similar exit latencies (F(1,30) = 0.004, p = 0.949). Results Free Exploration Trial (FET) MRCaMKCre and MR-Tg female mice. MRCaMKCre (defi- cient) female mice had a significantly longer latency to leave the center of the board when compared to their controls and also when compared to MR-Tg (overexpressing) female mice (861 vs 561; p = 0.039 and vs 361; p,0.0001, respectively). Further- more, MRCaMKCre female mice showed more perseveration than MR-Tg mice (2063 vs. 1162; p = 0.007). Exploratory behavior (% perseveration, % series, latency to leave the center, rim dips, rearing, stretched attends) and general activity (velocity, distance moved and number of holes visited) were comparable between MR-Tg mice and their control littermates (data not shown). Behavioral parameters were also comparable between control littermates of MRCaMKCre and MR-Tg female mice. General Procedure Each trial started by placing the mouse in a cylinder (Plexiglas; 25 cm high; 10 cm diameter) located at the center of the CHB. After 5 s the cylinder was lifted and the mouse could explore the January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e86236 2 Mineralocorticoid Receptors in Learning Strategies Figure 1. Experimental design of training and memory testing. doi:10.1371/journal.pone.0086236.g001 Figure 1. Experimental design of training and memory testing. doi:10.1371/journal.pone.0086236.g001 exit. Therefore, the mice had to use a stimulus-response navigation strategy to locate the exit. Analysis of Behavior Behavior was digitally recorded and analyzed with Ethovision XT 6.1 (Noldus Information Technology b.v., Wageningen, The Netherlands). This image analysis system sampled the position of the mouse 12.5 times per second. The CHB was virtually subdivided into subareas of special interest: center (start area), rim, zone of holes (area including all holes and the space between the holes, but excluding the center, middle and rim of the CHB) and four quadrants (covering three holes with the exit hole in the middle). We calculated preference values of the mice for the quadrant that contained the exit hole, and in the S-R task also for the quadrant that contained the exit hole of the previous trial. The following parameters were calculated by Ethovision: velocity (cm/ s), distanced moved (cm), latency of first visit to exit hole (exit latency, s) (and to former exit hole for the S-R task), and latency to the quadrant of the exit hole (s) and duration in quadrant of the exit hole (s). The experimenter hand scored the number of holes visited (mouse puts at least its nose in the hole), rim dips (looking over the edge of the board), stretched attends, % perseveration (visiting the same hole twice in a row or with one other hole between the two holes) and % serial hole visits (visiting at least three adjacent holes in a row). g MRCaMKCre male. MRCaMKCre males showed more persev- eration and reared less than their controls (MRCaMKCre vs g MRCaMKCre male. MRCaMKCre males showed more persev- eration and reared less than their controls (MRCaMKCre vs controls, % perseveration: 2765 vs 1064; p = 0.018; rearing: 0.0860.1 vs 1.2560.5; p = 0.034). Statistical Analysis Data are presented as mean 6 SEM. Statistical analysis included one-way ANOVA, MANOVA, T-tests and General Linear Model repeated measures. Post hoc tests (Tukey) for multiple comparisons were used when appropriate. Reported p values are two-tailed and statistical significance was accepted for p,0.05. Statistical calculations were performed with IBM SPSS Statistics (version 20; SPSS Inc.; Chicago, IL). The numbers of female mice in the different stages of the estrous cycle were too low for test-statistics per stage. Therefore, we included the stage of the estrous cycle as a covariate in the one-way ANOVA and General Linear Model repeated measures analyses. MRCaMKCre males. The performance did not differ in any respect between MRCaMKCre males and controls (Figure 2C; Table 1). MRCaMKCre and MR-Tg female mice. Both MRCaMKCre MRCaMKCre and MR-Tg female mice. Both MRCa C e female mice and control littermates spent more time in the exit (target) quadrant than in the other quadrants, but MRCaMKCre females showed a trend for longer latency to the exit quadrant compared to their controls (56615.4 vs 2169.5; p = 0.070). MRCaMKCre females spent less time in the holes zone and showed less perseveration than their controls (Table 2). Also the general activity parameters differed between MRCaMKCre females and their controls. This is clear from the lower number of holes visited, shorter distance moved and a slower walking velocity in MRCaMKCre females (Table 2). Parameters for exploration and general activity were comparable between MR-Tg mice and controls. C MKC female mice and control littermates spent more time in the exit (target) quadrant than in the other quadrants, but MRCaMKCre p g MRCaMKCre and MR-Tg female mice. MRCaMKCre mice showed impaired performance as evident from the longer latencies to the exit compared to their controls. On the first day of S-R training an interaction of genotypetrial was found (F(5,5) = 2.763, p = 0.040), with longer exit latencies in trials 3 and 5 in MRCaMKCre females than controls (trial 3: p = 0.031; trial 5: p = 0.027). On the second day the MRCaMKCre females had longer exit latencies than their controls over all trials (F(1,22) = 14.130, p = 0.001). In MR-Tg female mice exit latency was short and comparable to control littermates on both days. On the first day, exit latencies of MR-Tg and MRCaMKCre as well as their controls were comparable. On the second day MRCaMKCre females had longer latencies than MR-Tg females (F(1,29) = 8.441, p = 0.007), while their respective controls performed comparably. g MRCaMKCre and MR-Tg female mice. MRCaMKCre mice showed impaired performance as evident from the longer latencies to the exit compared to their controls. On the first day of S-R training an interaction of genotypetrial was found (F(5,5) = 2.763, p = 0.040), with longer exit latencies in trials 3 and 5 in MRCaMKCre females than controls (trial 3: p = 0.031; trial 5: p = 0.027). On the second day the MRCaMKCre females had longer exit latencies than their controls over all trials (F(1,22) = 14.130, p = 0.001). In MR-Tg female mice exit latency was short and comparable to control littermates on both days. MRCaMKCre and MR-Tg female mice. Both MRCaMKCre On the first day, exit latencies of MR-Tg and MRCaMKCre as well as their controls were comparable. On the second day MRCaMKCre females had longer latencies than MR-Tg females (F(1,29) = 8.441, p = 0.007), while their respective controls performed comparably. MRCaMKCre mice. The male MRCaMKCre mice showed a trend for longer latency to the exit quadrant compared to their controls (males: 64614.5 vs 30610.9; p = 0.080). Furthermore, searching behavior also differed between MRCaMKCre male mice and controls. MRCaMKCre males showed a more evenly distributed searching pattern while their controls spent significantly more time in the exit quadrant than in the other quadrants (Table 2). p p p y MRCaMKCre male mice. Also MRCaMKCre male mice showed impaired performance on the second day of the S-R training compared to their controls (day 2: F(1,22) = 6.981, p = 0.015). MRCaMKCre male mice. Also MRCaMKCre male mice showed impaired performance on the second day of the S-R training compared to their controls (day 2: F(1,22) = 6.981, p = 0.015). Spatial Memory Test –24 Hours Later with Closed Exit Behavioral parameters (averaged over the day) recorded during the spatial training trials in MR-Tg and MRCaMKCre mice and their control littermates. Table 1. Behavioral parameters (averaged over the day) recorded during the spatial training trials in MR-Tg and MRCaMKCre mice and their control littermates. The spatial task consisted of six trials. Data represent mean 6 SEM of all trials. For statistics a repeated measures ANOVA was used over the trials. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female control littermates of MRCaMKCre mice. doi:10 1371/journal pone 0086236 t001 The spatial task consisted of six trials. Data represent mean 6 SEM of all trials. For statistics a repeated measures ANOVA was used over the trials. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female control littermates of MRCaMKCre mice. doi:10.1371/journal.pone.0086236.t001 (Figure 2A) and between the MRCaMKCre mice and their controls (Figure 2B (females) and 2C (males)). one.0086236.t002 Spatial Memory Test –24 Hours Later with Closed Exit Spatial Memory Test –24 Hours Later with Closed Exit Spatial Memory Test –24 Hours Later with Closed Exit Latency to the exit hole was longer than in trial 6 the day before, but comparable between MR-Tg mice and their controls Latency to the exit hole was longer than in trial 6 the day before, but comparable between MR-Tg mice and their controls PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e86236 3 Mineralocorticoid Receptors in Learning Strategies Figure 2. Spatial task: latency to the exit hole over six training trials and spatial memory test. A) Female MR-Tg mice (n = 19) take less time to find the exit hole than controls (n = 20). B) Female MRCaMKCre mice take more time to find the exit hole than controls (n = 12 per group). C) Male MRCaMKCre and control mice (n = 12 per group) have comparable latencies to locate the exit hole. Bars show mean 6 SEM. p,0.05 over trials, vs. control littermates. n.s. = not significant. doi:10.1371/journal.pone.0086236.g002 Figure 2. Spatial task: latency to the exit hole over six training trials and spatial memory test. A) Female MR-Tg mice (n = 19) take less time to find the exit hole than controls (n = 20). B) Female MRCaMKCre mice take more time to find the exit hole than controls (n = 12 per group). C) Male MRCaMKCre and control mice (n = 12 per group) have comparable latencies to locate the exit hole. Bars show mean 6 SEM. p,0.05 over trials, vs. control littermates. n.s. = not significant. doi:10.1371/journal.pone.0086236.g002 January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org 4 Mineralocorticoid Receptors in Learning Strategies Table 1. Behavioral parameters (averaged over the day) recorded during the spatial training trials in MR-Tg and MRCaMKCre mice and their control littermates. Females Females Males MR-Tg Control MRCaMKCre Control MRCaMKCre Control Velocity (cm/s) 860 761 560* 661 561 560 Latency to leave center (s) 360 360 1063# 561$ 963 963 The spatial task consisted of six trials. Data represent mean 6 SEM of all trials. For statistics a repeated measures ANOVA was used over the trials. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female control littermates of MRCaMKCre mice. doi:10.1371/journal.pone.0086236.t001 Table 1. Data represent mean 6 SEM. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female MRCaMKCre; ,duration in exit quadrant vs average duration in other quadrants. doi:10 1371/journal pone 0086236 t002 PLOS ONE \| www.plosone.org 5 January 2014 \| Volume 9 \| Issue 1 \| e86236 doi:10.1371/journal.pone.0086236.t002 (Figure 2A) and between the MRCaMKCre mice and their controls (Figure 2B (females) and 2C (males)). C C (Figure 2A) and between the MRCaMKCre mice and their controls (Figure 2B (females) and 2C (males)). All groups decreased their exit latencies over the trials (MR-Tg females and their controls: day 1: F(5,185) = 13.314, p,0.0001; day 2: F(5,110) = 3.725, p = 0.015; Figure 3A; MRCaMKCre females and their controls: day 1: F(5,110) = 28.585, p,0.0001; day 2: F(5,110) = 3.406, p = 0.030; Figure 3B; MRCaMKCre males and their controls: day 1: F(5,110) = 25.426, p,0.0001; day 2: F(5,110) = 16.049, p,0.0001; Figure 3C). (Figure 2A) and between the MRCaMKCre mice and their controls (Figure 2B (females) and 2C (males)). All groups decreased their exit latencies over the trials (MR-Tg females and their controls: day 1: F(5,185) = 13.314, p,0.0001; day 2: F(5,110) = 3.725, p = 0.015; Figure 3A; MRCaMKCre females and their controls: day 1: F(5,110) = 28.585, p,0.0001; day 2: F(5,110) = 3.406, p = 0.030; Figure 3B; MRCaMKCre males and their controls: day 1: F(5,110) = 25.426, p,0.0001; day 2: F(5,110) = 16.049, p,0.0001; Figure 3C). ( g ( ) ( )) MRCaMKCre and MR-Tg female mice. Both MRCaMKCre 236.t002 Data represent mean 6 SEM. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCa MRCaMKCre; ,duration in exit quadrant vs average duration in other quadrants. Stimulus-response Learning C) Male MRCaMKCre mice take more time to locate the exit than controls on day 2. Bars show mean 6 SEM. p,0.05 vs. control littermates. doi:10.1371/journal.pone.0086236.g003 Figure 3. Stimulus-response (S-R) task: latency to the exit hole over six training trials on two days. A) Female MR-Tg mice and controls have short and similar latencies to the exit hole. B) Female MRCaMKCre mice take more time to locate the exit than controls in trials 3 and 5 on day 1, and in all trials on day 2. C) Male MRCaMKCre mice take more time to locate the exit than controls on day 2. Bars show mean 6 SEM. p,0.05 vs. control littermates. doi:10.1371/journal.pone.0086236.g003 bottle. We interpret a visit to the position of the exit hole of the previous trial as an indication that a mouse applied a spatial strategy to locate the exit hole. Therefore, for every trial we measured the latency to the previous exit hole, the latency to the quadrant of the previous exit hole and the percentage of time spent in this quadrant (Table 3). Latency to the spatial exit was comparable between MR-Tg females and their control littermates. MRCaMKCre females took longer to reach the quadrant of the spatial exit (p = 0.015) and spent less time in this quadrant (p = 0.018) while the latency to the spatial exit hole was similar to their controls (Table 4). MRCaMKCre males took significantly longer to reach the quadrant of the spatial exit hole (p = 0.042), spent less time in this quadrant (p = 0.043) and showed a trend of longer latency to the spatial exit hole than their controls (p = 0.095) (Table 4). On the first day of the S-R training, the time spent in the quadrant of the previous exit was significantly shorter for MRCaMKCre males and females than for the control littermates (males: F(1,22) = 7.615, p = 0.011; females: F(1,22) = 9.356, p = 0.006; Table 3). Latency to the quadrant of the previous exit was longer for MRCaMKCre female mice than for their control littermates (F(1,22) = 7.953, p = 0.010; Table 3) with no difference between MRCaMKCre male mice and controls. On the second day, latency to the previous exit hole was comparable between the MRCaMKCre mice and controls in both males and females. Stimulus-response Learning Latency to the exit hole and exit quadrant of the previous trial were also comparable between MR-Tg females and controls. Searching strategies and general activity in S-R trials. Searching strategies were defined as a way to find the exit hole, expressed by the order of holes visited. A serial hole visit was defined as visiting at least three adjacent holes in a row. Perseveration was defined as visiting the same hole twice in a row or with one other hole between the two holes. MRCaMKCre and MR-Tg female mice. On the first day, MRCaMKCre female mice visited less holes in a serial manner than their control littermates (% series: F(1,22) = 11.509, p = 0.003), while on the second day the genotypes had similar percentages of serial hole visits (Table 5). Both MR-Tg genotypes had a similar percentage of serial hole visits (Table 5). MRCaMKCre female mice moved slower than their controls on both days (velocity; day 1: F(1,22) = 14.592, p = 0.001; day 2: F(1,22) = 6.083, p = 0.022) and visited less holes on the first day (F(1,22) = 8.691, p = 0.007; Table 3). Velocity on both days and distance walked on the first day were similar between MR-Tg female mice and controls. On MRCaMKCre and MR-Tg female mice. On the first day, MRCaMKCre female mice visited less holes in a serial manner than their control littermates (% series: F(1,22) = 11.509, p = 0.003), while on the second day the genotypes had similar percentages of serial hole visits (Table 5). Both MR-Tg genotypes had a similar percentage of serial hole visits (Table 5). MRCaMKCre female mice moved slower than their controls on both days (velocity; day 1: F(1,22) = 14.592, p = 0.001; day 2: F(1,22) = 6.083, p = 0.022) and visited less holes on the first day (F(1,22) = 8.691, p = 0.007; Table 3). Velocity on both days and distance walked on the first day were similar between MR-Tg female mice and controls. On Long-term spatial memory in the S-R task. Before training in the S-R task, mice had received spatial training, with the exit in a fixed location in relation to the extra maze cues. These cues were still available in the S-R task, however now the exit was variable marked by a bottle, irrespective of spatial cues. We expected that spatial memory might influence the acquisition of the S-R task. Stimulus-response Learning Stimulus-response Learning Short-term spatial memory in the S-R task. In every trial the exit hole was in a different position and always marked by the Short-term spatial memory in the S-R task. In every trial the exit hole was in a different position and always marked by the Performance. One week after the spatial version of the CHB task, all animals were tested for stimulus-response (S-R) learning. Table 2. Behavioral parameters recorded during the spatial memory test 24 hours after six spatial training trials in MR-Tg and MRCaMKCre mice and their control littermates. Females Females Males Behavioral parameters MR-Tg Control MRCaMKCre Control MRCaMKCre Control General activity Distance moved (cm) 741649 741665 264629$# 527668 512690 501660 Velocity (cm/s) 661 661 2.560$# 4.561 461 461 Total hole visits 961 961 361$# 962 862 861 Searching Latency to exit quadrant (s) 2167 3569 56615 21610 64615 30611 Duration in exit quadrant (s) 5066, 4067 5961,$ 6967, 2968 4768, Average duration in other quadrants (s) 2162 2362 1464$ 1363 2663 2163 Duration in holes zone (s) 4165 3563 2368$# 4968 4667 4565 % Perseveration 1663 1363 563$# 1764 1264 1163 Data represent mean 6 SEM. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female MRCaMKCre; ,duration in exit quadrant vs average duration in other quadrants. doi:10.1371/journal.pone.0086236.t002 Table 2. Behavioral parameters recorded during the spatial memory test 24 hours after six spatial training trials in MR-Tg and MRCaMKCre mice and their control littermates. Table 2. Behavioral parameters recorded during the spatial memory test 24 hours after six spatial training trials in MR-Tg and MRCaMKCre mice and their control littermates. January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Mineralocorticoid Receptors in Learning Strategies PLOS ONE \| www.plosone.org 6 January 2 January 2014 \| Volume 9 \| Issue 1 \| e86236 January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Mineralocorticoid Receptors in Learning Strategies Figure 3. Stimulus-response (S-R) task: latency to the exit hole over six training trials on two days. A) Female MR-Tg mice and controls have short and similar latencies to the exit hole. B) Female MRCaMKCre mice take more time to locate the exit than controls in trials 3 and 5 on day 1, and in all trials on day 2. Stimulus-response Learning Therefore, we measured the latency to the exit hole of the spatial task during the first trial of the first S-R training day. Table 3. Behavioral parameters (averaged over the day) recorded during the stimulus-response task in MR-Tg and MRCaMKCre mice and their control littermates. Females Females Males MR-Tg Control MRCaMKCre Control MRCaMKCre Control Day 1 Velocity (cm/s) 860 860 561$ 861 761 861 Distance moved (cm) 367664 380659 296647 287648 313664 253646 Total hole visits 661 661 461 561 561 561 %Perseveration 863* 462 764 662 362 363 Latency to previous exit quadrant (s) 48612 46612 72614* 47614 58615 51615 Duration in previous exit quadrant (s) 1864 1864 1365* 2365 1462* 2163 Day 2 Velocity (cm/s) 961 961 761* 961 761 861 Distance moved (cm) 213631* 253638 249651 213649 245646 207638 Total hole visits 361 461 361 361 361 361 %Perseveration 562* 562 362 262 362 362 Latency to previous exit quadrant (s) 51612 45611 48613 48614 55616 61616 Duration in previous exit quadrant (s) 1464 1263 1765 2264$ 1462 1061 Both days of the stimulus-response task consisted of six trials. Data represent mean 6 SEM of all trials of one day. For statistics a repeated measures ANOVA was used over the trials of one day. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female MRCaMKCre. doi:10.1371/journal.pone.0086236.t003 eters (averaged over the day) recorded during the stimulus-response task in MR-Tg and MRCaMKCre mice Both days of the stimulus-response task consisted of six trials. Data represent mean 6 SEM of all trials of one day. For statistics a repeated measures ANOVA was used over the trials of one day. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg; $male vs female MRCaMKCre. January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org 7 Mineralocorticoid Receptors in Learning Strategies Table 4. Spatial exit memory during the first trial of the stimulus-response task on day 1 (one week after the spatial memory test) in MR-Tg and MRCaMKCre mice and their control littermates. Behaviour in a Novel Environment and Learning Being exposed to the CHB after a life in a small cage with conspecifics is a challenge for the mouse. Exploration is the natural response. We and others [6,18,26,27], have shown that activation or blockade of MRs in rats and mice alters the exploration pattern and behavioral flexibility but not general activity measures. We here show that MR-overexpressing female mice showed an exploration pattern comparable to their control littermates, while MR-deficient male and female mice showed different exploratory behavior compared to controls without alterations in general Behavioral parameters Data represent mean 6 SEM. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #fem doi:10.1371/journal.pone.0086236.t004 stimulus-response learning requires more training trials [22,25]. In previous studies we had tested performance and the use of memory systems of mice with manipulated MR expression in a dual-solution CHB task. Pharmacological blockade of MR as well as MR deficiency led to deterioration of performance in naı¨ve non-stressed male mice [17–19]. However, in this design the selected strategies were mutually exclusive (animals had to choose between the two strategies), so that we did not know to what extent MR expression specifically influences the two systems. Thus, the impairment in performance could have been the result of a dysfunction of the hippocampus, a dysfunction of the dorsal striatum or both, or the coordination in the behavior-controlling network. the second day, the MR-Tg female mice walked less than the controls (distance; F(1,37) = 5.000, p = 0.031; Table 3). During both days of the stimulus-response task, MR-Tg female mice showed more perseveration than the controls (day 1: F(1,37) = 4.594, p = 0.039; day 2: F(1,37) = 8.390, p = 0.006; Table 3). MRCaMKCre male mice. Searching strategies were different between the genotypes in MRCaMKCre male mice. On the first day, MRCaMKCre males visited less holes in a serial manner than their control littermates (% series: F(1,22) = 19.489, p,0.0001), while on the second day the genotypes had similar percentages of serial hole visits (Table 5). In contrast to the females, general activity was comparable between genotypes for MRCaMKCre male mice. We report here that the expression of MRs is critical for both memory systems. Thus, forebrain MR-deficient female mice showed both an impaired spatial and stimulus-response learning, while MR overexpression resulted in opposite effects, i.e. improved spatial performance while stimulus-response learning was not improved, probably due to a ceiling effect. Performance deficits in both tasks were more strongly expressed in MR-deficient female than male mice, revealing and supporting a sex-dependent effect of MR deficiency. The opposite results of MR deficiency and MR overexpression confirm and substantiate the impact of MR on acquisition of novel information, not only for spatial but also for stimulus-response learning. Behavior and the Estrous Cycle We determined the stage of the estrous cycle in female mice by vaginal cytology after each behavioral task. We did not encounter the metestrus stage. The numbers of MRCaMKCre female mice and controls in the proestrus stage in all behavioral tasks were too low for test-statistics. The numbers of MR-Tg females and controls in the proestrus stage in the FET were too low for test-statistics. The estrus stage was well represented in all behavioral tasks in all groups. Given this uneven distribution, we opted for including the stage of the estrous cycle as a covariate in the one-way ANOVA and General Linear Model repeated measures analyses. These analyses did not support a significant contribution of estrous cycle on performance. Stimulus-response Learning Females Females Males Behavioral parameters MR-Tg Control MRCaMKCre Control MRCaMKCre Control Latency to spatial exit hole (s) 68612 82611 88614 71613 96611 64615 Latency to spatial exit quadrant (s) 38612# 38611 77616 28610 73613* 36611 Duration in spatial exit quadrant (% of time on CHB) 3466 3666 2069* 5168 2366* 4568 Data represent mean 6 SEM. Behavioral parameters that differ significantly; p,0.05: vs same sex control littermates; #female MRCaMKCre vs MR-Tg. doi:10.1371/journal.pone.0086236.t004 Table 4. Spatial exit memory during the first trial of the stimulus-response task on day 1 (one week after the spatial memory test) in MR-Tg and MRCaMKCre mice and their control littermates. MRs Involved in Stimulus-response Learning In rodents, stimulus-response learning takes longer and requires more training than spatial learning: they have to overcome their natural tendency to use a spatial strategy [22,25]. We expected that training the mice in the spatial version of the CHB task preceding training in the stimulus-response task might even amplify the difficulty in acquiring stimulus-response learning. On the other hand, due to prior training, the environment is familiar and mice have learned that there is an exit hole. With this prior experience they now have to learn that the exit hole is marked by an intramaze cue, the bottle, positioned at a different location every trial. Thus, again, mice had to be flexible and adapt to a novel situation. Will mice express a spatial bias to the fixed location of the exit hole learned during spatial training? C MKC Overall, behavior in the absence of functional MRs might represent a restriction of behavioral flexibility [18], while selective activation of MR allows adequate explorative behavior and adaptation. We reproduced an earlier finding [19] that MRCaMKCre female mice remain longer in the center during spatial training. Conversely, MR-overexpressing mice displayed more entries into the central area of an open field and had shorter latencies entering the light compartment of a light/dark task, which was labeled by others as reduced anxiety-related behavior [8]. This suggests that staying longer in the center as reported for the MRCaMKCre mice is due to enhanced anxiety. Yet, unconditioned anxiety was not observed in MRCaMKCre mice [7,10,18,23]. The behavior of MRCaMKCre mice may therefore also point to a different coping style. g p g As expected, the control littermates of the MRCaMKCre male and female mice and the MR-Tg mice and their control littermates showed a spatial tendency (more time in the spatial exit quadrant) in the first trial of the stimulus-response task. On the first day of training, controls of the MRCaMKCre mice used more serial searches and switched to a preference of the stimulus- response strategy on the second day. MR-Tg female mice showed more perseverations but a comparable low percentage of serial searches as their control littermates. The spatial tendency was absent in MRCaMKCre male and female mice, which could e.g. be explained by their poorer earlier performance in the spatial task or by a higher propensity to switch to the stimulus-response strategy. Novel Situation of Closed Exit Hole 24 Hrs after Spatial Learning Exposing the mice 24 hrs after six spatial trials to the CHB with the exit hole closed can be compared to a probe (free exploration trial) in the water maze. Latency to the exit hole reflects retention of spatial memory, but their behavior is also the response to this novel situation with no exit available. Similar latency to exit indicates that retention is comparable between the groups. Time to leave the center was comparable between the groups. Other measures provide a wealth of information. In the water maze task, the time spent in quadrant is mainly used to demonstrate the strength of spatial memory. Earlier studies in the water maze showed that MR-overexpressing male mice spent significantly more time during the probe trial in the target quadrant than control mice [8,27]. Is this strong memory or perseveration which could be another kind of coping with novelty? In the present study, control mice of all groups, spent most time in the target quadrant as do MRCaMKCre female and MR-Tg female mice. MRCaMKCre male mice spent a comparable amount of time in all quadrants. Latency to the target quadrant was shorter in all control groups and MR-Tg mice than in MRCaMKCre mice. Moreover, MRCaMKCre female mice had the lowest number of hole visits, spent the least time in the zone with holes and moved the shortest distance compared to all groups. This may reflect a similar difference in coping style as observed on the first day. Overall, the MRCaMKCre mice seem to lack the high degree of behavioral flexibility that is required for optimal performance in the stimulus-response task. Mineralocorticoid Receptors in Learning Strategies We tentatively conclude that spatial memory - when expressed by latencies - is not affected by MR. However, coping with a novel situation seems to depend on MR. activity: persistent revisits of holes in the male mice and longer time spent in the center before exploring the CHB in the female mice. These behaviours appear to be characteristic for MRCaMKCre mice, as they are in line with behavioral responses previously seen when MR-deficient mice were exposed to conditions of novelty [7,10,18,19]. After acute pharmacological inhibition of MR in rats goal-directed search strategies in a water maze task were absent [6]. In contrast, selectively activating MRs in adrenalectomized rats normalized the exploration pattern [26]. In line with MR effects in rodents, the few studies that were done in humans with pharmacological blockade of MR reported a deficit of selective attention and impairment of working memory [28,29] indicating difficulties in the acquisition of novel informa- tion. MRs Involved in Stimulus-response Learning However, we can rule out the latter possibility because we could not detect a stimulus-response strategy on either day. The MRCaMKCre mice simply took more time to solve the task. In contrast, MR-Tg mice were as fast as their control littermates. Since mice of all control groups perform well and the latency to the exit is short, we suggest a ceiling effect in performance, which may explain why we could not detect further improvement of MR-overexpressing mice. Even when subtracting the time in center from the overall latency to the exit hole, the latencies of MRCaMKCre mice were longer than in control mice. It is likely that the different exploration pattern of MR-deficient mice seen during the initial exposure to the CHB underlies or at least contributes to their impaired performance in the spatial and stimulus-response tasks. Processing of information from the environment is depending on the MR function, as shown by behavior 24 hrs after spatial learning and during the stimulus- response task. Performance of MR-deficient mice was impaired compared to MR-overexpressing mice, but comparable to their controls during that first stimulus-response trial. As revealed by analysis over the trials of the first day of stimulus-response training, the performance of control mice appears to be guided by spatial tendency, which was absent in the MR-deficient mice. Therefore, we propose that MR deficiency exerts a more general effect on behavior by inhibiting or delaying the adaptation to novel requirements. The current behavioral set-up did not allow to distinguish between a strong spatial tendency and reduced behavioral flexibility, as both will result in long latencies in the first stimulus-response trial after spatial training. C C Discussion To further delineate the role of MRs in spatial and stimulus- response learning we subjected mice either lacking or overex- pressing MRs in the forebrain to a spatial and stimulus-response learning task. Solving these tasks requires hippocampal and dorsal striatum memory systems, respectively. Spatial learning is the predominant form of learning in male rodents; acquisition of Table 5. Percentage of serial hole visits averaged over the trials of the two days of the stimulus-response task. Females Females Males Stimulus-response task MR-Tg Control MRCaMKCre Control MRCaMKCre Control Day 1 1966 1865 1467 2768 1867 3869* Day 2 1165 1465 1366 1666 1166 1166 Both days of the stimulus-response task consisted of six trials. Data represent mean 6 SEM of all trials of one day. For statistics a repeated measures ANOVA was used over the trials of one day. *Significantly different between genotypes of the same group over the day; p,0.05. doi:10.1371/journal.pone.0086236.t005 tage of serial hole visits averaged over the trials of the two days of the stimulus-response task. Table 5. Percentage of serial hole visits averaged over the trials of the two days of the stimulu January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org 8 Mineralocorticoid Receptors in Learning Strategies January 2014 \| Volume 9 \| Issue 1 \| e86236 Memory Systems, Glucocorticoids and MR: Human Studies Memory Systems, Glucocorticoids and MR: Human Studies Previous research showed that glucocorticoids are involved in the stress-induced shift from hippocampus-dependent to dorsal striatum-dependent learning. In human studies, this role of glucocorticoids was extended from navigational to other forms of learning [21,36,37]. Recently, Schwabe et al. suggested that the relationship between glucocorticoid concentrations and the use of different memory systems may not be linear but more likely in the shape of an inverted u-shaped curve [37,38]. This reasoning is based on findings that humans exhibiting high stress-induced cortisol responses used more stimulus-response learning [39], while spatial learners had higher basal cortisol concentrations [40] and pharmacological elevations of cortisol led to use of more spatial learning [41]. Schwabe et al. discussed that functioning of the hippocampus and dorsal striatum may be affected differently by low, moderate and high levels of glucocorticoids, and thus, allowing different memory systems to be in control of behavior [37,38]. Also here we might deal with a differential contribution of MR and GR to the behavioral effects which have not been entangled yet. In addition to the overall sex-dependent differences, the phase of the estrous cycle may influence the behavior of female mice [19,20,34]. Recently we reported a specific interaction between MR and female sex hormones [19]. MRCaMKCre females showed impaired performance in the dual-solution CHB task specifically in the proestrus and estrus phase of the cycle. The phase of the estrous cycle had no effect on performance of C57Bl/6j and control littermates of MRCaMKCre female mice [19]. Due to the relatively low numbers per cycle stage, we could not incorporate the data on the estrus phases in the current study, which is a limitation. However, introducing the cycle stage as a co-variate in our analysis did not affect the outcome. Recently, blockade of MR was reported to prevent the stress- induced shift from hippocampal towards dorsal striatum-depen- dent learning in a classification task in humans, underlining the importance of MR for the use of multiple memory systems [42]. Furthermore, stress-induced facilitation of inhibitory control in a stop-signal task in humans was reported to depend on MR functioning, indicating that MRs are important for the balance between inhibition and excitation that underlies adaptive behavior [43]. Influence of Task-dependent Characteristics on Behaviour Previous studies using the dual-solution CHB and water maze tasks reported impaired performance, predominantly delayed learning, in MRCaMKCre male mice [7,18]. In the present study, MRCaMKCre male mice performed comparable to control mice. Task-specific characteristics might have influenced the perfor- mance in several ways. First, for mice the water maze is a more stressful task than a dry land maze task such as the CHB [30,31]. Acute stress impaired the performance of control mice in the dual- Task-specific characteristics might have influenced the perfor- mance in several ways. First, for mice the water maze is a more stressful task than a dry land maze task such as the CHB [30,31]. Acute stress impaired the performance of control mice in the dual- January 2014 \| Volume 9 \| Issue 1 \| e86236 January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org 9 Mineralocorticoid Receptors in Learning Strategies [7,10], but also elevated basal corticosterone levels have been reported [18]. A fast increase of corticosterone in response to stress indicates the lack of MR-dependent inhibitory control of the HPA axis and apparently more efficient negative feedback can be deduced from lower corticosterone measured directly after training in a circular holeboard task [18]. Elevated basal corticosterone in MR-deficient mice might reflect the sensitivity of the HPA axis to subtle environmental changes, which are under inhibitory control of MR in control mice. MR-overexpressing mice and their control littermates had comparable basal cortico- sterone levels and showed a comparable corticosterone response to restraint stress [8]. Like previously reported, we expect that the mice in the present study also show this initial MR-dependent disinhibition the HPA axis resulting in elevated concentrations of corticosterone. solution and spatial CHB task [17,22]. Interestingly, acute stress did not further impair performance of MRCaMKCre male mice in the dual-solution CHB task [18]. Second, the dual-solution version of the CHB task provides both intramaze and spatial cues. Since more cues are available, this requires a fine tuned coordination of hippocampal and dorsal striatal memory systems. The coordina- tion of memory systems might be affected by MR deficiency, resulting in delayed learning. In contrast to the MR-deficient mice, MR-overexpressing male mice showed no difference in spatial learning in water maze and Y-maze tasks [8,27]. Influence of Task-dependent Characteristics on Behaviour Moreover, a rather selective method, namely viral-mediated overexpression of MR in the hippocampus in male rats caused no differences in spatial performance in a water maze task [32]. The findings in MR-overexpressing mice suggest that the behavioral parameters of the tasks might not be sensitive enough to measure MR effects. Alternatively, we may conclude that MR manipulations do not directly affect the memory process. If and how these different-to-control corticosterone concentra- tions in MR-mutant mice affect and act via GRs: we don’t know yet. The characteristics and action of GR (low affinity, fast feedback) indicate an involvement of GR in MR-deficient mice in a time domain different from control mice. GRs are involved in HPA axis regulation and stress effects on cognition, specifically supporting memory consolidation [6,11–13]. GR mRNA is increased in the hippocampus of MRCaMKCre mice; however their memory is not improved [18]. GR mRNA and basal corticoste- rone levels in MR-Tg mice are comparable to controls, as is their memory [8]. Therefore, we cannot exclude that deficits in spatial and stimulus-response performance in the MR-deficient mice are linked to increased circulating basal corticosterone levels that may act via GR. Are Female Mice More Sensitive than Males to Changing Environments? Male C57Bl/6j mice and control littermates of MRCaMKCre male mice solve the dual-solution CHB task by using a spatial strategy, while female C57Bl/6j and control littermates of MRCaMKCre female mice use both, spatial and stimulus-response strategies [18–21]. MR deficiency led to impaired spatial performance in MRCaMKCre female mice, while the effect was less apparent in MRCaMKCre male mice. Similar results were found in other learning tasks. For example, MRCaMKCre female mice were unable to extinguish the contextual fear memory and could not discriminate between cue and context episodes of the task, while no effects of MR ablation were found in MRCaMKCre male mice [23]. In a radial arm maze task, MRCaMKCre female mice made more errors than males [7]. Sex-dependent differences in performance were aggravated by acute stress: male mice switched from a spatial to a stimulus-response strategy, while female mice switched to a spatial strategy [17,33]. These observations are in line with our present findings and underline that using subjects of both sexes increases the likelihood to detect effects of the experimental manipulation. In general, variations in MR appear to affect females more explicitly than males. Memory Systems, Glucocorticoids and MR: Human Studies Although the use of MR (and GR antagonists) contributes to the understanding of the function of either receptor, we are still confronted by the fact that the blockade of MR (or deficiency of MR) increases corticosterone concentrations that should allow the activation of GR. January 2014 \| Volume 9 \| Issue 1 \| e86236 References 23. ter Horst JP, Carobrez AP, van der Mark MH, de Kloet ER, Oitzl MS (2012) Sex differences in fear memory and extinction of mice with forebrain-specific disruption of the mineralocorticoid receptor. Eur J Neurosci 36: 3096–3102. 1. de Kloet ER, Joe¨ls M, Holsboer F (2005) Stress and the brain: From adaptation to disease. Nat Rev Neurosci 6: 463–47. 2. Joe¨ls M, Krugers HJ, Lucassen PJ, Karst H (2009) Corticosteroid effects on cellular physiology of limbic cells. Brain Res 1293: 91–100. p p J 24. Casanova E, Fehsenfeld S, Mantamadiotis T, Lemberger T, Greiner E, et al. (2001) A CamKIIalpha iCre BAC allows brain-specific gene inactivation. Genesis 31: 37–42. 3. Reul JM, de Kloet ER (1985) Two receptor systems for corticosterone in rat brain: Microdistribution and differential occupation. Endocrinology 117: 2505– 2511. 25. Packard MG, McGaugh JL (1996) Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning. Neurobiol Learn Mem 65: 65–72. 4. de Kloet ER, Oitzl MS, Joe¨ls M (1999) Stress and cognition: Are corticosteroids good or bad guys? Trends Neurosci 22: 422–426. g 26. Oitzl MS, Fluttert M, de Kloet ER (1994) The effect of corticosterone on reactivity to spatial novelty is mediated by central mineralocorticosteroid receptors. Eur J Neurosci 6: 1072–9. 5. Joe¨ls M, Karst H, de Rijk R, de Kloet ER (2008) The coming out of the brain mineralocorticoid receptor. Trends Neurosci 31: 1–7. 5. Joe¨ls M, Karst H, de Rijk R, de Kloet ER (2008) The 6. Oitzl MS, de Kloet ER (1992) Selective corticosteroid antagonists modulate specific aspects of spatial orientation learning. Behav Neurosci 106: 62–71. p J 27. Harris AP, Holmes MC, de Kloet ER, Chapman KE, Seckl JR (2013) Mineralocorticoid and glucocorticoid receptor balance in control of HPA axis and behaviour. Psychoneuroendocrinology 38: 648–58. specific aspects of spatial orientation learning. Behav Neuro 7. Berger S, Wolfer DP, Selbach O, Alter H, Erdmann G, et al. (2006) Loss of the limbic mineralocorticoid receptor impairs behavioral plasticity. Proc Natl Acad Sci USA 103: 195–200. y gy 28. Cornelisse S, Joe¨ls M, Smeets T (2011) A randomized trial on mineralocorticoid receptor blockade in men: Effects on stress responses, selective attention, and memory. Neuropsychopharmacology 36: 2720–8. 8. Lai M, Horsburgh K, Bae S, Carter RN, Stenvers DJ, et al. (2007) Forebrain mineralocorticoid receptor overexpression enhances memory, reduces anxiety and attenuates neuronal loss in cerebral ischaemia. References Packard MG, Knowlton BJ (2002) Learning and memory functions of the basal ganglia. Annu Rev Neurosci 25: 563–593. 37. Schwabe L, Wolf OT (2013) Stress and multiple memory systems: From ‘thinking’ to ‘doing’. Trends Cogn Sci 17: 60–68. 16. Lee AS, Duman RS, Pittenger C (2008) A double dissociation revealing bidirectional competition between striatum and hippocampus during learning. Proc Natl Acad Sci 105: 17163–17168. 38. Schwabe L (2013) Stress and the engagement of multiple memory systems: integration of animal and human studies. Hippocampus 23: 1035–1043. 17. Schwabe L, Scha¨chinger H, de Kloet ER, Oitzl MS (2010) Corticosteroids operate as a switch between memory systems. J Cogn Neurosci 22: 1362–72. 39. Schwabe L, Oitzl MS, Philippsen C, Bo¨hringer A, Richter S, et al. (2007) Stress modulates the use of spatial and stimulus-response learning strategies in humans. Learn Mem 14: 109–116. 18. ter Horst JP, van der Mark MH, Arp M, Berger S, de Kloet ER, et al. (2012) Stress or no stress: Mineralocorticoid receptors in the forebrain regulate behavioral adaptation. Neurobiol Learn Mem 98: 33–40. J p g ( ) Stress or no stress: Mineralocorticoid receptors in the forebrain regulate behavioral adaptation. Neurobiol Learn Mem 98: 33–40. 40. Bohbot VD, Gupta M, Banner H, Dahmani L (2011) Caudate nucleus- dependent response strategies in a virtual navigation task are associated with lower basal cortisol and impaired episodic memory. Neurobiol Learn Mem 96: 173–180. 19. ter Horst JP, Kentrop J, Arp M, Hubens CJ, de Kloet ER, et al. (2013) Spatial learning of female mice: A role of the mineralocorticoid receptor during stress and the estrous cycle. Front Behav Neurosci. 41. Schwabe L, Oitzl MS, Richter S, Scha¨chinger H (2009) Modulation of spatial and stimulus-response learning strategies by exogenous cortisol in healthy young women. Psychoneuroendocrinology 34: 358–366. 20. ter Horst JP, Kentrop J, de Kloet ER, Oitzl MS (2013) Stress and estrous cycle affect strategy but not performance of female C57BL/6J mice. Behav Brain Res 241: 92–95. 42. Schwabe L, Tegenthoff M, Ho¨ffken O, Wolf OT (2013) Mineralocorticoid receptor blockade prevents stress-induced modulation of multiple memory systems in the human brain. Biol Psychiatry 74: 801–808. 21. Schwabe L, Dalm S, Scha¨chinger H, Oitzl MS (2008) Chronic stress modulates the use of spatial and stimulus-response learning strategies in mice and man. Neurobiol Learn Mem 90: 495–503. 43. References Eur J Neurosci 25: 1832– 1842. 29. Otte C, Moritz S, Yassouridis A, Koop M, Madrischewski AM, et al. (2007) Blockade of the mineralocorticoid receptor in healthy men: Effects on experimentally induced panic symptoms, stress hormones, and cognition. Neuropsychopharmacology 32: 232–8. 9. Zhou M, Bakker EHM, Velzing EH, Berger S, Oitzl M, et al. (2010) Both mineralocorticoid and glucocorticoid receptors regulate emotional memory in mice. Neurobiol Learn Mem 94: 530–537. 30. Dalm S, Schwabe L, Schachinger H, Oitzl MS (2009) Post-training self administration of sugar facilitates cognitive performance of male C57BL/6J mice in two spatial learning tasks. Behav Brain Res 198: 98–104. 10. Brinks V, Berger S, Gass P, de Kloet ER, Oitzl MS (2009) Mineralocorticoid receptors in control of emotional arousal and fear memory. Horm Behav 56: 232–238. 31. Whishaw IQ, Tomie J (1997) Of mice and mazes: Similarities between mice and rats on dry land but not water mazes. Physiol Behav 60: 1191–1197. D S l k R ( ) O i f i l i id d 11. Lupien SJ, McEwen BS (1997) The acute effects of corticosteroids on cognition: Integration of animal and human model studies. Brain Res Rev 24: 1–27. 32. Ferguson D, Sapolsky R (2008) Overexpression of mineralocorticoid and transdominant glucocorticoid receptor blocks the impairing effects of glucocor- ticoids on memory. Hippocampus 18: 1103–1111. 12. Sandi C, Rose SPR (1994) Corticosterone enhances long-term retention in one- day-old chicks trained in a weak passive avoidance learning paradigm. Brain Res 647: 106–112. 33. Bettis TJ, Jacobs LF (2009) Sex-specific strategies in spatial orientation in C57BL/6J mice. Behav Processes 82: 249–255. J 34. ter Horst JP, de Kloet ER, Scha¨chinger H, Oitzl MS (2012) Relevance of stress and female sex hormones for emotion and cognition. Cell Mol Neurobiol 32: 725–35. 13. Roozendaal B, McReynolds JR, Van der Zee EA, Lee S, McGaugh JL, et al. (2009) Glucocorticoid effects on memory consolidation depend on functional interactions between the medial prefrontal cortex and basolateral amygdala. J Neurosci 29: 14299–14308. 35. Oitzl MS, Champagne DL, van der Veen R, de Kloet ER (2010) Brain development under stress: Hypotheses of glucocorticoid actions revisited. Neurosci Biobehav Rev 34: 853–866. 14. White NM, McDonald RJ (2002) Multiple parallel memory systems in the brain of the rat. Neurobiol Learn Mem 77: 125–184. 36. Schwabe L, Wolf OT (2012) Stress modulates the engagement of multiple memory systems in classification learning. J Neurosci 32: 11042–11049. 15. Acknowledgments We are grateful to S. Berger and G. Schutz, German Cancer Institute, Heidelberg, Germany and A. Fitch and J. Seckl, University of Edinburgh, U.K., for providing the original breeding stock of the MR mutant mice in 2008. Two Receptors for Adaptive Behavior: MR and GR Two Receptors for Adaptive Behavior: MR and GR Adaptive behavior depends on balanced MR and GR activation [1,35]. As documented previously [6,7,10,18] and extended by our current observations, MRs modulate the behavioral response pattern in novel situations. We cannot estimate the number of MRs that is necessary to induce the changes observed in MR- overexpressing mice. Lai et al (2007) report a brain-site-dependent increase of MR mRNA of 4 to 10 times in MR-Tg mice. MR deficiency in the forebrain affects the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Basal corticosterone levels of MR-deficient mice are either comparable to controls January 2014 \| Volume 9 \| Issue 1 \| e86236 January 2014 \| Volume 9 \| Issue 1 \| e86236 PLOS ONE \| www.plosone.org 10 Mineralocorticoid Receptors in Learning Strategies In summary: our current study demonstrates that MRs are relevant for spatial as well as stimulus-response learning. Deficits in both tasks were more strongly expressed in MR-deficient female than male mice, revealing and supporting a sex-dependent effect. We suggest that the common nominator of MR effects can be represented as behavioral flexibility which requires a critical balance between inhibitory and excitatory systems. The conse- quences of corticosteroid actions via MR can be observed at different levels, e.g., the switch between memory systems, strategies, selective attention, performance. In this manner MRs influence and coordinate the processing of information particularly under novel conditions, which is fundamental for behavioral adaptation. Author Contributions Conceived and designed the experiments: JMA JPTH MJ HJK MSO GF. Performed the experiments: JMA. Analyzed the data: JMA JPTH MSO. Contributed reagents/materials/analysis tools: JMA SK MSO. Wrote the paper: JMA JPTH MJ HJK MSO. References Schwabe L, Ho¨ffken O, Tegenthoff M, Wolf OT (2013) Stress-induced enhancement of response inhibition depends on mineralocorticoid receptor activation. Psychoneuroendocrinology 38: 2319–2326. 22. Schwabe L, Scha¨chinger H, de Kloet ER, Oitzl MS (2010) Stress impairs spatial but not early stimulus–response learning. Behav Brain Res 213: 50–55. PLOS ONE \| www.plosone.org January 2014 \| Volume 9 \| Issue 1 \| e86236 January 2014 \| Volume 9 \| Issue 1 \| e86236 11
https://openalex.org/W2969925561	https://link.springer.com/content/pdf/10.1007/s10147-019-01526-7.pdf	English	null	An open-label, phase 1 study of androgen receptor antagonist, apalutamide in Japanese patients with metastatic castration-resistant prostate cancer	International journal of clinical oncology	2,019	cc-by	6,459	Abstract Conclusion Apalutamide had manageable safety profile without any DLT or any new safety signals and favourable efficacy fi Methods In this open-label, multi-center study, patients received apalutamide 240 mg (once-daily, orally) for first 1 week (PK week) during which PK parameters were assessed. 1 week later (Cycle 1 Day1), after reassessing safety, continuous daily dosing (4 weeks/cycle; once-daily orally) was initiated. Endpoints evaluated were: safety, tolerability, PK and antitumour efficacy of apalutamide. Dose-limiting toxicities (DLTs) were evaluated during PK week and Cycle 1. fi Results All six patients received apalutamide. The most common treatment-emergent adverse events (TEAEs) were abdomi- nal discomfort, nasopharyngitis, dysgeusia, rash, and hot flush [2/6 patients (33.3%) each]. No death or DLTs were reported. Grade 3 TEAEs were spinal-cord compression and renal disorder (1/6 patient each). In continuous daily dosing period, PK steady-state of apalutamide was reached approximately by week 4. A significant accumulation of apalutamide was observed (mean accumulation index 3.55), based on AUC0–24. Median (range) serum prostate-specific antigen level decreased from 54.42 (8.92–310.11) ng/mL at baseline to 11.70 (0.37–47.74) ng/mL at week 12 with ≥ 50% reduction in 4/6 (66.7%) patients and 90% reduction in 2/6 (33.3%) patients. l Grade 3 TEAEs were spinal-cord compression and renal disorder (1/6 patient each). In continuous daily dosing period, PK steady-state of apalutamide was reached approximately by week 4. A significant accumulation of apalutamide was observed (mean accumulation index 3.55), based on AUC0–24. Median (range) serum prostate-specific antigen level decreased from 54.42 (8.92–310.11) ng/mL at baseline to 11.70 (0.37–47.74) ng/mL at week 12 with ≥ 50% reduction in 4/6 (66.7%) patients and 90% reduction in 2/6 (33.3%) patients.ifi Conclusion Apalutamide had manageable safety profile, without any DLT or any new safety signals, and favourable efficacy in Japanese mCRPC patients. Thus, it was ascertained to be an adequate dosage regimen in Japanese mCRPC patients. Trial registration ClinicalTrials.gov identifier: NCT02162836. rds Androgen receptor antagonist · Apalutamide · Metastatic castration-resistant prostate cancer · P International Journal of Clinical Oncology (2019) 24:1596–1604 https://doi.org/10.1007/s10147-019-01526-7 International Journal of Clinical Oncology (2019) 24:1596–1604 https://doi.org/10.1007/s10147-019-01526-7 ORIGINAL ARTICLE ORIGINAL ARTICLE Tomohiro Tsuchiya1 · Keiichiro Imanaka2 · Yuki Iwaki2 · Ryo Oyama2 · Katsuyoshi Hashine3 · Akito Yamaguchi4 · Hiroji Uemura5 Received: 26 March 2019 / Accepted: 30 July 2019 / Published online: 24 August 2019 © The Author(s) 2019 4 Harasanshin Hospital, Fukuoka, Japan 5 Yokohama City University Medical Centre, 4‑57, Urafune‑cho, Minami‑ku, Yokohama 232‑0024, Japan 5 Yokohama City University Medical Centre, 4‑57, Urafune‑cho, Minami‑ku, Yokohama 232‑0024, Japan Abstract Background Apalutamide, a nonsteroidal potent androgen receptor antagonist, was safe and effective in patients with non- metastatic castration-resistant prostate cancer (nmCRPC) and metastatic-CRPC (mCRPC) in global studies. In this phase 1 study, safety, pharmacokinetics (PK), and efficacy of apalutamide were evaluated in Japanese patients with mCRPC.i Background Apalutamide, a nonsteroidal potent androgen receptor antagonist, was safe and effective in patients with non- metastatic castration-resistant prostate cancer (nmCRPC) and metastatic-CRPC (mCRPC) in global studies. In this phase 1 study, safety, pharmacokinetics (PK), and efficacy of apalutamide were evaluated in Japanese patients with mCRPC. Methods In this open-label, multi-center study, patients received apalutamide 240 mg (once-daily, orally) for first 1 week (PK week) during which PK parameters were assessed. 1 week later (Cycle 1 Day1), after reassessing safety, continuous daily dosing (4 weeks/cycle; once-daily orally) was initiated. Endpoints evaluated were: safety, tolerability, PK and antitumour efficacy of apalutamide. Dose-limiting toxicities (DLTs) were evaluated during PK week and Cycle 1. Background Apalutamide, a nonsteroidal potent androgen receptor antagonist, was safe and effective in patients with non- metastatic castration-resistant prostate cancer (nmCRPC) and metastatic-CRPC (mCRPC) in global studies. In this phase 1 study, safety, pharmacokinetics (PK), and efficacy of apalutamide were evaluated in Japanese patients with mCRPC. Methods In this open-label, multi-center study, patients received apalutamide 240 mg (once-daily, orally) for first 1 week (PK week) during which PK parameters were assessed. 1 week later (Cycle 1 Day1), after reassessing safety, continuous daily dosing (4 weeks/cycle; once-daily orally) was initiated. Endpoints evaluated were: safety, tolerability, PK and antitumour efficacy of apalutamide. Dose-limiting toxicities (DLTs) were evaluated during PK week and Cycle 1. Results All six patients received apalutamide. The most common treatment-emergent adverse events (TEAEs) were abdomi- nal discomfort, nasopharyngitis, dysgeusia, rash, and hot flush [2/6 patients (33.3%) each]. No death or DLTs were reported. Grade 3 TEAEs were spinal-cord compression and renal disorder (1/6 patient each). In continuous daily dosing period, PK steady-state of apalutamide was reached approximately by week 4. A significant accumulation of apalutamide was observed (mean accumulation index 3.55), based on AUC0–24. Median (range) serum prostate-specific antigen level decreased from 54.42 (8.92–310.11) ng/mL at baseline to 11.70 (0.37–47.74) ng/mL at week 12 with ≥ 50% reduction in 4/6 (66.7%) patients and 90% reduction in 2/6 (33.3%) patients. 4 Harasanshin Hospital, Fukuoka, Japan An open‑label, phase 1 study of androgen receptor antagonist, apalutamide in Japanese patients with metastatic castration‑resistant prostate cancer Tomohiro Tsuchiya1 · Keiichiro Imanaka2 · Yuki Iwaki2 · Ryo Oyama2 · Katsuyoshi Hashine3 · Akito Yamaguchi4 · Hiroji Uemura5 iya1 · Keiichiro Imanaka2 · Yuki Iwaki2 · Ryo Oyama2 · Katsuyoshi Hashine3 · Akito Yamaguchi4 * Hiroji Uemura hu0428@yokohama‑cu.ac.jp Introduction Prostate cancer is the 6th leading cause of cancer death in men worldwide (307,000 deaths in 2012) [1]. Prostate can- cer is estimated as one of the most common type of can- cers in Japanese men and the sixth largest cause of cancer- related death [2–4]. Androgen deprivation therapy remains The results of this study were presented at the 56th annual meeting of Japan Society of Clinical Oncology, 18–20th October 2018, Japan. The results of this study were presented at the 56th annual meeting of Japan Society of Clinical Oncology, 18–20th October 2018, Japan. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10147-019-01526-7) contains supplementary material, which is available to authorized users. * Hiroji Uemura hu0428@yokohama‑cu.ac.jp 1 Gifu University Hospital, Gifu, Japan 2 Janssen Pharmaceutical K.K., Tokyo, Japan 3 National Hospital Organization Shikoku Cancer Centre, Matsuyama, Japan Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10147-019-01526-7) contains supplementary material, which is available to authorized users. * Hiroji Uemura hu0428@yokohama‑cu.ac.jp 1 Gifu University Hospital, Gifu, Japan 2 Janssen Pharmaceutical K.K., Tokyo, Japan 3 National Hospital Organization Shikoku Cancer Centre, Matsuyama, Japan Vol:.(123456789 1 3 :.(123456789 3 International Journal of Clinical Oncology (2019) 24:1596–1604 1597 inhibitors (e.g., abiraterone acetate, orteronel, galeter- one, and systemic ketoconazole). Patients with a history of seizure or condition that may predispose to seizure, or use of radiopharmaceutical agents (e.g., strontium-89) or investigational immunotherapy (e.g., sipuleucel-T) within 12 weeks or any investigational non-immunologic agent within 4 weeks before the first dose of study agent were also excluded. Patients who used concurrent therapy with medications known to lower the seizure threshold and strong CYP3A4 inhibitors or inducers, herbal, and non-herbal prod- ucts that could decrease serum PSA level, systemic (oral/IV/ IM) corticosteroids, or any other experimental treatment on another clinical trial within 4 weeks before the first dose of study agent were excluded. the mainstay of treatment for advanced castration-sensitive prostate cancer. Despite the effective blocking of androgen biosynthesis and androgen receptor (AR) signalling, most of patients eventually progress and the disease ultimately becomes castration-resistant [5]. Apalutamide, a second generation nonsteroidal potent AR antagonist, selectively binds to the ligand-binding domain of AR with seven–tenfold greater affinity than the clinically approved antiandrogen, bicalutamide. In addition, in contrast to bicalutamide, apalutamide lacks significant AR agonist activity in preclinical models of CRPC and is unable to induce AR nuclear translocation and DNA binding in pros- tate cancer cells [6]. Introduction Apalutamide has a major metabolite JNJ-56142060, a pharmacologically active AR antagonist, with approximately threefold less potency than apalutamide. Apalutamide, a second generation nonsteroidal potent AR antagonist, selectively binds to the ligand-binding domain of AR with seven–tenfold greater affinity than the clinically approved antiandrogen, bicalutamide. In addition, in contrast to bicalutamide, apalutamide lacks significant AR agonist activity in preclinical models of CRPC and is unable to induce AR nuclear translocation and DNA binding in pros- tate cancer cells [6]. Apalutamide has a major metabolite JNJ-56142060, a pharmacologically active AR antagonist, with approximately threefold less potency than apalutamide. In the previous phase 1/2 study [7], apalutamide was safe and well-tolerated at 30–480 mg dose in non-Japanese castration-resistant prostate cancer (CRPC) patients. In the phase 2 part of that study [7], apalutamide at the 240 mg daily dose was safe with promising efficacy [prostate-spe- cific antigen (PSA) response] in patients with non-meta- static-CRPC (nmCRPC) and metastatic-CRPC (mCRPC). The present study was conducted to evaluate the safety, tol- erability, pharmacokinetics (PK), and the antitumor effects of apalutamide after a single-dose (240 mg) and multiple- dose administration (240 mg once-daily) of apalutamide in Japanese men with mCRPC. Study design and treatment In the previous phase 1/2 study [7], apalutamide was safe and well-tolerated at 30–480 mg dose in non-Japanese castration-resistant prostate cancer (CRPC) patients. In the phase 2 part of that study [7], apalutamide at the 240 mg daily dose was safe with promising efficacy [prostate-spe- cific antigen (PSA) response] in patients with non-meta- static-CRPC (nmCRPC) and metastatic-CRPC (mCRPC). The present study was conducted to evaluate the safety, tol- erability, pharmacokinetics (PK), and the antitumor effects of apalutamide after a single-dose (240 mg) and multiple- dose administration (240 mg once-daily) of apalutamide in Japanese men with mCRPC. In this phase 1, open-label, multi-centre study, the safety, tolerability, PK, and preliminary evidence of the antitumor effects of apalutamide (240 mg, once-daily orally) was eval- uated in Japanese patients with mCRPC. This study com- prised 3 periods: a screening period (28 days before study treatment), a treatment period (PK week and continuous daily dosing), and a post-treatment safety follow-up period (30 days after treatment discontinuation). Dose-limiting tox- icity (DLT) evaluation period was defined as the PK week (1 week) and Cycle 1 (4 weeks) (Fig. 1). y g In PK week, patients received a single oral dose of apalutamide 240 mg on day 1. After a week, patients were reassessed for safety to check treatment-emergent adverse events (TEAEs), serious TEAEs, DLT, Grade 3 toxicities, and other safety parameters on Cycle 1 day 1 (day 8) and these safety profiles were compared with those of the phase 1 global study. If no new safety signals, no difference in the frequency or the degree of an event was reported, the safety profile was acceptable. The patients were then started on continuous daily oral therapy of apalutamide 240 mg until disease progression, unacceptable toxicity, withdrawal of consent, or death, whichever occurred first. If any DLT was reported, the treatment was stopped and restarted only after the toxicities were resolved to ≤ Grade 1 or baseline. After recovery, the dose was to be reduced accordingly (level 1: 180 mg/day, level 2: 120 mg/day) and restarted in accordance with the original schedule. Patients were to be withdrawn from the study if > 2 dose-level reduction was required or if the treatment did not restart within 28 days after occurrence of toxicity in principle. Intra-patient dose escalation was not allowed. Patients Japanese men aged ≥ 20 years with mCRPC who had his- tologically confirmed adenocarcinoma of the prostate without neuroendocrine differentiation or small cell fea- tures were included in this study. Inclusion criteria also involved maintained castrate levels of testosterone [< 50 ng/ dL (1.72 nmol/L)] within 4 weeks and serum PSA level of ≥ 2 ng/mL within 2 weeks before enrolment, which had risen on ≥ 2 successive occasions, ≥ 1 week apart. Patients who received first generation antiandrogen as part of an ini- tial combined androgen blockade therapy or as second-line hormonal therapy for continuing disease progression, or who progressed after AR antagonists, 5-α reductase inhibi- tors, estrogens, and any other anticancer therapy, including chemotherapy given in the adjuvant/neoadjuvant setting were required to be off those medications for ≥ 4 weeks before first dose administration. The study protocol and amendments were approved by the local Institutional Review Board and the study was con- ducted in accordance with the ethical principles outlined in the Declaration of Helsinki. The study was consistent with the International Conference on Harmonization, Good i The exclusion criteria were a history or current metasta- ses in the brain or untreated spinal-cord compression, pro- gressive epidural disease, prior treatment with the second generation antiandrogens (e.g., enzalutamide), or CYP17 1 3 1598 International Journal of Clinical Oncology (2019) 24:1596–1604 Fig. 1 Schematic overview of the study. D1–D7 day 1 to day 7, DLT dose-limiting toxicity, PK pharmacokinetics, PSA prostate-specific antigen of the study. D1–D7 day 1 to day 7, DLT dose-limiting toxicity, PK pharmacokinetics, PSA prostate-specific antigen Fig. 1 Schematic overview of the study. D1–D7 day 1 to day 7, DLT dose-limiting toxicity, PK pharmacokinetics, Clinical Practice guidelines, and applicable regulatory requirements. Written informed consent was obtained from all patients to participate in the study. The study was regis- tered at ClincalTrials.gov: NCT02162836. chromatography/mass spectrometry/mass spectrometry method. The plasma PK parameters for apalutamide, Cmax (maximum observed plasma concentration), tmax (time to reach the maximum observed plasma concentration), t1/2λ [elimination half-life associated with the terminal slope (λz) of the semi-logarithmic drug concentration–time curve, cal- culated as 0.693/λz], and AUC0–24 (area under the plasma concentration–time curve from time 0–24 h postdose) were determined via non-compartmental analysis with Phoenix WinNonlin (Version 6.4). Patients Parameters AUClast [AUC from time 0 to time the last observed quantifiable concentration (Clast)] and AUC∞ (AUC from time 0 to infinite time, cal- culated as the sum of AUClast and Clast/λz) were determined after a single oral administration (PK week). The parameters determined only after multiple oral administrations (Cycle 1 and later) were accumulation (Acc) index, effective half-life (EHL), percentage peak to trough fluctuation (PTF)%, and Ctrough (trough plasma concentration just before dose). The following plasma PK parameters were determined for the metabolite JNJ-56142060: Cmax, tmax, AUC0–24, Ctrough, Acc index, and metabolite-to-parent ratio (MPR). Antitumour efficacy assessment Assessment of antitumor activity of apalutamide was based on percentage change in serum PSA level from baseline reported at week 12 (or after treatment discontinuation, whichever occurred first). The maximal change was also reported for each patient using waterfall plots, at any time during the study. Serum PSA level was measured at screen- ing visit, day 1 of PK week, day 1 of every cycle, and the end of treatment (EOT) visit by a central laboratory. Soft-tissue disease (measured by CT scans) and bone disease (evaluated radionuclide bone scans) were assessed at screening visit, Safety and tolerability evaluation Safety assessments included results of TEAEs, clinical labo- ratory tests, vital signs (blood pressure, pulse rate, and body temperature) and Eastern Cooperative Oncology Group (ECOG) performance status, 12-lead electrocardiograms findings, and physical examination. All TEAEs were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 (NCI CTCAE v4.0). The tolerability of apalutamide 240 mg in Japanese patients was evaluated based on the incidence of DLT (Online Resource 1). A study evaluation team (SET) was involved for final evaluation of toxicities based on DLT cri- teria, and for final decision on the safety and tolerability of apalutamide (240 mg). DLTs were assessed during the PK week and Cycle 1. Safety and tolerability of apalutamide 240 mg in Japanese patients with mCRPC were confirmed, if ≤ 1/6 patients experienced DLT. TEAEs of special inter- est (seizure, skin rash, hypothyroid, fall, and fracture) were also assessed. Pharmacokinetics Venous blood samples (approximately 3 mL each) for the determination of plasma concentrations of apalutamide and JNJ-56142060 (active metabolite) were collected and analyzed using a validated, specific, and sensitive liquid 1 3 3 1599 International Journal of Clinical Oncology (2019) 24:1596–1604 week 12. Visible soft-tissue tumors [measurable (target) or non-measurable (non-target) lesions] were evaluated using RECIST Version 1.1. Radiographic progression was defined as soft-tissue progression evaluated by RECIST Version 1.1, which was confirmed on repeat imaging ≥ 6 weeks later or by bone progression by Prostate Cancer Clinical Trials Working Group (PCWG) 2 [8].i and day 1 of every 3 cycles (e.g., Cycles 4, 7, 10, and so on), and the EOT visit. Table 1 Demographic and baseline characteristics (all- treated population) BMI body mass index, ECOG Eastern Cooperative Oncology Group, PSA prostate-specific antigen, SD standard deviation Criteria for disease progression Apalutamide was continued until both PSA progression and radiographic progression were documented, or until clinical progression such as a skeletal-related event, or until the treating physician decided to initiate a new sys- temic anticancer therapy. PSA progression was defined as PSA of ≥ 25% and ≥ 2 ng/mL above the nadir, which was confirmed by a second value obtained ≥ 3 weeks later or defined as PSA of ≥ 25% and ≥ 2 ng/mL above baseline after Symptomatic clinical progression was defined by devel- opment of a skeletal-related event (pathologic fracture, spinal-cord compression, and need for surgery on bone or radiotherapy to bone) or progression of pain (decision was based on principal investigator’s judgement; and as pain status was an exploratory endpoint, it was not checked on regular basis). The appearance of ≥ 2 new lesions on day 1 of BMI body mass index, ECOG Eastern Cooperative Oncology Group, PSA prostate-specific antigen, SD Apalutamide 240 mg (N = 6) Age (years), median (range) 78.0 (70–85) BMI (kg/m2), mean (SD) 22.04 (0.973) Time from initial diagnosis to first dose (months), median (range) 68.9 (9.2–132.7) PSA at initial diagnosis (ng/mL), median (range) 112.20 (4.8–1169.3) Extent of disease at screening, n (%) Bone 4 (66.7) Bone only 1 (16.7) Soft tisssue 5 (83.3) Lymph node 3 (50.0) Prostate mass 4 (66.7) Other 2 (33.3) Tumor stage at initial diagnosis, n (%) T2, T2a, T2b, T2c 3 (50.0) T3, T3a, T3b 2 (33.3) T4 1 (16.7) Metastasis stage at initial diagnosis, n (%) M0 4 (66.7) M1, M1a, M1b, M1c 2 (33.3) ECOG performance status score at screening, n (%) 0 5 (83.3) 1 1 (16.7) Gleason score at initial diagnosis, n (%) < 7 1 (16.7) 7 1 (16.7) ≥ 8 4 (66.7) Prior therapy, n (%) Hormonal therapy 6 (100.0) Chemotherapy 0 Radiotherapy 1 (16.7) Surgery 2 (33.3) Prostate 1 (16.7) Orchiectomy 1 (16.7) Other 2 (33.3) Table 1 Demographic and baseline characteristics (all- treated population) BMI body mass index, ECOG Eastern Cooperative Oncology Group, PSA prostate-specific antigen, SD standard deviation 1 3 1600 International Journal of Clinical Oncology (2019) 24:1596–1604 Cycle 4 visit with ≥ 2 additional new lesions on confirmatory bone scan was considered as disease progression. compliance and 1 patient (16.7%) achieved < 80% compli- ance. The median duration of study agent from day 1 Cycle 1 (except PK week) was 169.0 (range 74–869) days. Patients A total of 15 patients were screened and 6 patients of those were enrolled and treated with apalutamide at 4 sites in Japan. At the time of clinical data cutoff (15 March 2017), 5/6 patients (83.3%) discontinued treatment and 1/6 patient (16.7%) was continuing the treatment. The treatment dis- continuation was primarily due to disease progression [3/6 patients (50.0%)], physician decision, and withdrawal of consent [1/6 patient (16.7%) each]. Patient demographics and baseline characteristics are shown in Table 1. Overall, the median age of the patients was 78.0 (range 70–85) years; mean (SD) body mass index (BMI) was 22.04 kg/m2 (0.973). All patients had an ECOG performance status of 0–1. All patients received prior hor- monal therapy and none of them received chemotherapy. Criteria for disease progression At the time of clinical cutoff, all the 6 patients had received at least 3 cycles and 2 patients (33.3%) received at least 30 cycles of study agent. The median average daily dose of study agent was 236.0 (range 170.3–240.0) mg/day. Statistical methods Six patients were to be enrolled in the study to evaluate the tolerability of apalutamide at 240 mg. However, sample size was not based on statistical considerations. All-treated population set was defined as all patients who received ≥ 1 dose of apalutamide, PK analysis population set was defined as all patients who received ≥ 1 dose of apalutamide and had ≥ 1 post-treatment sample collected for PK evaluation. All-treated population set was used for efficacy and safety analyses. Patients were excluded from the PK analysis if their data did not allow accurate assessment of the PK. Plasma concentrations and corresponding PK parameters for apalutamide and JNJ-56142060 were summarized using descriptive statistics. Adverse events All patients experienced at least one TEAE. The most frequently reported TEAEs (> 1 patient) were abdominal discomfort, nasopharyngitis, dysgeusia, rash, and hot flush [2/6 patients (33.3%) each] (Table 2). The drug-related TEAEs reported in > 1 patient were abdominal discom- fort, dysgeusia, and hot flush [2/6 patients (33.3%) each]. One patient (16.7%) reported serious TEAE (Grade 3 spinal-cord compression) and, therefore, discontinued the study agent. A total of 2/6 patients (33.3%) reported Grade 3 TEAEs [spinal-cord compression and renal dis- order, 1/6 patients (16.7%) each]. However, at data cutoff, renal disorder was resolved, while the event of spinal-cord Percentage change from baseline in serum PSA level at week 12 was summarized using descriptive statistics and presented graphically using waterfall plots. PSA and per- centage change from baseline was also presented graphically using individual case plot. Time to PSA progression was summarized using descriptive statistics. The median time of PSA progression was estimated by Kaplan–Meier method. Table 2 Overview of treatment-emergent adverse events through week 24 (all-treated population) Table 2 Overview of treatment-emergent adverse events through week 24 (all-treated population) Results TEAEs treatment-emergent adverse events aTEAE f i l i i l d i ki h h h id Apalutamide 240 mg (N = 6) N (%) Patients with ≥ 1 TEAEs 6 (100) Serious TEAEs 1 (16.7) Grade 3 or higher TEAEs 2 (33.3) Spinal-cord compression 1 (16.7) Renal disorder 1 (16.7) TEAEs leading to discontinuation of study agent 1 (16.7) Dose-limiting toxicity 0 Death 0 TEAEs ≥ 2 patients Abdominal discomfort 2 (33.3) Nasopharyngitis 2 (33.3) Dysgeusia 2 (33.3) Rash 2 (33.3) Hot flush 2 (33.3) TEAEs of special interesta Rash 2 (33.3) Rash generalized 1 (16.7) Rib fracture 1 (16.7) TEAEs treatment-emergent adverse events Clinical laboratory evaluation compression was resolving. No death and no DLTs were reported. Dose interruption was reported in 2/6 patients (33.3%) due to TEAEs [Grade 2 rash generalized and Grade 1 toxic skin eruption, 1/6 patients (16.7%) each]. Three patients (50.0%) were reported with TEAEs of special interest (rash in 2 patients and rib fracture in 1 patient). The Grade 1 rash of the special interest TEAE resolved in both the patients without any action on dose of the study agent; however, it reoccurred in one patient as Grade 2 rash generalized on day 55. The event of rash generalized resolved on day 74 and reoccurred on day 83 with similar grade. It was reported to be resolving after dose interruption at time of clinical cutoff. Grade 1 rib fracture was observed in 1/6 patient (16.7%), no action on dose of the study agent was taken and the event was not recovered at clinical cutoff date. No seizure, hypothyroid, or fall was reported (Table 2). compression was resolving. No death and no DLTs were reported. Dose interruption was reported in 2/6 patients (33.3%) due to TEAEs [Grade 2 rash generalized and Grade 1 toxic skin eruption, 1/6 patients (16.7%) each]. Three patients (50.0%) were reported with TEAEs of special interest (rash in 2 patients and rib fracture in 1 patient). The Grade 1 rash of the special interest TEAE resolved in both the patients without any action on dose of the study agent; however, it reoccurred in one patient as Grade 2 rash generalized on day 55. The event of rash generalized resolved on day 74 and reoccurred on day 83 with similar grade. It was reported to be resolving after dose interruption at time of clinical cutoff. Grade 1 rib fracture was observed in 1/6 patient (16.7%), no action on dose of the study agent was taken and the event was not recovered at clinical cutoff date. No seizure, hypothyroid, or fall was reported (Table 2). The Grade 3 hematology laboratory abnormalities were lym- phocyte count decreased and lymphocyte count increased [1/6 patient (16.7%) each]. Treatment compliance and extent of exposure Overall, median (range) of drug compliance was 98.3% (69.3–100.0%). Of total, 3/6 patients (50%) achieved 100% compliance, 2/6 patients (33.3%) achieved ≥ 80 to ≤ 100% a TEAEs of special interest includes seizure, skin rash, hypothyroid, fall, and fracture 3 International Journal of Clinical Oncology (2019) 24:1596–1604 1601 Acc accumulation, CV coefficient of variation, EHL effective half-life, MPR metabolite-to-parent ratio, PK p Acc accumulation, CV coefficient of variation, EHL effective half-life, MPR metabolite-to-parent ratio, PK pharmacokinetics, PTF peak to trough fluctuation, SD standard deviation aN=5 patients Pharmacokinetic assessment After single-dose administration, individual t1/2λ of apalutamide ranged from 110 to 231 h; however, the data should be cautiously inter- preted owing to the short sampling period (up to 168 h). After multiple-dose administration, mean EHL was 50.2 h and mean PTF% was 177% for apalutamide. A steady state of JNJ-56142060 was reached approximately by day 1 of Cycle 2 (week 5) (data not shown). The mean Cmax of JNJ-56142060 was approximately 19-fold higher after multiple-dose administration (7.11 μg/mL at Cycle 1 day 22) compared with single-dose administration (0.366 μg/ mL at PK week day 1). Mean Acc index based on AUC0–24 of JNJ-561402060 was 57.3. Mean MPR after single-dose administration and multiple-dose administration for AUC 0–24 was 0.0814 and 1.24, respectively (Table 3). multiple-dose administration. The mean Cmax of apalu- tamide was approximately twofold higher after multiple- dose administration (7.57 μg/mL at Cycle 1 day 22) com- pared with single-dose administration (3.88 μg/mL at PK week day 1). On Cycle 1, day 22, mean Acc index based on AUC0–24 of apalutamide was 3.55. After single-dose administration, individual t1/2λ of apalutamide ranged from 110 to 231 h; however, the data should be cautiously inter- preted owing to the short sampling period (up to 168 h). After multiple-dose administration, mean EHL was 50.2 h and mean PTF% was 177% for apalutamide. A steady state of JNJ-56142060 was reached approximately by day 1 of Cycle 2 (week 5) (data not shown). The mean Cmax of JNJ-56142060 was approximately 19-fold higher after multiple-dose administration (7.11 μg/mL at Cycle 1 day 22) compared with single-dose administration (0.366 μg/ mL at PK week day 1). Mean Acc index based on AUC0–24 of JNJ-561402060 was 57.3. Mean MPR after single-dose administration and multiple-dose administration for AUC 0–24 was 0.0814 and 1.24, respectively (Table 3). administered at 240 mg once-daily dose showed manage- able toxicity and overall TEAEs were consistent with the safety profile reported in global phase 1 studies of apaluta- mide [9–12]. No deaths, drug-related serious TEAEs, any Grade 4 or higher TEAEs, DLTs or seizure were reported in this study. Two patients required dose interruptions due to TEAEs. Pharmacokinetic assessment After single-dose administration of apalutamide 240 mg, mean Cmax and AUC0–24 (3.88 μg/mL and 33.6 μg h/mL, respectively) in Japanese patients with mCRPC were higher than Cmax and AUC0–24 (2.97 μg/mL and 21.9 μg h/mL, respectively; unpublished data) observed in non-Japanese patients with mCRPC (N = 3). However, similar mean Cmax (7.57 vs 7.55 μg/mL) and AUC0–24 (122 vs 127 μg h/mL) were observed in Japanese vs non-Japanese patients with mCRPC, after multiple-dose administration of apalutamide 240 mg [9]. Moreover, mean MPR for AUC0–24 in Japanese and non-Japanese patients with mCRPC were also compa- rable (1.24 vs 1.09; unpublished data).fi The efficacy results of this phase 1 study demonstrated durable decline in PSA with apalutamide 240 mg once-daily dose, suggesting that the drug was effective and showed comparable trend with the result of global phase 1/2 study (NCT01171898) [7, 12]. In this study, a ≥ 50% reduction in serum PSA level from baseline was confirmed in 4/6 patients (66.7%) at week 12. Apalutamide 240 mg once-daily dose was approved in US [13] and Japan [14] for nmCRPC fol- lowing positive results from the phase 3 SPARTAN study, where apalutamide confirmed significant improvements in metastasis-free survival (P < 0.001) and progression-free Efficacy assessment The median serum PSA level decreased from 54.42 (range 8.92–310.11) ng/mL at baseline to 11.70 (range 0.37–47.74) ng/mL at week 12. Most of the patients [4/6 patients (66.7%)] achieved at least 50% reduction from baseline in serum PSA level at week 12 and a few patients could also achieve 90% reduction [2/6 patients (33.3%)] (Fig. 2). The median maximum reduction of percentage change from baseline in serum PSA level during treatment was − 88.3% (range − 41.6 to − 99.8%) (Table 4).f Fig. 2 Waterfall plots of prostate-specific antigen percent change from baseline at week 12 (all-treated population) At clinical cutoff, 4/6 patients showed progression of dis- ease [PSA progression and radiographic progression: 2/6 patients (33.3%), clinical progression: 1/6 patients (16.7%), disease progression (PSA was reported to be > 40 and no pain progression was observed) confirmed by the investiga- tor: 1/6 patients (16.7%)]. One patient with radiographic dis- ease progression had bone metastases and the other patient had metastatic progression of prostate mass at periaortic lymph node and both sides of internal iliac artery lymph node. The patient who did not meet predefined progression of disease criteria showed increase in serum PSA level with no pain progression. Pharmacokinetic assessment PK analysis was performed in all the six patients; however, one patient was excluded from the descriptive statistical analysis due to vomiting after the first dose. Nonetheless, after multiple doses, parameters not affected by vomiting were included. A steady state of apalutamide was reached approximately by day 22 of Cycle 1 (week 4), as indicated by the mean trough concentration–time profile (data not shown). The median tmax of apalutamide was reached at 1.58 h after single-dose administration and 1.44 h after Table 3 Summary of PK parameters of apalutamide and its metabolite JNJ-56142060 in plasma (PK analysis population) Acc accumulation, CV coefficient of variation, EHL effective half-life, MPR metabolite-to-parent ratio, PK pharmacokinetics, PTF peak to trough fluctuation, SD standard deviation Apalutamide 240 mg JNJ-56142060 PK week day 1 (N = 5) Cycle 1 day 22 (N = 6) PK week day 1 (N = 5) Cycle 1 day 22 (N = 6) Cmax (μg/mL) Mean (SD) 3.88 (0.793) 7.57 (1.19) 0.366 (0.0751) 7.11 (0.551) CV% 20.4 15.7 20.5 7.80 tmax (h) Median 1.58 1.44 168 3.68 Range (1.00–2.05) (0.950–4.00) (95.5–168) (0.00–23.8) AUC0–24 (h μg/mL) Mean (SD) 33.6 (4.78) 122 (17.5) 2.69 (0.307) 150 (15.6) CV% 14.2 14.3 11.4 10.4 AUClast (h μg/mL) Mean (SD) 116 (14.1) – 45.4 (8.59) – CV% 12.1 – 18.9 – Acc index Mean (SD) – 3.55 (0.139)a – 57.3 (6.41)a CV% – 3.90 – 11.2 EHL (h) Mean (SD) – 50.2 (2.34)a – – CV% – 4.70 – – PTF% (%) Mean (SD) – 177 (23.5) – – CV% – 13.2 – – MPR for Cmax Mean (SD) – – 0.0987 (0.0314) 0.951 (0.104) CV% – – 31.8 10.9 MPR for AUC0–24 Mean (SD) – – 0.0814 (0.0159) 1.24 (0.159) CV% – – 19.6 12.8 rameters of apalutamide and its metabolite JNJ-56142060 in plasma (PK analysis population) Table 3 Summary of PK parameters of apalutamide and its metabolite JNJ-56142060 in plasma (PK analysis pop 1 International Journal of Clinical Oncology (2019) 24:1596–1604 1602 multiple-dose administration. The mean Cmax of apalu- tamide was approximately twofold higher after multiple- dose administration (7.57 μg/mL at Cycle 1 day 22) com- pared with single-dose administration (3.88 μg/mL at PK week day 1). On Cycle 1, day 22, mean Acc index based on AUC0–24 of apalutamide was 3.55. Discussion This open-label study assessed the safety, tolerability, PK, and antitumor effects of apalutamide after single and mul- tiple doses in Japanese men with mCRPC. Apalutamide Fig. 2 Waterfall plots of prostate-specific antigen percent change from baseline at week 12 (all-treated population) 1 3 International Journal of Clinical Oncology (2019) 24:1596–1604 1603 International Journal of Clinical Oncology (2019) 24:1596–1604 Table 4 Summary of prostate- specific antigen assessment (all-treated population) Apalutamide 240 mg (N = 6) CI confidence interval, NE not evaluable, PSA prostate-specific antigen PSA (ng/mL) Baseline, median (range) 54.420 (8.92–310.11) 12 weeks, median (range) 11.700 (0.37–47.74) Percent change from baseline at 12 weeks, median (range) − 75.04 ( − 97.9 to − 2.0) Maximum reduction from baseline, median (range) − 29.630 ( − 295.45 to − 3.77) Maximum reduction of percent change from baseline, median (range) − 88.26 ( − 99.8 to − 41.6) PSA progression Number of events (%) 2 (33.3) Number of censored (%) 4 (66.7) Time to event (months) Median (95% CI) 27.89 (5.82, NE) CI confidence interval, NE not evaluable, PSA prostate-specific antigen the final decision about where to publish these data, and approved submission to the journal. the final decision about where to publish these data, and approved submission to the journal. survival (P < 0.001) as compared to placebo [15]. Apaluta- mide also showed a very good risk–benefit balance in phase 3 TITAN study involving Japanese patients with metastatic castration-sensitive prostate cancer (mCSPC), where apalu- tamide confirmed significant improvements in radiographic progression-free survival (P < 0.001) and overall survival (P < 0.005) as compared to placebo [16]. Funding This work was supported by Janssen Pharmaceutical K.K., Tokyo, Japan. Compliance with ethical standards Conflict of interest Dr. Hiroji Uemura received grants from Jans- sen Pharmaceutical K.K.(during the conduct of the study) and from Bayer Yakuhin, Limited, Takeda Pharmaceutical Company Limited, AstraZeneca K.K., TAIHO Pharmaceutical Company Limited, Astel- las Pharma Inc. and Pfizer Inc (outside the research work, lecture, or subsidies). Dr. Katsuyoshi Hashine received grants from Janssen Phar- maceutical (during the conduct of the study) and from Takeda Pharma- ceutical (subsidies). Dr. Tomohiro Tsuchiya and Dr. Akito Yamaguchi received grants from Janssen Pharmaceutical K.K. during the conduct of the study. Dr. Keiichiro Imanaka, Ms. Yuki Iwaki, and Mr. Ryo Oy- ama are employees of Janssen Pharmaceutical K.K. The small sample size of the study restrains reliable dem- onstration of the efficacy. However, the small sample size is commonly observed in early development studies and is sufficient to allow clinical judgment of safety and toler- ability, and assessment of PK profile. Furthermore, as this was a single-dose study, it was difficult to determine dose proportionality. In conclusion, apalutamide 240 mg was well-tolerated without any DLT or any new safety signals with a favour- able efficacy in Japanese mCRPC patients. In addition, the pharmacokinetic profile of apalutamide 240 mg once-daily in Japanese mCRPC patients was consistent with the estab- lished profile in the non-Japanese population. Thus, apalu- tamide 240 mg (once-daily, orally) was ascertained to be an adequate dosage regimen in Japanese mCRPC patients. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Acknowledgements Rukhsar Wasta, M Pharm and Varkha Agrawal, PhD provided writing assistance and Sangita Patil, PhD, CMPP (all from SIRO Clinpharm Pvt. Ltd., India) provided additional editorial support for this manuscript. Authors also thank the study participants, without whom this study would never have been accomplished, and the investigators for their participation in this study. Mr. Hidehisa Noguchi (ex-employee of Janssen pharmaceutical KK, Japan) provided substan- tial contribution at the manuscript development stage. References 1. Bray F, Jerlay J, Soerjomataram I et al (2018) Global cancer sta- tistics 2018: GLOBOCAN estimates of incidence and mortaility worldwide for 36 caancers in 185 countries. CA Cancer J Clin 68(6):394–424 2. Foundation for promotion of cancer research (2014) Cancer statis- tics in Japan—2014. Available via DIALOG. https://ganjoho.jp/ data/reg_stat/statistics/brochure/2014/cancer_statistics_2014_ fig_E.pdf. Accessed Nov 2018 Author contributions KI, YI, and RO contributed to conception and design of the study. YI was involved in data analysis. All authors were involved in data interpretation, development, and critical revision of the manuscript for intellectual content. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors meet ICMJE criteria and all those who fulfilled those criteria are listed as authors. All authors provided direction and comments on the manuscript, made i 3. Hori M, Matsuda T, Shibata A et al (2015) Cancer incidence and incidence rates in Japan in 2009: a study of 32 population-based cancer registries for the Monitoring of Cancer Incidence in Japan (MCIJ) project. Jpn J Clin Oncol 45(9):884–891 1 3 1604 International Journal of Clinical Oncology (2019) 24:1596–1604 11. Posadas EM, Chi KN, Wit RD et al (2017) Phase Ib study of apalutamide (APA) with abiraterone acetate (AA) and prednisone (P) in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC): update on safety and efficacy. J Clin Oncol 35(6_suppl):173 4. Matsubara N, Mukai H, Hosono A et al (2017) Phase 1 study of darolutamide (ODM-201): a new-generation androgen receptor antagonist, in Japanese patients with metastatic castration-resistant prostate cancer. 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References Eur Urol 70(6):963–970 14. List of approved products in Pharmaceuticals and Medical Devices Agency (PMDA). https://www.pmda.go.jp/files/00022 9856.pdf#page=8. Accessed June 2019 15. Smith MR, Saad F, Chowdhury S et al (2018) Apalutamide treat- ment and metastasis-free survival in prostate cancer. N Engl J Med 378(15):1408–1418 8. Scher HI, Halabi S, Tannock I et al (2008) Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: recommendations of the prostate cancer clinical trials working group. J Clin Oncol 26(7):1148–1159 16. Chi KN, Agarwal N, Bjartell A et al (2019) Apalutamide for meta- static, castration-sensitive prostate cancer. N Engl J Med. https:// doi.org/10.1056/NEJMoa1903307 9. Rathkopf DE, Morris MJ, Fox JJ et al (2013) Phase I study of ARN-509, a novel antiandrogen, in the treatment of castration- resistant prostate cancer. J Clin Oncol 31(28):3525–3530 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 10. Rathkopf DE, Slovin SF, Morris MJ et al (2017) Targeting recip- rocal feedback inhibition: Apalutamide and everolimus in patients with metastatic castration-resistant prostate cancer (mCRPC). J Clin Oncol 35(6_suppl):204 1 3 3
https://openalex.org/W4361845770	https://figshare.com/ndownloader/files/39867581	English	null	Supplementary Figures from Multiparametric MRI and Coregistered Histology Identify Tumor Habitats in Breast Cancer Mouse Models	null	2,023	cc-by	1,843	SUPPLEMENTARY FIGURES SUPPLEMENTARY FIGURES Supplementary Figure S1. Pimonidazole and carbonic anhydrase IX (CA-IX) staining patterns in 4T1 and MDA-MB-231 breast tumors. (A and G) Histological samples stained with CA-IX and respective masked images, showing the CA-IX-positive pixels in yellow; (B and H) Histological samples stained with pimonidazole and respective masked images, showing the pimonidazole-positive pixels in red; (C and I) Masked image combining CAI-IX (yellow) and pimonidazole (red). Cyan represents the intersection of markers, that is, pixels positive for both CA-IX and pimonidazole. (D and J) Quantification of tissue area (%) for each marker. p-values were obtained using one-way analysis of variance (ANOVA) followed by the Tukey test for comparison of mean values between regions (p<0.05; p<0.001; *p<0.0001) (n=6 histological samples for 4T1 and n=8 histological samples for MDA-MB-231). Graphs represent mean and SD. (E and K) Quantification of CA-IX-positive and CA-IX-negative areas in the pimonidazole-positive area. (F and L) Quantification of pimonidazole-positive and pimonidazole-negative areas in the CA-IX-positive area Supplementary Figure S1. Pimonidazole and carbonic anhydrase IX (CA-IX) staining patterns in 4T1 and MDA-MB-231 breast tumors. (A and G) Histological samples stained with CA-IX and respective masked images, showing the CA-IX-positive pixels in yellow; (B and H) Histological samples stained with pimonidazole and respective masked images, showing the pimonidazole-positive pixels in red; (C and I) Masked image combining CAI-IX (yellow) and pimonidazole (red). Cyan represents the intersection of markers, that is, pixels positive for both CA-IX and pimonidazole. (D and J) Quantification of tissue area (%) for each marker. p-values were obtained using one-way analysis of variance (ANOVA) followed by the Tukey test for comparison of mean values between regions (p<0.05; *p<0.001; *p<0.0001) (n=6 histological samples for 4T1 and n=8 histological samples for MDA-MB-231). Graphs represent mean and SD. (E and K) Quantification of CA-IX-positive and CA-IX-negative areas in the pimonidazole-positive area. (F and L) Quantification of pimonidazole-positive and pimonidazole-negative areas in the CA-IX-positive area. Supplementary Figure S2. Breast tumor stained with hematoxylin & eosin (H&E), showing different stages of necrosis in the black squares, which were masked as non-viable regions. Red square represents an area of viable tumor cells, surrounding a blood vessel. Supplementary Figure S2. Breast tumor stained with hematoxylin & eosin (H&E), showing different stages of necrosis in the black squares, which were masked as non-viable regions. Red square represents an area of viable tumor cells, surrounding a blood vessel. Supplementary Figure S2. Breast tumor stained with hematoxylin & eosin (H&E), showing different stages of necrosis in the black squares, which were masked as non-viable regions. Red square represents an area of viable tumor cells, surrounding a blood vessel. Supplementary Figure S3. Representative examples of the corresponding habitat maps from MRI and histology. 4T1 tumor is shown in (A) and MDA-MB-231 is shown in (G). (A and G) Six MRI parameter maps were obtained [T2-map, T2-map, Apparent diffusion coefficient (ADC), Slope, Area under the curve (AUC) and time to max (TTM)]. Field of view 30 X 30 mm2; image size 256 X 256; Tumor volume of interest (VOI) is shown in red. (B and H) These six MRI parameter maps were clustered by using a Gaussian Mixture Model (GMM) to create the Habitat Maps.. Green cluster shows high enhancement in DCE and the blue cluster shows low enhancement in DCE. Magenta and yellow Clusters show moderate enhancement in DCE. Histological images ((C and I) H&E; (D and J) pimonidazole; (E and K) CD31) were used to create a downsampled ground truth habitat-map from histology (F and L) in the same resolution of MR images. Ground truth habitat maps are delineated by viable cells region in green, non-viable cells regions in blue and hypoxic areas in viable or non-viable regions in magenta and yellow, respectively. Supplementary Figure S3. Representative examples of the corresponding habitat maps from MRI and histology. 4T1 tumor is shown in (A) and MDA-MB-231 is shown in (G). (A and G) Six MRI parameter maps were obtained [T2-map, T2-map, Apparent diffusion coefficient (ADC), Slope, Area under the curve (AUC) and time to max (TTM)]. Field of view 30 X 30 mm2; image size 256 X 256; Tumor volume of interest (VOI) is shown in red. (B and H) These six MRI parameter maps were clustered by using a Gaussian Mixture Model (GMM) to create the Habitat Maps.. Green cluster shows high enhancement in DCE and the blue cluster shows low enhancement in DCE. Magenta and yellow Clusters show moderate enhancement in DCE. Supplementary Figure S2. Breast tumor stained with hematoxylin & eosin (H&E), showing different stages of necrosis in the black squares, which were masked as non-viable regions. Red square represents an area of viable tumor cells, surrounding a blood vessel. Histological images ((C and I) H&E; (D and J) pimonidazole; (E and K) CD31) were used to create a downsampled ground truth habitat-map from histology (F and L) in the same resolution of MR images. Ground truth habitat maps are delineated by viable cells region in green, non-viable cells regions in blue and hypoxic areas in viable or non-viable regions in magenta and yellow, respectively. Supplementary Figure S4. Histograms showing the pixels values distribution of Area under the curve (AUC), slope and time to max (TTM) from DCE-MRI in each cluster of representative MRI habitat maps for 4T1 and MDA-MB-231 tumor samples (field of view 30 X 30 mm2; image size 256 X 256). (A and E) Clusters green classified as viable tumor cell regions; (B and F) Clusters magenta, classified as hypoxic viable; (C and G) Clusters yellow, classified as hypoxic non-viable and (D and H) Clusters blue, classified as non-viable tumor cell regions. Supplementary Figure S4. Histograms showing the pixels values distribution of Area under the curve (AUC), slope and time to max (TTM) from DCE-MRI in each cluster of representative MRI habitat maps for 4T1 and MDA-MB-231 tumor samples (field of view 30 X 30 mm2; image size 256 X 256). (A and E) Clusters green classified as viable tumor cell regions; (B and F) Clusters magenta, classified as hypoxic viable; (C and G) Clusters yellow, classified as hypoxic non-viable and (D and H) Clusters blue, classified as non-viable tumor cell regions. Supplementary Figure S5. Representative examples showing that the DCE-MRI maps have visual similarity with pimonidazole staining in breast samples. (A) DCE-MRI derived maps [Slope, Area under the curve (AUC) and time to max (TTM)] (field of view 30 X 30 mm2; image size 256 X 256). (B) Histological slices stained with pimonidazole and masked images showing the pimonidazole-positive pixels in (C). Supplementary Figure S5. Representative examples showing that the DCE-MRI maps have visual similarity with pimonidazole staining in breast samples. (A) DCE-MRI derived maps [Slope, Area under the curve (AUC) and time to max (TTM)] (field of view 30 X 30 mm2; image size 256 X 256). (B) Histological slices stained with pimonidazole and masked images showing the pimonidazole-positive pixels in (C). Supplementary Figure S6. Supplementary Figure S2. Breast tumor stained with hematoxylin & eosin (H&E), showing different stages of necrosis in the black squares, which were masked as non-viable regions. Red square represents an area of viable tumor cells, surrounding a blood vessel. Representative examples of MRI habitat maps, which were created by clustering five MRI-parameters [T2-map, T2-map, Slope, Area under the curve (AUC) and time to max (TTM)] using a Gaussian Mixture Model (GMM) Expectation-Maximization algorithm. Field of view 30 X 30 mm2; image size 256 X 256. Note that these maps are from the same tumors/slices shown in figure 4 and supplementary fig. S3 are visually similar the maps created by clustering six parameters, which also included the ADC maps. Supplementary Figure S6. Representative examples of MRI habitat maps, which were created by clustering five MRI-parameters [T2-map, T2-map, Slope, Area under the curve (AUC) and time to max (TTM)] using a Gaussian Mixture Model (GMM) Expectation-Maximization algorithm. Field of view 30 X 30 mm2; image size 256 X 256. Note that these maps are from the same tumors/slices shown in figure 4 and supplementary fig. S3 are visually similar the maps created by clustering six parameters, which also included the ADC maps. Supplementary Figure S7. Mean values of each parameter from habitat maps generated clustering five MRI-parameters [T2-map, T2-map, Slope, Area under the curve (AUC) and t to max (TTM)]. Mean values of each parameter were compared between clusters (habitats) the (A) 4T1 (n=12 samples) and (B) MDA-MB-231 (n=4 samples) tumor samples. Gra represent mean and SD. p-values were obtained using one-way analysis of variance (ANOV followed by the Tukey test for comparison of mean values between regions (a-c indicate p<0 between groups). Supplementary Figure S7. Mean values of each parameter from habitat maps generated by clustering five MRI-parameters [T2-map, T2-map, Slope, Area under the curve (AUC) and time to max (TTM)]. Mean values of each parameter were compared between clusters (habitats) for the (A) 4T1 (n=12 samples) and (B) MDA-MB-231 (n=4 samples) tumor samples. Graphs represent mean and SD. p-values were obtained using one-way analysis of variance (ANOVA) followed by the Tukey test for comparison of mean values between regions (a-c indicate p<0.05 between groups). Supplementary Figure S7. Mean values of each parameter from habitat maps generated by clustering five MRI-parameters [T2-map, T2-map, Slope, Area under the curve (AUC) and time to max (TTM)]. Mean values of each parameter were compared between clusters (habitats) for the (A) 4T1 (n=12 samples) and (B) MDA-MB-231 (n=4 samples) tumor samples. Graphs represent mean and SD. Supplementary Figure S2. Breast tumor stained with hematoxylin & eosin (H&E), showing different stages of necrosis in the black squares, which were masked as non-viable regions. Red square represents an area of viable tumor cells, surrounding a blood vessel. p-values were obtained using one-way analysis of variance (ANOVA) followed by the Tukey test for comparison of mean values between regions (a-c indicate p<0.05 between groups). tary Figure S8. Histograms showing the distribution of the Apparent ADC) values in the cluster classified as non-viable tumor cells (blue) in Field of view 30 X 30 mm2; image size 256 X 256. (A) Representative ex MDA-MB-231 tumor samples. Supplementary Figure S8. Histograms showing the distribution of the Apparent diffusion coefficient (ADC) values in the cluster classified as non-viable tumor cells (blue) in the MRI habitat map. Field of view 30 X 30 mm2; image size 256 X 256. (A) Representative examples of 4T1 and (B) MDA-MB-231 tumor samples. Supplementary Figure S8. Histograms showing the distribution of the Apparent diffusion coefficient (ADC) values in the cluster classified as non-viable tumor cells (blue) in the MRI habitat map. Field of view 30 X 30 mm2; image size 256 X 256. (A) Representative examples of 4T1 and (B) MDA-MB-231 tumor samples. Supplementary figure S9. Representative images of the immunohistochemistry for perilipin-1 in breast tumor samples. (A) Positive stained adipocytes in 4T1 and (B) MDA-MB-231 breast tumor samples. Breast tumor cells were negative for perilipin-1. 20X magnification. Supplementary figure S9. Representative images of the immunohistochemistry for perilipin-1 in breast tumor samples. (A) Positive stained adipocytes in 4T1 and (B) MDA-MB-231 breast tumor samples. Breast tumor cells were negative for perilipin-1. 20X magnification.
https://openalex.org/W4389488385	https://bmcnurs.biomedcentral.com/counter/pdf/10.1186/s12912-023-01636-8	English	null	A study on the types of disaster awareness in nursing students: Q methodology	BMC nursing	2,023	cc-by	8,878	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background Research for the development of nursing education strategies to enhance the competency of the nursing students on disaster safety are needed. This study aimed to identify the types of perceptions on disaster safety in nursing students, and to analyze and describe the characteristics of each type of disaster safety perception of nursing students in South Korea. Methods An exploratory study design applying Q methodology, a research method designed to study subjectivity. Participants were 30 nursing students in their 20s who are living in C city. This P-set was selected to best reveal the disaster safety awareness of nursing students. Participants provided their subjective viewpoints by sorting 30 statements into a grid. Analyses involved correlation and factor analysis. The study was carried out from June to December, 2020. Results In this study, four types of disaster safety awareness of nursing students were uncovered. The characteristics of each type were confirmed as follows: Type 1 was national responsibility, type 2 was individual responsibility, type 3 was preparedness-oriented, and type 4 was education-oriented. Conclusion This study shows that the types of perceptions on disaster safety in Korean nursing students were national responsibility, individual responsibility, preparedness-oriented, and education-oriented. The findings from this study can be implied as fundamental data in nursing education of disaster safety. Keywords Nursing student, Disaster, Safety, Q methodology Background Disaster is an event that occurs when demand exceeds immediately available resources [1, 2]. It can also be defined as an event that overwhelms local capacity and requires a request from external resources at the national or international level [3]. Disasters occur frequently all over the world, and large-scale disaster events are con stantly occurring in South Korea. Furthermore, the num ber of occurrences is increasing every year [4]. Correspondence: Sohyune Sok 5977sok@khu.ac.kr 1DKMediinfo Nursing Information Research Institute, Changwon-si, Gyeongsangnam-do, Republic of Korea 2Department of Nursing, Graduate School, Kyung Hee University, Seoul, Republic of Korea 3College of Nursing Science, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea Nurses provide nursing care relevant to each disaster stage by helping those who cannot solve their own health problems in an environment impacted by the disaster [5]. Before a disaster occurs, it is necessary to identify and prepare for individual-level and community-level © The Author(s) 2023. 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. BMC Nursing BMC Nursing Seong et al. BMC Nursing (2023) 22:469 https://doi.org/10.1186/s12912-023-01636-8 Open Access Mihyeon Seong1, Dajung Ryu2 and Sohyune Sok3 Mihyeon Seong1, Dajung Ryu2 and Sohyune Sok3* Mihyeon Seong1, Dajung Ryu2 and Sohyune Sok3* Seong et al. BMC Nursing (2023) 22:469 Page 2 of 10 Page 2 of 10 Page 2 of 10 Seong et al. BMC Nursing necessary for nursing students to be aware of the para digm that reflects social and medical changes, to prepare for the future society, and to establish a value system for humanities and nursing professions in the undergraduate course of the university in order to develop the ability to cope with various problem situations to be faced in the nursing field [20]. It is important to first explore the per ception of disaster safety recognized by nurses who will work in the nursing field in the future.h problems, and establish a plan to save as many lives as possible. Nurses are important health care workers in disasters, as they are responsible for identifying addi tional needs of people and play a pivotal role in all stages of a disaster [6]. Due to the severity of disasters that are increasing around the world, the role of nurses to patients in disas ters is being emphasized, and the demand for improving the ability of nurses to respond to disasters is increasing [7, 8]. Nursing capacity, which requires nurses to have knowledge and skills that are unique to disasters, is also required for nurses providing care during a disaster when they may have scarce resources and work in suboptimal environments during disaster situations [9–11]. Unlike clinical trials with well-equipped resources and well- trained nurses, most disaster sites are in suboptimal envi ronments that require a response with limited resources and often in the absence of advanced systematic disaster safety preparedness education. i Therefore, this study seeks to understand the disaster safety as it is perceived by nursing students. The study uses Q methodology to reveal subjective perspectives about disaster safety from the perspective of nursing students.h The purpose of this study was to uncover the types of subjectivities held by nursing students relative to disas ters and to describe each type of perspective. The results will provide the fundamental data necessary for research and development of nursing education strategies that would be effective in cultivating nurses who could pre pare for various disaster situations by confirming the diverse perspectives of the nursing students and enhanc ing their understanding of disaster. Accordingly, the aims of this study were to identify the types of awareness of disaster in nursing students, and to analyze and describe the characteristics of each type of disaster awareness of nursing students. Labrague et al. [7] reported that disaster response capa bilities of nurses were closely related to previous disaster nursing experience or disaster-related education. How ever in particular, nursing students, who will become nurses in the future, lack awareness of disasters and disaster nursing, and they do not fully understand the required role of nurses in disasters [12]. For this reason, it is essential for nurses to acquire knowledge and skills in order to respond promptly to disaster situations. The need to provide the basics for disaster nursing to nurs ing students has also been raised [12, 13]. Disaster educa tion should be included as an essential part of the nursing curriculum because all disasters start locally, and nurses must be aware of the various disaster situations that can occur, as well as have adequate and effective response capabilities [14]. Study designh This study is an exploratory study that applied Q meth odology, a subjective research method, to explore disaster safety awareness of nursing students more systematically and scientifically. Q methodology is a methodology that uncovers and describes the multiple viewpoints about a topic or event. That is, it is a way to find correlations between people across subjective attributes. Q method ology assumes intra-individual differences in meaning, so that differences in views among participants. Thus, Q focuses on the relationship between a variable and a stimulus. In addition, factor analysis performed in paral lel for this purpose is generally a method of classifying variables. In the R methodology, the variable becomes a test item or characteristic, but in the Q methodology, the respondent himself models it by comparing and ordering stimuli (usually statements), and eventually expresses his or her own opinion [21]. Nevertheless, disaster education is covered only in some nursing schools, and research on disasters knowl edge among nursing students is insufficient [14–16]. Nurses or nursing students in Korea have a vague con ception of disaster or disaster nursing and may fall into a panic when an actual disaster occurs [15]. At a time when various data are needed to organize a systematic disaster preparedness curriculum, it is neces sary to develop disaster awareness through systematic and appropriate training in undergraduate courses in order to respond with sufficient capacity to sudden disas ter situations [9, 17]. According to a study by Woo, et al. [18], it is argued that disaster awareness should be started from undergraduate courses because disaster awareness is correlated with disaster preparedness. Also, according to Kwon, et al. [19], it was suggested that nursing stu dents should increase their awareness of disasters and cultivate their ability to prepare and cope with disasters through disaster education. This disaster education is Composition of concourse and Q-sample In this study, a literature review on disaster and in-depth interviews with the nursing students were conducted in order to form the concourse. In Q, the concourse repre sents a universe of subjective items related to the topic. A Page 3 of 10 Seong et al. BMC Nursing (2023) 22:469 Seong et al. BMC Nursing interview, were recruited to form the P-set. The purpose and procedure of the study were explained to the subjects who participated voluntarily, and informed consent was obtained. In addition, it was explained that the subject can withdraw his/her intention to participate in the study at any time if he/she does not want it, and there will be no disadvantages. semi-structured questionnaire was administered in order to establish various subjective statements through in- depth interviews; subjects that could have different opin ions were selected [21] and proceeded until the data was saturated. To obtain a concourse about disaster safety of nursing students, the subjects for an in-depth interview were randomly selected from a total of 30 first, second, and third-year nursing college students in their twenties residing in city C, and then individual in-depth inter views were conducted.h Q-sort Q sample classification is a process in which a subject classifies statements derived from the normal distribu tion of the Q distribution table and assigns a score to each item. A normal distribution reflects participants’ obser vations of a very strong positive or negative, for which a limited number of items can be ranked at the poles of the distribution. At the same time, a relatively large number of suborders are ranked toward the center of the distribu tion based on their relative indifference [21–23].h The in-depth interview on disaster awareness of nurs ing students was conducted for about two weeks from August 1, 2020 to August 15, 2020, and 86 of the subjec tive items (concourses) were extracted through the in- depth interview process. However, a Q sample is typically between 40 and 60 items [21]. Duplicate questions from the concourse were deleted, as each topic had a common meaning and value. In addi tion to the researcher, three nursing professors partici pated to readjust the sentences. Thirty items made up the Q-sample and were selected through the process of exchanging opinions several times and revising whether or not they were composed of statements that clearly reveal the subjectivity of the study participants (Table 1). f The time required for Q-sorting, investigation on gen eral characteristics, and interview was about 1 h. Data collection lasted about one month in September 2020. For the Q-sorting, Q-sample items were written in one statement on a paper card (Q-card) and numbered from 1 to 30 (the last number). Each statement card prepared in this way was read by the study participant and clas sified into three categories: agreement (+), neutral (0), and disagreement (-). After completing the classifica tion into three groups, the statements of the Q-statement P-set selection Based on this, a convenience sample of a total of 30 peo ple, including those who had already participated in the Table 1 Q statement 1. Disaster management is difficult due to the shortage of young adults due to aging 16. The individual’s luck in the event of a disaster is important. 2. Disaster management is difficult if the emergency contact and cooperation system among residents is insufficient. 17. It is possible to keep personal property in case of a disaster. 3. Disaster management is difficult due to lack of local government’s equip ment, manpower, and budget support. 18. When the disaster holds resources, it is stockpiling in the home. 4. Disaster management is difficult due to the lack of evacuation sites and facilities. 19. I know evacuation tips or situations when many disasters occur. 5. Disaster management is difficult if there are insufficient fire stations, health centers, and hospitals. 20. National and municipal responsibilities are important for safety and disaster management. 6. Disaster safety education should be essential in the school curriculum. 21. Talking about a disaster in the media is helpful for disaster safety. 7. Disasters happen. 22. Disaster training, training is necessary. 8. Disaster can kill me too. 23. I have a habit of checking emergency exits and emergency routes when in any place. 9. Disasters occur frequently and are growing in scale. 24. I have an emergency contact network in case of a disaster. 10. Disaster acts as an impediment to the country’s economic development. 25. I can induce disaster evacuation to those around me in the event of a disaster. 11. The State strives to prevent disasters and protect the people from the dangers. 26. The responsibility of each citizen is important for safety and disaster management. 12. It is enough to educate and prepare in advance through disaster education. 27, In most safety accidents and disasters, it is ignorance to nurture further. 13. Disaster policy and legislation aid in disaster safety guidelines. 28. You can pay more taxes for disaster and safety management. 14. Insensitivity to safety can lead to more disasters. 29. Government disasters and safety accidents are the result of negligence and oversight by agencies. 15. Most safety accidents and disasters are the result of irresponsibility and greed. 30. Korea has an efficient administrative system and legal basis for disaster management. Ethical considerationsh The study was approved by the Institutional Research Board of K University (KHSIRB-19-012). The purpose and procedure of the study were explained to the subjects who voluntarily participated, and consent was obtained for the study. In addition, it was explained to the subjects that they could withdraw their intention to participate in the study at any time if they did not want to take part in the study, and that there would be no disadvantages. P-set selection 27, In most safety accidents and disasters, it is ignorance to nurture further. 28. You can pay more taxes for disaster and safety management. 29. Government disasters and safety accidents are the result of negligence and oversight by agencies. 30. Korea has an efficient administrative system and legal basis for disaster management. Page 4 of 10 Seong et al. BMC Nursing (2023) 22:469 Seong et al. BMC Nursing component analysis and Varimax rotation. Then, iden tify the eigen value and cumulative variance of each fac tor and select them as appropriate factors. We confirmed that they were distinguished and identified detailed characteristics of each type. After identifying the char acteristics of each type, the type name is named and interpreted. When interpreting the type, questions with an absolute standard score (Z-score) of 1.0 or higher are used to interpret the meaning of agreement and disagree ment and determine the name of the type [24]. card group classified as agreement were re-read and re- classified in order from the strongest agreement (+ 4) to neutral (0) among the Q-statement cards. Similarly, the Q-statement card group classified as disagreement was classified in order from the strongest disagreement (-4) to neutral (0), and arranged according to the card arrangement distribution table shown in Fig. 1. After the classification was completed, we tried to obtain use ful information for interpreting the Q-factors and gain ing a broader understanding of the phenomenon through questions related to the reason or feeling of classification, and additionally disaster regarding the statements placed on both ends. The time required for the Q-sorting, ques tionnaire preparation, and interview was about 30 min to 1 h, on average, about 40 min. The study was carried out from June to December, 2020. Data analysis Th d This study was analyzed by using the principal compo nent extraction with varimax rotation analysis within the PQ Method program (GNU general public license). The collected data were entered by assigning a point to the 30 Q-statements starting from − 4 for the strongest disagreement item to 0 for the neutral item, and 4 for the strongest agreement item depending on the level of agreement or disagreement for each participant’s Q-sort. The analysis in the PQ method is based on correlation and factor analysis. The determination of the number of factors is based on operancty. In practice, there are many cases where there is a difference between the statisti cal standard based on Eigen value 1 and the theoretical number of factors. It is more desirable to consider con ceptual meaning and theoretical aspects rather than rely ing only on statistical criteria [23]. In this study, Q factor analysis was conducted for typing by applying principal Result analysis BMC Nursing Table 2 General characteristics and factor weights of P samples by type Type ID Factor weights Gender Age Disaster education Religion Disaster experience and type Desired type of disaster education Type1 P-6 0.52 F 26 Yes None Typhoon ③ (n = 13) P-7 0.60 F 21 Yes None None ②, ③, ⑤ P-11 0.68 F 21 Yes None Typhoon ④ P-12 0.84 F 21 Yes None Typhoon, Flood ②, ③ P-13 0.88* F 21 Yes Buddhism Typhoon, Flood, Earthquake ②, ③ P-14 0.67 F 21 Yes Presbyterian Typhoon, Earthquake ②, ③, ④, ⑤ P-15 0.50 F 21 Yes Catholic Typhoon, Flood and Earthquake ③, ④ P-16 0.65 F 21 Yes None Typhoon, Earthquake ④, ⑤ P-17 0.60 F 22 Yes None Typhoon, Earthquake ③, ④ P-18 0.55 F 21 Yes None None ②, ③, ④ P-21 0.60 F 21 Yes None Typhoon, Earthquake ②, ④ P-22 0.71 F 21 Yes Presbyterian Typhoon ②, ③ P-26 0.64 F 21 Yes Presbyterian Typhoon ②, ③ Type2 P-2 0.72 F 23 Yes None Typhoon ⑤ (n = 6) P-10 0.47 F 21 Yes None Typhoon ③, ④ P-20 0.74* M 24 None Presbyterian None ③, ④ P-24 0.70 F 23 Yes None None ③, ④ P-28 0.62 F 21 Yes Catholic Typhoon ③, ④ P-29 0.60 F 21 Yes Buddhism Typhoon ⑤ Type3 P-3 0.73* F 23 Yes None Typhoon ⑤ (n = 2) P-9 0.72 F 21 Yes None None ② Type4 P-1 0.70 F 23 Yes None Earthquake ①, ③, ⑤ (n = 6) P-4 0.57 F 23 Yes None None ①, ③ P-5 0.63* F 23 Yes None None ①, ③, ④, ⑤ P-8 0.53 F 21 Yes None None ④, ⑤ P-23 0.59 F 21 Yes None Earthquake ②, ③, ④ P-30 0.62 F 22 Yes None Typhoon ③, ④ * Typical of types ① University lecture ② Public advertisement ③ Regular education ④ Field training ⑤ Private training ① University lecture ② Public advertisement ③ Regular education ④ Field training ⑤ Private training Table 4 Correlations among the types Type1 Type2 Type3 Type4 Type1 1.00 Type2 0.48 1.00 Type3 0.13 0.04 1.00 Type4 0.48 0.50 0.19 1.00 Table 3 Eigen value and variance by type Type1 Type2 Type3 Type4 Eigen values 10.80 2.76 2.44 2.19 Variance (%) 24 16 8 13 Cumulative (%) 24 40 48 61 earthquakes, and floods. Result analysis Among the 30 subjects in this study, during the classifi cation process, 3 P samples (P-19, P-25, and P-27) that could not be explained by type characteristics as two or more factors were classified into complex types were excluded from the factor analysis. In addition, 26 out of 27 subjects were female and had experience in disaster education, whereas 1 male subject had no experience in disaster education. The age of the subjects ranged from 21 to 26 years old. In regard to religion, 2 subjects are Buddhists, 2 subjects are Catholics, 4 subjects are Protestants, and 19 subjects are non-religious. Those with experience of disasters had experienced typhoons, Fig. 1 Card arrangement distribution table Fig. 1 Card arrangement distribution table Fig. 1 Card arrangement distribution table Page 5 of 10 Seong et al. BMC Nursing (2023) 22:469 Seong et al. Type 1: National responsibility typei yp p y yp Subjects of type 1 were identified with 13 out of 27 par ticipants, and all were female. The age ranged from 21 to 26, and all had experience in disaster education. Religion was Buddhist 1, Catholic 1, Protestant 3, and non-reli gious 8 (Table 2). It was characteristic that the subjects of Type 1 recognized that the government’s role and respon sibility played an important role in disaster safety aware ness (Table 5). In particular, they thought that in order to protect the rights of the people, the country should expand facilities, manpower, and resources. However, individual disaster capabilities were somewhat insuffi cient. The most important statements as of type 1 are as follows (Table 2): “Isn’t it even in the constitution? There is a country to protect me. However, looking at what the country is doing with coronavirus these days, I think, as a nursing student, there seems to be a shortage of medical personnel. In particular, there are not enough nurses, but I am upset because the country seems to talk only about the simple idea of lack of manpower (P = 13).” In addition, other participants in Type 1 also made statements such as wanting to receive a fair price from the state as a tax- paying citizen. Based on this, type 1 with these charac teristics was named ‘national responsibility type’ in this study. Analysis of the types Type 1: National responsibility typei from 21 to 24 years, and 5 subjects had experience in disaster education, but P-20, which is typical of Type 2, had no experience in disaster education. Religion was 1 Buddhist, 1 Catholic, 1 Protestant, and 3 non-religious (Table 2). As a result of factor analysis, the subjects of type 2 recognized that personal responsibility and role were important in disaster safety. They said that regular education, personal conscience, morality, etc. are neces sary to develop individual disaster safety capabilities, and that personal ability and regular training are important when surviving in a disaster (Table 5). However, even though most of them received disaster education, they recognized that the individual’s capabilities were still insufficient, which could lead to death. Looking at this in detail, the statements of P-20 with the representativeness, the most important statement as of type 2 are as follows (Table 2): “When I see it all the time, it seems that the cause of the damage is due to the greed of a bad company or individual to save a penny. So I think regular training is necessary.” Another participant of the same type stated that the disaster situation was worsening due to the prev alence of individualism, and that we should help each other and make concessions. Based on this, type 2 with these characteristics was named ‘individual responsibility type’ in this study. from 21 to 24 years, and 5 subjects had experience in disaster education, but P-20, which is typical of Type 2, had no experience in disaster education. Religion was 1 Buddhist, 1 Catholic, 1 Protestant, and 3 non-religious (Table 2). As a result of factor analysis, the subjects of type 2 recognized that personal responsibility and role were important in disaster safety. They said that regular education, personal conscience, morality, etc. are neces sary to develop individual disaster safety capabilities, and that personal ability and regular training are important when surviving in a disaster (Table 5). However, even though most of them received disaster education, they recognized that the individual’s capabilities were still insufficient, which could lead to death. Result analysis The subjects also said that disas ter education was necessary in university lectures, public service advertisements, formal education, field educa tion, and private education (Table 2). correlation between types showing similarity by type, the correlation coefficient between type 2 and type 4 was the highest at r = 0.50, and the correlation coefficient between type 2 and type 3 was lower at r = 0.04 (Table 4). The rea son Type 2 and Type 4 are similar is inferred because Korea’s disaster education is an elective subject, and the reason the correlation between Type 2 and Type 3 is low is that Type 3 refers to the preparation of infrastructure. In this study, four types of disaster awareness of nurs ing students according to the characteristics of each type are as follows: Type 1 was ‘national responsibility type’ in 13 students, type 2 was ‘individual responsibility type’ in 6 students, type 3 was ‘preparedness-oriented type’ in 2 students, and type 4 was ‘education-oriented type’ in 6 students. The Eigenvalue of this type is shown in Tables 3, and the explanatory power was 61%. Moreover, in the Page 6 of 10 Page 6 of 10 Seong et al. BMC Nursing (2023) 22:469 Seong et al. BMC Nursing Blanks and deleted statements were not significant Analysis of the types Type 1: National responsibility typei Looking at this in detail, the statements of P-20 with the representativeness, the most important statement as of type 2 are as follows (Table 2): “When I see it all the time, it seems that the cause of the damage is due to the greed of a bad company or individual to save a penny. So I think regular training is necessary.” Another participant of the same type stated that the disaster situation was worsening due to the prev alence of individualism, and that we should help each other and make concessions. Based on this, type 2 with these characteristics was named ‘individual responsibility type’ in this study. Discussion particular, they said that it was necessary to stockpile supplies or resources in preparation for a disaster, and recognized that both individuals and the country need to reserve facilities, resources, and manpower for this pur pose. They believed that even in the event of a disaster, they could survive with sufficient preparation (Table 5). Looking at this in detail, the statements of P-3 with the representativeness, the most important statement as of type 3 are as follows (Table 2): “I am still young, so I can’t die blindly. I check every time this happens, but I also have a survival bag at home and boxes of masks.” Other par ticipants of the same type Also, disasters can strike unex pectedly and countries may not be prepared for them. Therefore, it was stated that it is necessary for individuals to prepare and check in advance, and to avoid confusion in preparation for various disaster situations lurking in Korean society. Based on this, type 3 with these charac teristics was named ‘preparedness-oriented type’ in this study. particular, they said that it was necessary to stockpile supplies or resources in preparation for a disaster, and recognized that both individuals and the country need to reserve facilities, resources, and manpower for this pur pose. They believed that even in the event of a disaster, they could survive with sufficient preparation (Table 5). Looking at this in detail, the statements of P-3 with the representativeness, the most important statement as of type 3 are as follows (Table 2): “I am still young, so I can’t die blindly. I check every time this happens, but I also have a survival bag at home and boxes of masks.” Other par ticipants of the same type Also, disasters can strike unex pectedly and countries may not be prepared for them. Therefore, it was stated that it is necessary for individuals to prepare and check in advance, and to avoid confusion in preparation for various disaster situations lurking in Korean society. Based on this, type 3 with these charac teristics was named ‘preparedness-oriented type’ in this study. In the undergraduate course of nursing college, educa tion is needed to develop the ability to cope with various situations encountered in the field by establishing a value system for the nursing profession with a sense of human ity and nursing ethics [25]. The study by Goniewicz et al. Type 4: education-oriented type yp yp Type 4 subjects were 6 out of 27, all of whom were female, aged 21 to 23 years, and all were non-educated (Table 2). The type 4 subjects were aware of the impor tance of education in disaster. They recognized that they needed to receive disaster education and train regularly, and that education and training should be systematically formed and provided at the national or local government level. They said that if proper education and training were given, it would increase the survival rate during emergen cies, and people should always be aware that life could be lost. However, they also said that disaster education in the nursing department curriculum was insufficient. The type 4 subjects said that disaster training should be given through various curriculums, such as university, elemen tary, middle, and high schools, and private level (Table 5). Looking at this in detail, the statements of P-5 with the representativeness, the most important statement as of type 2 are as follows (Table 2): “I think that not only nurs ing education, but also disaster education, should be con ducted regularly starting from childhood and become a regular course. The education I am receiving poorly at the moment is insufficient and forgotten over time.” Another participant of the same type also stated that since disaster education is provided only to certain occupations, aware ness improvement education or experiential education for all citizens should be provided, and a system should be prepared so that they can receive it consistently. Based on this, type 4 with these characteristics was named ‘edu cation-oriented type’ in this study. As a result of this study, all four types indicate that indi viduals lack disaster capacity, and recognize the need for disaster education. This means that nursing students are acutely feeling the need for disaster education as in the current situation of the COVID-19 pandemic. Nursing students are not ready to respond to a disaster situation, but showing their willingness to participate in disaster response will greatly contribute to the development of nursing science in the future. Looking at the results of this study, Type 1 place impor tance on the responsibilities and roles of the country. Discussion [26] also emphasized that disaster education and training is a key component, and that exercises, as well as training and graduate studies focused on disaster response, are an important aspect of education and training activities. International organizations such as WHO and ICN criti cize the undergraduate nursing education curriculum, which is insufficient to prepare nursing graduates to par ticipate in disaster relief despite the increasing number of disasters worldwide [27, 28]. Kim and Lidia [27] stated that nurses respond to disasters at the forefront and that undergraduate education is necessary to equip them with basic competencies and knowledge to respond quickly in case of disasters. It is important that education prop erly prepares nurses to act in difficult situations due to unexpected circumstances [26]. Through this study, it is expected that the concept of disaster perceived by nurs ing students will be explored and will serve as basic data to develop the disaster nursing competency of nursing students. Type 2: individual responsibility type BMC Nursing (2023) 22:469 Seong et al. BMC Nursing (2023) 22:469 Type 2: individual responsibility type y y Type 3 subjects were 2 out of 27, female, 21 and 23 years old, and all were non-religious (Table 2). The type 3 sub jects recognized that disaster requires preparedness. In Type 2 subjects were represented by 6 out of 27 partici pants, including 1 female and 1 male. The age ranged Table 5 Z score of type Table 5 Z score of type Type1 Type2 Type3 Type4 1. Disaster management is difficult due to the shortage of young adults due to aging -1.20 -1.37 3. Disaster management is difficult due to lack of local government’s equipment, manpower, and budget support. -1.09 4. Disaster management is difficult due to the lack of evacuation sites and facilities. 1.12 1.86 5. Disaster management is difficult if there are insufficient fire stations, health centers, and hospitals. 1.85 7. Disasters happen. 1.46 2.23 -1.24 8. Disaster can kill me too. 1.87 1.40 -1.86 1.16 9. Disasters occur frequently and are growing in scale. 1.02 11. The State strives to prevent disasters and protect the people from the dangers. 1.24 1.86 1.85 12. It is enough to educate and prepare in advance through disaster education. 1.51 13. Disaster policy and legislation aid in disaster safety guidelines. -1.11 14. Insensitivity to safety can lead to more disasters. 1.22 1.55 16. The individual’s luck in the event of a disaster is important. -1.12 -1.56 -2.63 18. When the disaster holds resources, it is stockpiling in the home. 1.24 -1.58 20. National and municipal responsibilities are important for safety and disaster management. -1.24 1.01 22. Disaster training, training is necessary. 1.49 1.68 23. I have a habit of checking emergency exits and emergency routes when in any place. -1.87 24. I have an emergency contact network in case of a disaster. -1.62 -1.52 -1.24 -1.30 25. I can induce disaster evacuation to those around me in the event of a disaster. -1.50 28. You can pay more taxes for disaster and safety management. -1.21 -1.66 29. Government disasters and safety accidents are the result of negligence and oversight by agencies. -2.03 Blanks and deleted statements were not significant 1. Disaster management is difficult due to the shortage of young adults due to aging 8. Disaster can kill me too. 9. Disasters occur frequently and are growing in scale. Page 7 of 10 Seong et al. BMC Nursing (2023) 22:469 Page 7 of 10 Seong et al. Type 4: education-oriented type l Based on the results of the present study, in the under graduate course of the nursing college, it is necessary to establish a value system for the nursing profession with a sense of humanity and nursing ethics, and to cultivate the ability to cope with various situations to be faced in the field [25]. Disaster nursing should prepare for disasters affecting the entire community, and provide nursing care not only for individuals and families, but also for groups and the entire community. Also, systematic analysis and education on learning goals or concepts for disasters are necessary [38–40].h Therefore, it is expected that this study will recognize the importance of nurses in a disaster situation, explore the concept of disaster recognized by the nursing stu dents first, and become the basic data for nurturing the disaster nursing competency of the nursing students. In addition, confirming the change in disaster safety aware ness of nursing students through the prepared education will provide insight in countless crises.h Type 3 recognized the importance of active prepara tion for survival. Type 3 places importance on the roles of both the government and individuals in disaster pre paredness, which includes establishing plans, organiza tion, equipment, education, training, and cooperation systems [33]. It also implies that there is a demand from the people to diversify political, social, and economic structures, and develop strategies to prepare for damage caused by disasters [34]. The study results can be used for research and devel opment of nursing education strategies that would be effective in cultivating nurses who could prepare for vari ous disaster situations. Furthermore, it can be used as an institutional device to enhance the competency of the nursing students. In further studies, experimental stud ies need to be conducted to develop an effective nurs ing education strategies or program, and to prove its effectiveness. As a result of this study, Type 4 recognized education related to disasters as important. The National Student Nurses’ Association (NSNA) emphasizes that nursing students must prepare for disasters in their individual, family, and nursing education courses and emphasizes education for this purpose [7, 35]. This seems to be the reason why nursing students feel that disaster education is insufficient, even though most of them have answered in this study that they have received disaster education. Type 4: education-oriented type In the event of a major disaster, the belief system of the peo ple changes differently, which may change their reactions based on their surroundings, thereby suggesting that a change in the disaster management paradigm is neces sary [6, 29]. This means that accountability is imposed on the government and stakeholders, and the needs of the people in an interdependent relationship should be reflected with the common goal of minimizing damage in the disaster management system [6, 29]. In this study, the nursing students said that the government should prepare a systematic education program with human and material resources and systems in order to cultivate disaster awareness and disaster nursing competency. Therefore, in line with the needs of the nursing students, the country should take the initiative to prepare various systems for nurturing nurses who will be at the forefront of disaster response. Page 8 of 10 Page 8 of 10 Seong et al. BMC Nursing (2023) 22:469 Seong et al. BMC Nursing (2023) 22:469 Seong et al. BMC Nursing In the results of this study, Type 2 recognized that personal responsibility, competence, and morality were important. A nurse’s competency refers to the ability to integrate nursing care knowledge, ethical knowledge, personal knowledge, and aesthetic knowledge [30]. Labrague, et al. [7] reported that disaster-related expe riences and awareness influence the ability to perform disaster nursing. In this study, recognizing the nursing students’ personal competence as important is a positive part of disaster management, and this supports the previ ous studies that nursing students want to actively respond to a disaster [31]. According to a study by Eom and Hwang [32] those who directly suffered or experienced disasters understood it as personal responsibility. There fore, in future research, it is necessary to analyze people with personal responsibility in depth. Taken together, it is thought that preparing a program to improve individual factors such as personality, morality, and self-resilience is one way to raise awareness of disaster safety and enhance disaster nursing capacity. by disasters in the future. As such, the characteristics of the types of disaster awareness of nursing students identified in this study have confirmed once again that it is urgent to prepare a disaster education program that reflects the diverse needs of undergraduate students. Type 4: education-oriented type Therefore, it is necessary to identify the educational needs of nursing students for disaster education and develop a systematic curriculum based on this [13]. The study by Jung et al. [36] and the study by Kang and Piao [37] confirmed that the nursing students’ competency for disaster nursing increased through the disaster education program, and a number of previous studies also reported that the ability and knowledge to perform disaster nurs ing improved after education. It can be said that disaster education is essential for the nursing students who will play a pivotal role in the future potential disaster site. Therefore, in Korea, efforts are currently being made to develop disaster nursing education content in the com munity [38]. Each year, millions of people are affected by major events such as disasters, and disaster nursing edu cation can have positive consequences for those affected Limitationsh This study had some limitations. Since this study was conducted on nursing students living in a city, it is nec essary to be careful when generalizing [41, 42] the study results, and there is a limitation in extending the inter pretation to nursing students all in South Korea. There fore, it is necessary to repeat and expand the study in the future while taking into consideration the sampling of the subjects. Also, the environmental situation, such as the frequency of disasters in the environment of the city in which the research was carried out, might affect the results of this study. Declarations 19. Kwon YS, Choi EY, Park SJ, Park SY. Content analysis on the Disaster Crisis and countermeasures recognized by nursing students. J Korean Nurs Res. 2019;3(3):49–59. 19. Kwon YS, Choi EY, Park SJ, Park SY. Content analysis on the Disaster Crisis and countermeasures recognized by nursing students. J Korean Nurs Res. 2019;3(3):49–59. Data Availability The datasets generated and/or analyzed during the current study are not publicly available due no new data were created or analyzed in this study, but are available from the corresponding author on reasonable request. 17. Yang S. Convergence analysis of the factors influencing core competencies on Disaster nursing among nursing students. J Convergence Inform Technol. 2018;8(3):39–51. 18. Woo JH, Yu JY, Park JY. Experience, awareness and preparedness of Disaster among nursing College Students. Crisisonomy. 2015;11(11):19–25. 18. Woo JH, Yu JY, Park JY. Experience, awareness and preparedness of Disaster among nursing College Students. Crisisonomy. 2015;11(11):19–25. Acknowledgements g The authors are very thankful to all the participants for their valuable contribution. 13. Kim HJ. A study on Disaster preparedness, core competencies and educa tional needs on Disaster nursing of nursing students. J Korea Acad Indust coop Soc. 2015;16(11):7447–55. 13. Kim HJ. A study on Disaster preparedness, core competencies and educa tional needs on Disaster nursing of nursing students. J Korea Acad Indust coop Soc. 2015;16(11):7447–55.f Funding The authors have no funding to disclose. 16. Song H, Kim HS. Convergence of the factors influencing of Disaster percep tion, Disaster attitudes and self-resilience on core competencies on Disaster nursing. J Convergence on Culture Technol. 2020;6(1):351–9.l 16. Song H, Kim HS. Convergence of the factors influencing of Disaster percep tion, Disaster attitudes and self-resilience on core competencies on Disaster nursing. J Convergence on Culture Technol. 2020;6(1):351–9.l Conclusions International Council of Nurse. ICN Core Competencies in Disaster nursing version 2.0. Retrieved from. Geneva, Swiss. WHO: World Health Organization & International Council of Nurses; 2019. https://www.icn.ch/sites/default/files/ inline-files/ICN_Disaster-Comp-Report_WEB_final.pdf.ICN. 12. Öztekin SD, Larson EE, Yüksel S, Altun Uğraş G. Undergraduate nursing students’ perceptions about Disaster preparedness and response in Istanbul, Turkey, and Miyazaki, Japan: a cross sectional study. Japan J Nurs Sci. 2015;12(2):145–53. https://doi.org/10.1111/jjns.12058. 12. Öztekin SD, Larson EE, Yüksel S, Altun Uğraş G. Undergraduate nursing students’ perceptions about Disaster preparedness and response in Istanbul, Turkey, and Miyazaki, Japan: a cross sectional study. Japan J Nurs Sci. 2015;12(2):145–53. https://doi.org/10.1111/jjns.12058. Author contributions Conceptualization, M.S., D.R., and S.S.; methodology, M.S., D.R., and S.S.; validation, M.S. and S.S.; formal analysis, M.S., D.R., and S.S.; data curation, M.S. and S.S.; writing-original draft preparation, M.S. and S.S.; writing-review and editing, M.S., D.R., and S.S.; supervision, S.S.; All authors reviewed the manuscript. p 14. Park AS, Yang JH. The effect of Disaster awareness, attitude, preparedness on the basic competencies of Disaster nursing among nursing students. J Korean Data Analysis Soc. 2017;19(3):1625–36. 14. Park AS, Yang JH. The effect of Disaster awareness, attitude, preparedness on the basic competencies of Disaster nursing among nursing students. J Korean Data Analysis Soc. 2017;19(3):1625–36.f 15. Kim NH. The effect of Disaster care program on Disaster perception, attitude and Disaster nursing capacity of nursing students. Asia-pacific J Multi Serv Convergent with Art Humanities and Sociol. 2019;9(11):553–62.l 15. Kim NH. The effect of Disaster care program on Disaster perception, attitude and Disaster nursing capacity of nursing students. Asia-pacific J Multi Serv Convergent with Art Humanities and Sociol. 2019;9(11):553–62.l Consent for publication 23. Brown SR. Q methodology and communication: theory and application. Electr J Commun. 1990; 1(September). 23. Brown SR. Q methodology and communication: theory and application. Electr J Commun. 1990; 1(September). 24. Watts S, Stenner P, Doing. Q Methodological Research: theory, Method and Interpretation. Los Angeles: SAGE; 2012. 24. Watts S, Stenner P, Doing. Q Methodological Research: theory, Method and Interpretation. Los Angeles: SAGE; 2012. Competing interests The authors report no actual or potential conflicts of interest. g 25. Kim HJ, Lee YM. Influence of ageing knowledge and family strengths on age ism among the nursing students. Korean J Rehabil Nurs. 2018;21(2):83–90. 25. Kim HJ, Lee YM. Influence of ageing knowledge and family strengths on age ism among the nursing students. Korean J Rehabil Nurs. 2018;21(2):83–90. Received: 19 October 2022 / Accepted: 4 December 2023 Received: 19 October 2022 / Accepted: 4 December 2023 26. Goniewicz K, Goniewicz M, Burkle FM, Khorram-Manesh A. Cohort research analysis of Disaster experience, preparedness, and competency-based train ing among nurses. PLoS ONE. 2021;16(1):e0244488. https://doi.org/10.1371/ journal.pone.0244488. 27. Kim U, Lidia M. Disaster nursing: a descriptive survey of Australian under graduate nursing curricula. Australasian Emerg Nurs J. 2011;14(2):75–80. 27. Kim U, Lidia M. Disaster nursing: a descriptive survey of Australian under graduate nursing curricula. Australasian Emerg Nurs J. 2011;14(2):75–80. Ethics approval and consent to participate All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Institutional Review Board of Kyung Hee University (IRB No. KHSIRB-19-012), and consent to participate was obtained using written informed consent from study participants all. 20. Kim YS, Jang I. The factors associated with care willingness towards older adults among nursing students. J Korean Data Analysis Soc. 2017;19(3):1669–83. 21. Kim HK. Q methodology: Philosophy, theories, analysis, and application. Seoul, Korea, Seoul Commun. Books; 2008. 21. Kim HK. Q methodology: Philosophy, theories, analysis, Seoul, Korea, Seoul Commun. Books; 2008. 22. Akhtar-Danesh N, Baumann A, Cordingley L. Q-methodology in nursing research: a promising method for the study of subjectivity. Western J Nurs Res. 2008;30(6):759–73. https://doi.org/10.1177/0193945907312979. 22. Akhtar-Danesh N, Baumann A, Cordingley L. Q-methodology in nursing research: a promising method for the study of subjectivity. Western J Nurs Res. 2008;30(6):759–73. https://doi.org/10.1177/0193945907312979. Conclusions Millions of people are affected by major events such as disasters each year, and disaster nursing education can have positive outcomes for those affected by disasters in the future. As such, the characteristics of the type of disaster awareness of nursing students confirmed in this study result confirmed once again the urgent need to Seong et al. BMC Nursing (2023) 22:469 Seong et al. BMC Nursing (2023) 22:469 Page 9 of 10 Page 9 of 10 Seong et al. BMC Nursing prepare a disaster education program that reflects the diverse needs of students in the undergraduate course. It is expected that this study can be used as an institu tional device and basic data to meet the public’s expec tations and enhance the competency of nursing students at a time when the importance of disaster awareness increases and the public’s demand and interest increase. Through this study, the following points are suggested. First, it is proposed to develop a disaster nursing edu cation program considering the types derived through this study and to apply it usefully in nursing. Second, an in-depth study on the types derived from this study is needed. Third, in addition to national disasters such as the current COVID-19 pandemic, natural disasters have different regional variations, so it is suggested to conduct repeated research that supplements these points. 7. Labrague LJ, Hammad K, Gloe DS, McEnroe-Petitte DM, Obeidat AA, Leocadio MC. Disaster preparedness among nurses: a systematic review of literature. Int Nurs Rev. 2017;65(1):41–53. 8. Loke AY, Fung WM, Liu X. Mainland China nurses’ willingness to report to work in Disaster. J Emerg Manag. 2013;11(5):345–4. 9. Park JE. A convergence study on Disaster awareness, Disaster preparedness, and ego-resilience of nursing students. J Convergence Inform Technol. 2019;9(11):38–46. 9. Park JE. A convergence study on Disaster awareness, Disaster preparedness, and ego-resilience of nursing students. J Convergence Inform Technol. 2019;9(11):38–46. 10. World Health Organization & International Council of Nurses. International Council of Nurses Framework of Disaster Nursing Competencies. 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https://openalex.org/W2943988472	http://www.periodicos.ufc.br/rene/article/download/39638/pdf_1/	English	null	Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome	Rev Rene	2,019	cc-by	5,643	1Universidade Federal da Paraíba. João Pessoa, PB, Brazil. DOI: 10.15253/2175-6783.20192039638 www.revistarene.ufc.br DOI: 10.15253/2175-6783.20192039638 www.revistarene.ufc.br DOI: 10.15253/2175-6783.20192039638 www.revistarene.ufc.br DOI: 10.15253/2175-6783.20192039638 www.revistarene.ufc.br Article Review Received: Nov. 21st 2018; Accepted: Apr. 16th 2019. Correspoding author: Sergio Vital da Silva Júnior Correspoding author: Sergio Vital da Silva Júnior Rua Daura Saraiva, 508, Jardim Aeroporto, Bayeux, CEP: 58.308.130. João Pessoa, PB, Brazil. E-mail: sergioenfe1@gmail.com Qualidade de vida de pessoas vivendo com o vírus e síndrome da imunodeficiência humana Sergio Vital da Silva Júnior1, Wilton José de Carvalho Silva1, Natália Silva Lourenço1, Jordana Almeida Nogueira1, Ana Cristina de Oliveira e Silva1, Maria Eliane Moreira Freire1 Objective: to characterize the Brazilian scientific production on the quality of life of people living with human immunodeficiency virus/Acquired Immunodeficiency Syndrome. Methods: bibliometric study with 20 dissertations and 18 theses available at the Brazilian Digital Library of Theses and Dissertations. The empirical material was submitted to descriptive and inferential statistics using the R-Project software version 3.5.1a. Results: studies on the theme increased from 2006 to 2012. The majority came from the Northeast and Southeast regions and was mainly conducted in the Nursing and Psychology areas. The use of specific instruments to evaluate the quality of life of people living with human immunodeficiency virus or acquired immunodeficiency syndrome was evident. Conclusion: there is a significant Brazilian scientific production on the theme, with evidence regarding the physical, social and psychoemotional dimensions of the quality of life of people living with human immunodeficiency virus/Acquired Immunodeficiency Syndrome to the detriment of the cultural and spiritual/religious dimension. Descriptors: Acquired Immunodeficiency Syndrome; HIV; Quality of Life; Nursing; Bibliometri Objetivo: caracterizar a produção científica brasileira sobre qualidade de vida de pessoas vivendo com vírus da imunodeficiência humana/Síndrome da Imunodeficiência Adquirida. Métodos: estudo bibliométrico, com 20 dissertações e 18 teses, disponíveis na Biblioteca Digital Brasileira de Teses e Dissertações. O material empírico foi submetido à estatística descritiva e inferencial, utilizando-se o software R-Project, versão 3.5.1a. Resultados: observou-se incremento de estudos sobre a temática, no período de 2006 a 2012; a maioria oriundo das Regiões Nordeste e Sudeste do Brasil; realizados, principalmente, pelas áreas de Enfermagem e Psicologia. Evidenciou- se utilização de instrumentos específicos de avaliação da qualidade de vida de pessoas que vivem com vírus ou síndrome da imunodeficiência adquirida. Conclusão: existe significativa produção científica brasileira sobre a temática, com evidências quanto à avaliação das dimensões física, social, psicoemocional da qualidade de vida de pessoas vivendo com vírus da imunodeficiência humana/Síndrome da Imunodeficiência Adquirida, em detrimento da dimensão cultural e espiritual/religiosa. ome de Imunodeficiência Adquirida; HIV; Qualidade de Vida; Enfermagem; Bibliometria. Descritores: Síndrome de Imunodeficiência Adquirida; HIV; Qualidade de Vida; Enfermagem; B Correspoding author: Sergio Vital da Silva Júnior Received: Nov. 21st 2018; Accepted: Apr. 16th 2019. Rev Rene. 2019;20:e39638. Silva Júnior SV, Silva WJC, Lourenço NS, Nogueira JA, Oliveira e Silva AC, Freire MEM Introduction the field of health sciences in this context. This term generally involves the perception of health and of the impact of physical, social, psychological and spiritual aspects on health, including health aspects in an ex panded perspective(8). This concept is broad and en compasses a variety of conditions that can influence the way the individuals perceive their daily functio ning, sometimes affecting feelings and behaviors, but not limited to the physical health condition(9). Human Immunodeficiency Virus (HIV) infec tion and its manifestation through the Acquired Im munodeficiency Syndrome (AIDS) arose in the 1980s, bringing to the world population numerous clinical and social concerns. According to data from the Bra zilian Ministry of Health, in 2017, 42,420 new cases of HIV infection and 37,791 of AIDS were reported in Brazil. In recent years, the rate of detection of the syn drome has decreased(1). These numbers may be a re flection of advances in the prevention and, especially, in the treatment of the disease brought about with the introduction of efficient therapies. Since the beginning of the epidemic, resear chers around the world have outlined studies to as sess the impact of the problem on the quality of life of people living with HIV and AIDS in order to improve the provision of health care for this population. This fact justifies the present review, because its goal is to enable health professionals to plan and implement care for these people, based on scientific evidence, regarding the dimensions of quality of life affected by HIV infection and by the treatment. With the evolution in the effectiveness of anti retroviral therapy, HIV infection has become a mana geable chronic condition in which people with timely diagnosis and adequate treatment are able to have their life expectancy considerably prolonged(2). In order to know the state of the art about the subject in evidence, the present study aimed to cha racterize the Brazilian scientific production on the quality of life of people living with Human Immunode ficiency Virus and AIDS. However, the increase in the years of life of pe ople living with HIV and AIDS has brought undesira ble aspects related to the side effects of antiretroviral therapy and the issues of stigma and prejudice that cannot be neglected since they negatively affect the quality of life of this population(3-4). Rev Rene. 2019;20:e39638. Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome exclusion criteria to be rigorously followed(14). In the second stage, empirical material was collected. To do so, in June 2018, dissertations and doctoral theses were surveyed in the Brazilian Digital Library of Theses and Dissertations, linked to the Bra zilian Institute of Information in Science and Techno logy. The terminology in health based on the Health Sciences Descriptors (DeCS) was used in this step. In the third stage, from June to July 2018, data were collected using an instrument prepared by the researchers addressing information about the studies, such as year of publication, place of origin of the stu dy according to the geographic region of Brazil and higher education institution of publication, area of academic training of the author, study design, instru ments used to measure quality of life, and dimensions of quality of life assessed in the studies on the topic HIV/AIDS. It should be noted that, in order to observe the state of the art from sensu stricto Brazilian graduate programs on the subject studied, it was decided to in vestigate the theses and dissertations instead of origi nal articles because the dissemination of the articles coming from the gray literature hardly takes place in a timely manner after the defense of these works(13). Then, the fourth stage was started, consisting of the processing of the data in the R-Project software, version 3.5.1. This is a free software environment for statistical and graphical computation. The analyses included descriptive (frequency, mean and median of publications) and inferential statistical analyses, using the non-parametric Mann-Whitney test, which allows verifying whether there are differences in the variables related to the production of dissertations and theses. For the search of studies, the descriptors ‘HIV’, ‘AIDS’ and ‘quality of life’ were used, combined with the Boolean operator AND, with the condition of pre sence of the word in the title of the study, in order to refine the search with a focus on the target thematic area, resulting in the identification of 105 studies (dis sertations and theses). The data were presented in texts and graphs, analyzed in light of the literature pertinent to the subject investigated, contemplating the fifth and final operational stage of this study. Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome The inclusion criteria to select the sample were: studies published from 2005 to 2018, because this is considered a period in which scientific and techno logical advances related to the treatment of HIV and AIDS were disseminated by the scientific community; studies discussing the quality of life of people living with HIV and AIDS; and studies available online in the Brazilian Digital Library of Theses and Dissertations. As exclusion criterion: studies that did not use general or specific instruments to evaluate quality of life. As this was a bibliometric review study, there was no need for appreciation by an Ethics Committee in Research involving human beings, and the ethical principles from current resolutions and the Code of Ethics of Nursing Professionals were rigorously con sidered. After a thorough reading of the abstracts of the works and in compliance with the established criteria, 31 dissertations and 36 theses were excluded from the present review. Therefore, the sample consisted of 38 studies, among 20 dissertations and 18 theses, which were organized and grouped into a file folder in the Windows 10 operating system. Methods Quality of life involves several human dimen sions, including physical, psychological, social and spiritual aspects, which can have important reper cussions on the reality of each individual. They may be affected by interference from the bipolarity that results from the positive and negative influences that permeate the everyday life of people, but also from the subjectivity of thoughts, feelings and emotions typical of the human being(5). Bibliometric study using quantitative techni ques capable of showing indicators that represent the state of the art of knowledge produced on a given topic. Bibliometrics has been considered an impor tant tool for studies aimed at the characterization of scientific production, allowing greater visibility to me tric studies of recorded information, such as products resulting from scientific activity, articles and scientific productions in general(10-11). The development of the study followed operational stages(12); the first step consisted in defining the theme and elaborating the guiding question: how is the scientific production addressing the quality of life of people living with HIV and AIDS characterized, as published in dissertations and theses in Brazil? The perception of quality of life of each person is subject to the diverse interpretations and influences of the reality in which he/she is inserted, undergoing changes throughout the life(6). Quality of life scores of people living with HIV and AIDS are lower when com pared to people without the disease or people affected by other chronic conditions(7). The term ‘health-related quality of life’ arises in Rev Rene. 2019;20:e39638. Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome Results Regarding the characterization of the analyzed studies, it was observed that there was a greater per centage of publications in the year of 2009 (20.0%), followed by the years of 2011 and 2013 (15.0% in each year), 2008, 2010 and 2012 (10.0% in each year), 2005, 2006, 2007 and 2016 (5.0% in each year). There were no publications in the years 2014, 2015, 2017 and 2018. It should be noted that the sample was selec ted by two authors, as recommended by the Preferred Reporting Items for Systematic Reviews and Meta -Analysis (PRISMA) statement, allowing inclusion and Rev Rene. 2019;20:e39638. Silva Júnior SV, Silva WJC, Lourenço NS, Nogueira JA, Oliveira e Silva AC, Freire MEM In relation to the publication of theses, the highest number of publications occurred in 2011 (22.2%), followed by 2010 (16.6%), 2007, 2009, 2012 and 2013 (11.1% each year), 2006, 2008 and 2016 (5.6% in each year). In 2005, 2017 and 2018, no pu blications of theses were identified. With regard to the year of publication of the studies, the annual production of dissertations and theses was compared between the first seven and the last seven years of the research, as shown in the box plot presented in Figure 1. Figure 1 – Median of the annual academic production of dissertations and theses (n=38) Figure 1 – Median of the annual academic production of dissertations and theses (n=38) Between 2005 and 2018, the highest number of dissertations was in the Northeast Region (40.0%), followed by the Southeast (30.0%), South (20.0%) and Center-West (10.0% %). With respect to the the ses, a greater number of productions was observed in the Southeast (44.0%), followed by the Northeast and South (22.0%, respectively), and Central-West Region (12.0%). For the purpose of sampling, it is relevant to note that there was no scientific production related to the quality of life of people living with HIV and AIDS from institutions in the Northern Region of Brazil. Discussion One limitation of this study was the non-appli cation of the bibliometrics laws in view of the redu ced number of items in the sample, which hindered a more accurate analysis of the characteristics of the analyzed studies. Regarding the methodological aspects des- cribed in the dissertations and theses investiga- ted, 58.0% were descriptive, 26.0% exploratory, and 16.0% descriptive-exploratory. Regarding the ap proach, 66.0% of the studies were quantitative, 29.0% used the quantitative and qualitative approach, and 5.0% of the studies were qualitative. With regard to the publications of disserta tions, in the time period from 2005 to 2018, it was ob served in Figure 1 that, according to the sample me dian, there was a greater publication of dissertations in the early years of academic production, whereas in the final years the median was zero, indicating that in some years there was no production of dissertations on the subject. Regarding the technical procedures, most of the studies were field surveys (76.0%) followed by quasi experimental studies (10.0%), cohort studies (5.0%), procedures of the research-action type, cli- nical non-randomized trials, and case studies (3.0%), respectively. The analysis of the production of theses during the time interval studied showed that a greater num ber of works was published in the initial years. In the final years, there was no production, being the maxi mum value of publication of theses in the entire pe riod equal to two. The Mann-Whitney test was applied to the data, assuming that the samples were indepen dent, and revealed that the medians of the variables were equal at a significance level of 5%. Regarding the approach to health-related quali ty of life of people living with HIV/AIDS, regarding the dimensions of quality of life assessed in the studies that composed the sample, the physical dimension was the most cited in most studies (27.3%); followed by the emotional/psychological dimension (26.6%); so cial dimension (22.7%); cultural dimension (14.1%); and spiritual/religious dimension (9.4%). In order to investigate differences in the me dians of production of studies according to geographi cal region, values above the level of significance were observed. This indicates that there was no difference in the medians and the production of dissertations and theses by geographic region can be considered equal. Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome -QoL) instrument. In 52.7% of the investigations, generic instruments for assessment of quality of life were used (WHOQOL-100 or WHOQOL-bref); and in 34.2%, instruments for evaluation of anxiety, depres sion and self-esteem (Beck Depression Inventory, An xiety and Depression Scale, Rosemberg Self-esteem Scale, and Hope Scale) were used. The studies also used instruments for collection of additional informa tion on sociodemographic data, laboratory test data, as well as semi-structured interview scripts. Considering the significance level of 5%, the Mann-Whitney test was applied to identify differen- ces in the medians of production of studies by geo graphic region, and p-values higher than the level of significance were found, as in the Northeast: p=0.479; Southeast: p=0.879; South: p=0.999; and Center-West: p=0.999. Regarding the authorship of the dissertations and theses investigated, it was seen that nurses were the ones that most published, appearing in 39.0% of the studies, followed by psychologists, in 34.0%; phy- sical educators, in 8.0%; physical therapists and phy sicians, in 5.0% each; and dentists, economists and social workers, in 3.0% each. Results With regard to the origin of the studies that composed the sample of this research, scientific productions related to the quality of life of people living with HIV/AIDS were identified in the following higher education institutions located in the four macroregions of the country: University of São Paulo (29,.0%), Federal University of Paraiba (16%), Catholic Pontifical University of Rio Grande do Sul (11%), Federal University of Paraíba (16.0%), University of Brasília, Federal University of Rio Grande do Norte, Federal University of Rio Grande do Sul and Federal University of Ceará (8.0% each institution); Paulista State University, Federal University of Santa Catarina, State University of Campinas, Júlio de Mesquita Filho São Paulo State University, and Catholic University of Brasília (3.0% each institution). The Mann-Whitney non-parametric test was used to observe whether there was equality between the medians of the variables addressed in this review, assuming that the samples were independent, with a significance level of 5%. A p=0.775 was found. Rev Rene. 2019;20:e39638. Discussion Regarding the specificity of the instruments for assessment of quality of life contained in the sample, 89.0% of the studies used general tools for assess ment of quality of life of people living with HIV and AIDS, namely: the World Health Organization Quality of Life (WHOQOL-120 HIV or WHOQOL-HIV Bref) or the AIDS-Targeted Quality of Life Instrument (HAT Rev Rene. 2019;20:e39638. Silva Júnior SV, Silva WJC, Lourenço NS, Nogueira JA, Oliveira e Silva AC, Freire MEM Bibliometric analyses are an important metho dological resource for health sciences as they reveal patterns of research and identify trends(15). According to the results of this bibliometric study, an advance ment of the research on the theme in Brazil was ob served from 2006 to 2012, with a clear significant de crease in recent years. Despite the decrease of studies published at the sensu stricto level between 2014 and 2015, a concern of researchers with the evaluation of the quality of life of people living with HIV and AIDS was observed. chronicity of AIDS may interfere in the physical and mental health and the well-being of people who use it, leading to negative situations in the quality of life of individuals. Coping with this problem can bring with it circumstances of exclusion and stigma on the part of society, causing a rupture in interpersonal rela tionships of affection, sexuality and socialization(19-20). This corroborates studies analyzed in this re view, which identified in their evaluation of quality of life that the physical dimension was the most present. This shows that the manifestations imposed by the di sease related to HIV infection and AIDS can interfere with people’s quality of life, affecting activities of daily living and making individuals feel less socially impor tant(21). Regarding the place of origin of the studies, there was a predominance of institutions from the Northeast and Southeast Regions of Brazil. This result may be justified by the need for development of re search and strategies to support decision making in these regions, given the greater occurrence of notifi cations of HIV infection there(16). Discussion The result also corro borates a review study that explains the high number of productions on the quality of life of people living with HIV and AIDS in the Brazilian Southeastern Re gion based on the concentration of graduate programs in the health area, besides health services and a high number of cases of infection in this region(17). The emotional/psychological dimension may be affected by the fact that the individual has a chronic and incurable condition, as well as by the stigma that still exists due to the lack of knowledge about the di sease and the treatment. In the social dimension, the detachment from friends and family stands out, hin dering family interactions, making individuals to iso late in society and have low self-esteem(4). The feeling of guilt can also occur when a condition is considered a social deviation from the heteronormative idea that still prevails in the current collective imagination(18). It was observed in this bibliometric review that professionals from various areas of knowledge have shown interest in the theme in focus. However, a des criptive study carried out in Bahia, Brazil, involving professionals from health and social sciences who worked directly with people living with HIV/AIDS de monstrated that despite the advances related to the approach and management in the treatment of this disease, the prejudice among these professionals still persists and can negatively influence their practice of care provision. Thus, it is necessary to strengthen interdisciplinary actions in order to promote com prehensive care, free from any stereotype, with a view to promoting the quality of life of people living with HIV/AIDS(18). The present study showed that Nursing and Psychology are responsible for most of the publi cations on the topic of Quality of Life related to the Health of people living with HIV and AIDS. This may be associated with the fact that the diagnosis of HIV infection has biopsychosocial implications, since it is a chronic infection with an emotional impact on its effective control, rasing new challenges for patients and health professionals(22). Rev Rene. 2019;20:e39638. Discussion Due to the fact that the nursing category deals with people in hospital institutions in a daily basis and also because in the discussion of how to train profes sionals capable of understanding the complexity of aids, based on the psychosocial dimensions, there is a clear importance and need for research in this theme, In view of the above, although antiretroviral treatment is a positive aspect, when allied with the Rev Rene. 2019;20:e39638. Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome sing the concept of quality of life in a well defined way, researchers could delineate studies with the greatest possible accuracy, conferring reliability to the theory and later application in the praxis of health professio nals and related areas. in the attempt to improve nursing care in the area of care for people living with HIV and AIDS(23). This con firms the findings of the present study, in which nur sing has been leading the Brazilian scientific produc tion on the quality of life of infected people. It is, therefore, important to highlight the di fferent ways of measuring the quality of life, consi dering the subjectivity of the evaluation. Instruments that have scientific and technical rigor must be used. Scales for measuring the dimensions of quality of life should follow scientific precision and well defined sta tistical parameters in order to allow a reliable result to the study findings. Therefore, instruments in scien tific research must be related to the purpose of what is expected from the findings, where a specific purpose is essential, with validation of the applicability to the object of the study(26). In this discussion, in view of the complexity of the health care for people living with HIV and AIDS, interdisciplinary and intersectoral work is imperati ve in the search for strengthening a network of care for HIV/AIDS, with an integrated approach and com prehensive assistance to people living with HIV/AIDS. This can be achieved through a joint intervention plan involving professionals, health service teams and ma nagers and sectors of society in the attempt to res pond positively to the needs presented by users(24). It is pointed out that the studies developed on the theme at the national level are characteristic of the health knowledge field. Discussion It is, therefore, urgent that Nursing and Psychology professionals seek evidence about facts or circumstances that affect the quality of life of these people in order to demonstrate the con cern of the categories in developing more humanized care, focused on biopsychosocial issues of people li ving with HIV/AIDS. In the present survey, the analyzed studies eva luated the quality of life of people living with HIV and AIDS using general and specific instruments validated in Brazil. Some of these studies also used instruments of evaluation of depression, anxiety and self-esteem. In particular, it is noteworthy that it is impor tant to use valid and reliable instruments for what the study proposes to measure, so as to effectively reveal the knowledge about the living conditions of people who suffer with the chronic infection, according to the requirements of the disease and its physical, psycho logical, social, and spiritual repercussions. The later can lead to significant social changes, compromising interpersonal relationships and causing low self-es teem and lifestyle changes, with a negative impact on the quality of life of these people(27). Health care consists in the practice of caring for individuals in a comprehensive way, embracing them by listening to them and with a sensitive perspective during therapy, making it possible to care for indivi duals in their subjectivity, culture and history, not only focusing on the disease, but also on the persons live with the problem(21). A study on the quality of life of women living with HIV demonstrated the need for well-structured social support to enable positive qua lity of life in people living with HIV and AIDS(25). It is necessary, therefore, to apply instruments in the methodological course of the research that eva luate the dimensions of the human being with a view to reverse situations of negativity that interfere with the daily lives of people living with HIV infection. In the content of this trend, the studies should address the quality of life to be measured with tech nical and scientific rigor, focusing on the cultural, so cial, political and economic dimensions of the human being. This can have an impact on the care of patients, solidifying the clinical practice of health professionals based on the scientific literature produced. By addres The results found in this study also showed an incipient production focused on the spirituality/reli giosity of people living with HIV and AIDS. Religiosity Rev Rene. References 1. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Boletim Epidemiológico HIV/Aids [Internet]. 2017 [citado 2019 jan. 23]; 48(1):1- 52. Disponível em: http://portalarquivos2.saude. gov.br/images/pdf/2017/janeiro/05/2016_034- Aids_publicacao.pdf 1. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Boletim Epidemiológico HIV/Aids [Internet]. 2017 [citado 2019 jan. 23]; 48(1):1- 52. Disponível em: http://portalarquivos2.saude. gov.br/images/pdf/2017/janeiro/05/2016_034- Aids_publicacao.pdf The relevance of the implementation of spiritu ality as an adjunct to drug therapy is therefore clear, for it allows the provision of comprehensive health care and meeting the needs in all the dimensions of the people living with HIV and AIDS, which can con substantially improve their quality of life. 2. Okuno MFP, Gomes AC, Meazzini L, Scherrer Júnior G, Belasco Junior D, Belasco AGS. Qualidade de vida de pacientes idosos vivendo com HIV/ Aids. Cad Saúde Pública. 2014; 30(7):1551-9. doi: http://dx.doi.org/10.1590/0102-311X00095613 2. Okuno MFP, Gomes AC, Meazzini L, Scherrer Júnior G, Belasco Junior D, Belasco AGS. Qualidade de vida de pacientes idosos vivendo com HIV/ Aids. Cad Saúde Pública. 2014; 30(7):1551-9. doi: http://dx.doi.org/10.1590/0102-311X00095613 3. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Manual técnico para o diagnóstico da infecção pelo HIV em adultos e crianças [Internet]. 2018 [citado 2019 jan. 23]. Disponível em: http:// www.aids.gov.br/pt-br/node/57787 3. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Manual técnico para o diagnóstico da infecção pelo HIV em adultos e crianças [Internet]. 2018 [citado 2019 jan. 23]. Disponível em: http:// www.aids.gov.br/pt-br/node/57787 Collaborations is important in the treatment of the infection because it may contribute to the process of adherence to drug therapy. Thus, it seems necessary that professionals develop care practices related to spirituality in order to improve the health care for people living with HIV and AIDS and, consequently, the quality of life of pa tients(28). Silva Júnior SV, Silva WJC, Lourenço NS, Noguei ra JA, Oliveira e Silva AC and Freire MEM participated in the planning and designing or analysis and inter pretation of data, writing of the article, critically re view of the intellectual content, and final approval of the version to be published. In the practice, the psycho-spiritual dimension (although referred to as support for the treatment of people living with HIV and AIDS) is still little incor porated into professional practices in specialized care services. Therefore, more studies exploring the con texts in which spirituality is involved in the treatment of HIV infection are necessary(29). Discussion 2019;20:e39638. Silva Júnior SV, Silva WJC, Lourenço NS, Nogueira JA, Oliveira e Silva AC, Freire MEM Conclusion In recent years, there has been a significant pro duction in Brazilian graduate studies on the quality of life of people living with HIV and AIDS. The Southeast and Northeast regions lead the scientific production at the sensu stricto level, with the Federal University of São Paulo and the Federal University of Paraíba being the institutions that published more works. The ten dency to believe that this theme belongs only to the health area was confirmed. 4. Domingues JP, Oliveira DC, Marques SC. Quality of life social representations of people living with HIV/Aids. Texto Contexto Enferm. 2018; 27(2):e1460017. doi: dx.doi.org/10.1590/0104- 070720180001460017 5. Freire MEM, Sawada NO, França ISX, Costa SFG, Oliveira CDB. Health-related quality of life among patients with advanced cancer: an integrative review. Rev Esc Enferm USP. 2014; 48(2):357-67. doi: 10.1590/S0080-6234201400002000022 The set of findings of this study brings evidence regarding the evaluation of the physical, social, emo tional and psychological dimensions of the quality of life of people living with HIV and AIDS, as opposed to the cultural and spiritual/religious dimension, which is therefore a gap of knowledge in most of the studies analyzed. 6. Soares ASF, Amorim MISPL. Qualidade de vida e espiritualidade em pessoas idosas instituciona lizadas. Rev Port Enferm Saúde Mental [Inter net]. 2015 [citado 2019 jan. 23]; 2(spe2):45-51. Disponível em: http://www.scielo.mec.pt/pdf/ rpesm/nspe2/nspe2a08.pdf Rev Rene. 2019;20:e39638. Quality of life of people living with the human immunodeficiency virus and acquired immunodeficiency syndrome 7. Alemayehu M, Wubshet M, Mesfin N, Tamiru A, Gebayehu A. Health-related quality of life of HIV infected adults with and without Visceral Leishmaniasis in Northwest Ethiopia. Health Qual Life Outcomes. 2017; 15(65):65. doi: dx.doi. org/10.1186/s12955-017-0636-6 15. Luna IT, Silva KL, Oliveira SKP, Lima FET, Araújo TL, Damasceno MMC. Analysis of references of the brazilian nursing doctoral thesis: bibliometric study. Rev Enferm UFPE on line [Internet]. 2015 [cited Jan. 13, 2019]; 9(5):7753-9. Available from: periodicos.ufpe.br/revistas/revistaenfermagem/ article/view/10521/0 8. Costa JM, Nogueira LT. Fatores associados à quali dade de vida relacionada à saúde de receptores de transplantes renais em Teresina, Piauí, 2010. Epi demiol Serv Saúde. 2014; 23(1):121-9. doi: dx.doi. org/10.5123/S1679-49742014000100012 16. Ministério da Saúde (BR). Secretaria de Vigilância em Saúde. Boletim Epidemiológico HIV/Aids [Internet]. 2016 [citado 2019 jan. 23]. Disponível em: http://www.aids.gov.br/pt-br/pub/2016/ boletim-epidemiologico-de-aids-2016 9. Medeiros B, Silva J, Saldanha AAW. Determinantes biopsicossociais que predizem qualidade de vida em pessoas que vivem com HIV/Aids. Estud Psicol (Natal). 2013; 18(4):543-50. doi: http://dx.doi. org/10.1590/S1413-294X2013000400001 17. Conclusion Silva FS, Oliveira FBM, Mendes ACC, Silva G, Santos SO, Pessoa RMC. Preditores associados à qualidade de vida em pessoas vivendo com HIV/ Aids: revisão integrativa. Rev Prev Infecç Saude [Internet]. 2015 [citado 2019 jan. 23]; 1(2):53- 63. Disponível em: http://www.ojs.ufpi.br/index. php/nupcis/article/view/3589/pdf 10. Agra MAC, Freitas TCS, Caetano JÁ, Alexandre ACS, Sá GGM, Galindo Neto NM. Nursing dissertations and theses on the mobile emergency care services: a bibliometric study. Texto Contexto Enferm. 2018; 27(1):e3500016. doi: dx.doi.org/10.1590/0104- 07072018003500016 18. Suto CSS, Oliveira JF, Paiva MS. Social Repre sentations of health care professionals on Ac quired Immune Deficiency Syndrome. Rev Bras Enferm. 2018; 71(4):1934-9. doi: http://dx.doi. org/10.1590/0034-7167-2015-0001 11. Portal SG. Una nueva perspectiva teórica de la bibliometría basada en su dimensión históri ca y sus referentes temporales. Investig Bibl. 2016; 30(70):11-6. doi: dx.doi.org/10.1016/j.ib bai.2016.10.001 19. Villela WV, Monteiro S. Gênero, estigma e saúde: reflexões a partir da prostituição, do aborto e do HIV/Aids entre mulheres. Epidemiol Serv Saude. 2015; 24(3):531-40. doi: http://dx.doi. org/10.5123/S1679-49742015000300019 12. Vanz SAS, Stumpf IRC. Colaboração científica: re visão teórico-conceitual. Perspect Ciênc Inf. 2010; 15(2):42-55. doi: http://dx.doi.org/10.1590/ S1413-99362010000200004 20. Araújo MAD, Carvalho DMS, Oliveira HJP, Nasci mento LG, Santos ACC. A abordagem atual a mu- lher soropositiva para o HIV frente a maternidade: revisão integrativa. Rev Saúde [Internet]. 2017 [citado 2019 jan. 23]; 11(1): 41. Disponível em: http://revistas.ung.br/index.php/saude/article/ view/3141/2264 13. Costa ICP, Costa SFG, Andrade CG, Oliveira RC, Abrão FMS, Silva CRL. Scientific production on workplace bullying/harassment in dissertations and theses in the Brazilian scenario Rev Esc Enferm USP. 2015; 49(2):265-74. doi: dx.doi. org/10.1590/S0080-623420150000200012 21. Calvetti PÜ, Giovelli GRM, Gauer GJC, Moraes JFD. Níveis de ansiedade, estresse percebido e suporte social em pessoas que vivem com HIV/Aids. Psicol Teor Pesq. 2016; 32(4):e324317. doi: http:// dx.doi.org/10.1590/0102.3772e324317 14. Moher D, Liberati A, Tetzlaff J, Altman DG, PRISMA Group. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med. 2009; 151(4):264–9. doi: http://dx.doi.org/10.7326/0003-4819-151- 4-200908180-00135 22. Camillo SO, Maiorino FT, Chaves LC. Nursing teaching on HIV/Aids in the perspective of citizenship. Rev Gauch Enferm. 2013; 34(3): 117-23. doi: http://dx.doi.org/10.1590/S1983- 14472013000300015 Rev Rene. 2019;20:e39638. Silva Júnior SV, Silva WJC, Lourenço NS, Nogueira JA, Oliveira e Silva AC, Freire MEM 27. Hipolito RL, Oliveira DC, Costa TL, Marques SC, Pereira ER, Gomes AMT. Quality of life of people living with HIV/Aids: temporal, socio- demographic and perceived health relationship. Rev Latino-am Enfermagem. 2017; 25:e2874. doi: https://doi.org/10.1590/1518-8345.1258.2874 23. Conclusion Lopes LM, Magnabosco GT, Andrade RLP, Ponce MAZ, Wysocki AD, Ravanholi GM, et al. Coordenação da assistência prestada às pessoas que vivem com HIV/Aids em um município do Estado de São Paulo, Brasil. Cad Saúde Pública. 2014; 30(11): 2283-97. doi: dx.doi.org/10.1590/0102-311X00091213. 28. Oliveira ADF, Vieira MCA, Silva SPC, Mistura C, Jacobi CS, Lira MOSC. Effects of HIV in daily life of women living with AIDS. Rev Pesqui Cuid Fundam On line. 2015; 7(1):1975-86. doi: 10.9789/2175- 5361.2015.v7i1.1975-1986 24. Fernandes ES, Almeida IS, Costa CCP, Ribeiro IB. A vivência do ser-adolescente com HIV/Aids: um estudo fenomenológico para a enfermagem. Rev Enferm Prof [Internet]. 2014 [citado 2019 mar. 22]; 1(2):447-61. Disponível em: https://docplayer. com.br/36947218-Rev-enf-profissional-jul-dez- 1-2-447-issn.html 29. Espírito Santo CC, Gomes AMT, Oliveira DC, Marques SC. Antiretroviral treatment adherence and the spirituality of people with HIV/Aids: social representations study. Rev Enferm UERJ [Internet]. 2013 [cited Jan. 21, 2019]; 21(4):458- 63. Available from: https://www.e-publicacoes. uerj.br/index.php/enfermagemuerj/article/ view/10005/8130 25. Bellini JM, Reis RK, Reinato LAF, Magalhães RLB, Gir E. Quality of life of HIV seropositive women. Acta Paul Enferm. 2015; 28(4):350-4. doi: http:// dx.doi.org/10.1590/1982-0194201500059 26. Machado CJ, Pereira CA. Instrumentos de cole ta de dados na saúde pública: resenha crítica da obra de Monteiro e Horta. Cad Saúde Co letiva. 2014; 22(4):437-8. doi: http://dx.doi. org/10.1590/1414-462X201400040002 Rev Rene. 2019;20:e39638.
https://openalex.org/W2898880563	https://hal.sorbonne-universite.fr/hal-01926829/document	English	null	Characterization of the TLR Family in Branchiostoma lanceolatum and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus	Frontiers in immunology	2,018	cc-by	13,701	To cite this version: Jie Ji, David Ramos-Vicente, Enrique Navas-Pérez, Carlos Herrera-Úbeda, José Miguel Lizcano, et al.. Characterization of the TLR Family in Branchiostoma lanceolatum and Discovery of a Novel TLR22- Like Involved in dsRNA Recognition in Amphioxus. Frontiers in Immunology, 2018, 9, pp.2525. 10.3389/fimmu.2018.02525. hal-01926829 Characterization of the TLR Family in Branchiostoma lanceolatum and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus Jie Ji, David Ramos-Vicente, Enrique Navas-Pérez, Carlos Herrera-Úbeda, José Miguel Lizcano, Jordi Garcia-Fernàndez, Hector Escriva, Alex Bayes, Nerea Roher Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01926829 https://hal.sorbonne-universite.fr/hal-01926829v1 Submitted on 19 Nov 2018 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 ORIGINAL RESEARCH published: 02 November 2018 doi: 10.3389/ﬁmmu.2018.02525 Edited by: L. Courtney Smith, George Washington University, United States Toll-like receptors (TLRs) are important for raising innate immune responses in both invertebrates and vertebrates. Amphioxus belongs to an ancient chordate lineage which shares key features with vertebrates. The genomic research on TLR genes in Branchiostoma ﬂoridae and Branchiostoma belcheri reveals the expansion of TLRs in amphioxus. However, the repertoire of TLRs in Branchiostoma lanceolatum has not been studied and the functionality of amphioxus TLRs has not been reported. We have identiﬁed from transcriptomic data 30 new putative TLRs in B. lanceolatum and all of them are transcribed in adult amphioxus. Phylogenetic analysis showed that the repertoire of TLRs consists of both non-vertebrate and vertebrate-like TLRs. It also indicated a lineage-speciﬁc expansion in orthologous clusters of the vertebrate TLR11 family. We did not detect any representatives of the vertebrate TLR1, TLR3, TLR4, TLR5 and TLR7 families. To gain insight into these TLRs, we studied in depth a particular TLR highly similar to a B. belcheri gene annotated as bbtTLR1. The phylogenetic analysis of this novel BlTLR showed that it clusters with the vertebrate TLR11 family and it might be more related to TLR13 subfamily according to similar domain architecture. Transient and stable expression in HEK293 cells showed that the BlTLR localizes on the plasma membrane, but it did not respond to the most common mammalian TLR ligands. However, when the ectodomain of BlTLR is fused to the TIR domain of human TLR2, the chimeric protein could indeed induce NF-κB transactivation in response to the viral ligand Poly I:C, also indicating that in amphioxus, speciﬁc accessory proteins are needed for downstream activation. Based on the phylogenetic, subcellular localization and functional analysis, we propose that the novel BlTLR might be classiﬁed as an antiviral receptor sharing at least partly the functions performed by vertebrate TLR22. Characterization of the TLR Family in Branchiostoma lanceolatum and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus Jie Ji 1, David Ramos-Vicente 1,2, Enrique Navas-Pérez 3, Carlos Herrera-Úbeda 3, José Miguel Lizcano 4, Jordi Garcia-Fernàndez 3, Hector Escrivà 5, Àlex Bayés 1,2 and Nerea Roher 1* 1 Department of Cell Biology, Animal Physiology and Immunology, Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra, Spain, 2 Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain, 3 Department of Genetics, School of Biology and Institute of Biomedicine (IBUB), University of Barcelona, Barcelona, Spain, 4 Department of Biochemistry and Molecular Biology, Institute of Neurosciences, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 CNRS, Biologie Intégrative des Organismes Marins, BIOM, Sorbonne Université, Banyuls-sur-Mer, France TLR22 is thought to be viral Reviewed by: Katherine Buckley, Carnegie Mellon University, United States Loriano Ballarin, Università degli Studi di Padova, Italy Reviewed by: Katherine Buckley, Carnegie Mellon University, United States Loriano Ballarin, Università degli Studi di Padova, Italy Correspondence: Nerea Roher nerea.roher@uab.cat Correspondence: Nerea Roher nerea.roher@uab.cat Correspondence: Nerea Roher nerea.roher@uab.cat Specialty section: This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology Received: 11 July 2018 Accepted: 12 October 2018 Published: 02 November 2018 INTRODUCTION nucleic acids (9, 11–13). Recently, the sequencing of the genome in ﬁve bony ﬁsh species has allowed the discovery of at least 16 TLR types in teleosts (14). There are two types of immunity in vertebrates. One is the innate immunity, which is genetically programmed to detect invariant features of invading microbes. The other is the adaptive immunity, which employs antigen receptors that are not encoded in the germ line but are generated de novo (1). The innate immune system is the ﬁrst line of defense against infectious diseases (2). Immediately after infection, the innate response is activated to combat pathogens and synthesize inﬂammatory mediators and cytokines (3). However, the primary challenge of the innate immune system is how to discriminate a countless number of pathogens using a restricted number of receptors (2). As a response, a variety of receptors can recognize conserved motifs on pathogens (4). These conserved motifs are known as Pathogen-Associated Molecular Patterns (PAMPs) (5) and their recognition partners, are called Pattern Recognition Receptors (PRRs) (6). There are two structural types of TLRs according to the TLR ectodomain structure: sccTLRs and mccTLRs. The sccTLRs are characterized by the presence of a single cysteine cluster on the C-terminal end of LRRs (a CF motif), which is juxtaposed to the plasma membrane. Most TLRs found in deuterostomes have this domain organization. The mccTLRs are characterized by an ectodomain with two or more CF motifs and another cysteine cluster on the N-terminal side of the LRRs (NF motif). They are systematically found in protostomes but have also been identiﬁed in the invertebrate deuterostome S. purpuratus and the cnidarian N. vectensis (15). Both sccTLR and mccTLR share a common TLR structure: LRR+TM+TIR. According to the ectodomain architecture and phylogenetic criteria, vertebrate TLRs can be classiﬁed into six families: 1, 3, 4, 5, 7 and 11. TLR1 family includes TLR1/2/6/10/14/18/24/25 as well as TLR27; TLR3, 4 and 5 families only include TLR3, 4 and 5 itself; TLR7 family includes TLR7/8/9; TLR11 family includes two subfamilies: 11 (TLR11/12/16/19/20/26) and 13 (TLR13/21/22/23) (16, 17). Toll-like receptors (TLRs), among the most extensively studied PRRs, are type-I transmembrane proteins consisting of an ectodomain, a transmembrane (TM) domain and an intracellular Toll/interleukin-1 receptor (TIR) domain (7). The ectodomain, which functions as a PAMPs recognition domain, is arranged in tandem leucine-rich repeat (LRR), from one to many depending on the receptor type. INTRODUCTION The LRR contains a segment of 11 conserved residues with the consensus sequence LxxLxLxxNxL, where x can be any amino acid, L is a hydrophobic residue (leucine, valine, isoleucine, or phenylalanine) and N can be asparagine or cysteine (8). The TIR domain is present in the cytosol and is required for downstream signal transduction (9). Upon PAMP recognition, TLRs recruit TIR-domain containing adaptor proteins such as MyD88, TRIF, TIRAP/MAL, or TRAM, which initiate signal transduction pathways that culminate in the activation of NF-κB, IRFs, or MAP kinases regulating the expression of cytokines, chemokines, or type I interferons (IFN), which ﬁnally protect the host against infections (10). A variety of TLRs are capable of recognizing viruses. Among human TLRs, the envelope proteins from viruses are mainly recognized by TLR2 and TLR6. Viral nucleic acids are recognized by TLR3 (ssRNA or dsDNA), TLR7 (ssRNA), TLR8 (ssRNA), and TLR9 (dsDNA or CpG motifs) (18). In teleosts, it has been reported that Poly I:C could be recognized by diﬀerent TLRs. Teleost TLR13 was ﬁrstly reported in Miiuy croaker (Miichthys miiuy) which showed cytoplasmic localization in HeLa cells. It could respond to both Vibrio anguillarum and Poly I:C injection in vivo and Poly I:C stimulation in leukocytes (19). In fugu (Takifugu rubripes), TLR3 localizes in the endoplasmic reticulum and recognizes relatively short dsRNA, whereas TLR22 recognizes long dsRNA on the cell surface (20). Grass carp (Ctenopharyngodon idella) TLR22 is expressed in many tissues and is highly abundant in the gills. Infection of grass carp with grass carp reovirus (GCRV), a dsRNA virus, induces a rapid up- regulation of TLR22 gene expression in the spleen (21). Japanese ﬂounder (Paralichthys olivaceus) TLR22 is mainly expressed in peripheral blood leukocytes (PBL) and could be induced by both peptidoglycan and Poly I:C (22), whereas TLR3 gene expression in PBLs increased upon stimulation with Poly I:C and CpG ODN 1668 (23). Both TLR3 and TLR22 gene transcription had also been studied in large yellow croaker. Basal gene transcription was high in several immune organs and could be up-regulated after injection of Poly I:C in the anterior kidney (TLR22), spleen (TLR3 and 22), liver (TLR3) and blood (TLR3) (23). In the common carp (Cyprinus carpio L.), TLR22 was transcripted in TLRs are expressed in innate immune cells such as dendritic cells and macrophages as well as non-immune cells like ﬁbroblast and epithelial cells (10). Citation: Citation: Ji J, Ramos-Vicente D, Navas-Pérez E, Herrera-Úbeda C, Lizcano JM, Garcia-Fernàndez J, Escrivà H, Bayés À and Roher N (2018) Characterization of the TLR Family in Branchiostoma lanceolatum and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus. Front. Immunol. 9:2525. doi: 10.3389/ﬁmmu.2018.02525 November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 1 Ji et al. Toll-Like Receptor in B. lanceolatum teleost-speciﬁc TLR but here we demonstrate that teleosts and amphioxus TLR22-like probably shared a common ancestor. Additional functional studies with other lancelet TLR genes will enrich our understanding of the immune response in amphioxus and will provide a unique perspective on the evolution of the immune system. Keywords: toll-like receptor, TLR, evolution, amphioxus, Poly I:C, TLR22 Frontiers in Immunology \| www.frontiersin.org INTRODUCTION TLR3 is found both in mammals and teleost whereas TLR22 is present in many ﬁsh species and Xenopus, but absent from birds and other terrestrial animals (25). The origin of the TLRs involved in dsRNA virus recognition is still under study. The current hypothesis is that speciﬁc ﬁsh TLR duplication results from the ﬁsh speciﬁc Whole Genome Duplication (WGD) (26–28), but here we show that, in amphioxus, exists an ortholog of the TLR11 subfamily possessing TLR22 functional similarities, pointing out that a TLR22-like function was present in the ancestor of chordates. Amphioxus belongs to an ancient chordate lineage which shares key anatomical and developmental features with vertebrates and tunicates (also known as urochordates) (29). All chordates have a similarly organized genome though amphioxus has relatively little duplication (30). Thus amphioxus, with its phylogenetic position diverging at the base of chordates and its genomic simplicity, is a good non-vertebrate model to understand the evolution of vertebrates (31). Branchiostoma lanceolatum (Mediterranean amphioxus) has been extensively studied together with other amphioxus species such as Branchiostoma belcheri (Asian amphioxus), Branchiostoma japonicum (Asian amphioxus) and Branchiostoma ﬂoridae (Florida amphioxus) (32). To date, genomic data have revealed that B. ﬂoridae has 48 TLRs (33). However, only one full-length TLR, annotated as bbtTLR1, was functionally characterized in B. belcheri tsingtauense until now. The experimental data supports the immunological function of this TLR that together with MyD88 is involved in the activation of NF-κB signaling pathway (34). Further studies of TLRs in amphioxus are required to better understand the ancestors and functional evolution of vertebrate TLRs. To study the phylogenetic relationship of B. lanceolatum and vertebrate TLRs, we performed the maximum-likelihood analysis. Drosophila melanogaster Toll sequences and vertebrate TLR protein sequences were obtained from the National Center for Biotechnology Information (NCBI, https://www. ncbi.nlm.nih.gov/) and UniProt (http://www.uniprot.org/) (Supplementary Table 2). TLR sequences of Lytechinus variegatus (35) and Saccoglossus kowalevskii (36) were obtained from online repositories and a search similar to the one carried on in B. lanceolatum (Supplementary Data 1). In all the phylogenetic analysis, we only included the sequences that have a complete TIR domain. For full-length protein, sequences were aligned with MAFFT (37) choosing L- INS-i method which optimizes alignments for sequences containing hypervariable regions ﬂanked by one alignable domain. For TIR domain, sequences were aligned with MAFFT choosing G-INS-i method which allows to align the entire region with a global conservation. INTRODUCTION The alignment was trimmed using TrimAL (38) with “Automated 1” mode. The phylogenetic reconstruction was done using IQ-TREE (39) and its built-in ModelFinder software (40). Branch support was calculated running 1,000 replicates of the SH-like approximate likelihood ratio test (SH-aLRT) (41) and ultrafast bootstrap (42). In this study, we investigated the total number of TLR genes in B. lanceolatum and studied their phylogenetic and evolutionary relationships with vertebrate and invertebrate TLRs. We also examined the total number of TLR genes in B. ﬂoridae and B. belcheri according to our deﬁnition of a true TLR. We studied the basal gene expression of all the TLRs in adult amphioxus (B. lanceolatum). Moreover, we cloned the full length of a novel TLR in B. lanceolatum and we further investigated its subcellular localization and PAMP binding speciﬁcity using NF-κB luciferase assay in a mammalian expression system. Exhaustive phylogenetic analysis combined with functional data has allowed us to explore the evolution and function of this novel TLR compared with vertebrate TLRs. The TLR sequences of B. ﬂoridae and B. belcheri were obtained from the databases of JGI (http://genome.jgi.doe.gov/Braﬂ1/ Braﬂ1.home.html) and LanceletDB (http://genome.bucm.edu. cn/lancelet/index.php), respectively. The open reading frame was identiﬁed through sequence translation with ExPASy software (http://web.expasy.org/translate/). Transmembrane regions were predicted using TMHMM server v2.0 (http://www.cbs.dtu. dk/services/TMHMM/). The number of LRR domains was INTRODUCTION TLRs are largely divided into two subfamilies based on their subcellular localizations: cell surface or intracellular. Ten and twelve functional TLRs have been identiﬁed in humans and mice, respectively. Human TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed on the cell surface and recognize mainly microbial membrane components such as lipids, lipoproteins and proteins. Human TLR3, TLR7, TLR8, TLR9 and murine TLR11, TLR12, TLR13, which are expressed in intracellular vesicles such as those in the endoplasmic reticulum (ER), endosomes, lysosomes and endolysosomes, and recognize November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 2 Toll-Like Receptor in B. lanceolatum Ji et al. B. lanceolatum derived from several adult tissues and embryonic stages. The transcriptome data were obtained from an exhaustive collection of 52 RNA-Seq datasets using the Illumina technology. From 15 embryonic stages, one pre-metamorphosis stage and 9 adult organs, a total of 4.2 billion Illumina reads with a volume of 871 Gbp were obtained. These embryonic stages are eggs, 32 cells, blastula, 7, 8, 10, 11, 15, 18, 21, 24, 27, 36, 50, and 60 hpf. The adult tissues are neural tube, gut, hepatic tissue, gills, epidermis, muscle, female and male gonads, and cirri. For the transcriptome assembly, Tophat2 was used mapping each strand-speciﬁc RNA-seq sample against the recently assembled B. lanceolatum genome. Gene models were built using Cuﬄinks and each annotation merged using Cuﬀmerge to produce a single collection of transcripts. The transcriptome was translated into predicted proteins using the TransDecoder suite v3.0.1. From the PFAM database v30.0, we downloaded the hidden Markov models proﬁle collection (Pfam-A.hmm.gz) and extracted the two proﬁles for the protein domains that we were looking for, the TIR and the LRR domains. HMMER 3.1b was then used with the hmmsearch mode to identify the predicted proteins with these domains. Finally, a manually curated annotation was performed. Speciﬁc primers for each B. lanceolatum TLR were designed using NCBI primer designing tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/) and are shown in Supplementary Table 1. almost all the tissues. When ﬁsh was challenged with Poly I:C or Aeromonas hydrophila, the transcription of this TLR was up-regulated in a variety of tissues (24). Overall, TLRs with immune function have been found from cnidarians to mammals which imply a conserved evolution. Animals Branchiostoma lanceolatum adults were collected in the bay of Argelès-sur-Mer, France (latitude 42◦32′ 53′′ N and longitude 3◦03′ 27′′ E) with a speciﬁc permission delivered by the Prefect of Region Provence Alpes Côte d’Azur. B. lanceolatum is not a protected species. Amphioxus were kept in the laboratory in 60- l glass tanks with ∼50-l seawater and 5 cm height of sand on the bottom. Water temperature was maintained around 17◦C and the salinity ranged between 40 and 45 PSU. The photoperiod was set to 14 h light/10 h dark. The animals were not fed with extra food during the experiment. TABLE 1 \| Primers used for cloning and RT-qPCR. Category Primer Sequence (5′-3′) Product size (bp) Fragment Forward GGGACGATCCAGTCACGCTG 2,190 Reverse GACACCAACGGCTGCGCAG 5′RACE Reverse1 GAGTGAAGAACAGTGA 684 Reverse2 GTCATTCCCTCCAAGGTTCAAAGAAGTC 3′RACE Forward CGAAGACAGGCGATGGGTT 1,119 Full-length Forward AGAGAGAGAAAACTGCCAGCC 3,077 Reverse TTTCTGTCTCGACGGTCCTT RT-qPCR Forward TCACACGCTTTCTACGGCTT 122 Reverse AGGCTTAGGTCCAGTACGGT GAPDH Forward CCCCACTGGCCAAGGTCATCA 154 Reverse GCTGGGATGATATTCTGGTGGGC Full-Length cDNA Cloning of BLTLR Full-Length cDNA Cloning of BLTLR A DNA BLAST search of NCBI database was conducted using BbtTLR1 sequence from B. belcheri (GenBank: DQ400125.2). We obtained a sequence (GenBank: AF391294.1) from B. ﬂoridae showing 82% identity. In addition, a DNA BLAST search using bbtTLR1 was performed in the genome scaﬀold of B. lanceolatum and we identiﬁed a short sequence (ContigAmph29716) showing 83% identity. The forward primer (Table 1) was designed based on the conserved region between bbtTLR1 B. belcheri and B. ﬂoridae sequence. The reverse primer (Table 1) was designed based on the ContigAmph29716 sequence. We cloned a fragment of around 2,000 bp by PCR using the cDNA prepared from the whole animal. The 5′-end was obtained by 5′ RACE (Invitrogen) using gene speciﬁc primers (Table 1). A fragment of ∼600 bp was obtained. The 3′-end was obtained by 3′ RACE (Invitrogen) using gene speciﬁc primer (Table 1). A fragment of ∼1,000 bp was obtained. Finally, a PCR ampliﬁcation was carried out to obtain the full-length sequence with Expand high ﬁdelity PCR system (Roche) using the full-length primers (Table 1) designed in the non-coding regions from both 5′ to 3′-ends. All the fragments were separated by electrophoresis and cloned into the pGEM-T Easy Vector (Promega). Sequencing was carried out using T7 and SP6 primers (Servei de Genòmica i Bioinformàtica, IBB-UAB). The phylogenetic analysis of three Branchiostoma species (B. ﬂoridae, B. belcheri and B. lanceolatum) TLRs was performed using TIR domain sequences. The TIR domain sequences of vertebrates, S. kowalevskii and D. melanogaster were included. All the TIR domain sequences were identiﬁed from the full- length protein using SMART software. Prior to the analysis, sequences were aligned with MAFFT choosing G-INS-i method. The alignment was trimmed using TrimAL with “Automated 1” mode. The phylogenetic analysis was done using IQ-TREE and its built-in ModelFinder software. Branch support was calculated running 1,000 replicates of the SH-like approximate likelihood ratio test and ultrafast bootstrap. The phylogenetic analysis of BlTLR and BbtTLR1 was performed with the full-length protein using IQ-TREE software. The D. melanogaster Toll and the vertebrate TLR sequences were included in the analysis. The sequences were aligned with MAFFT choosing L-INS-i method. The alignment was trimmed using TrimAL with “Automated 1” mode. In the analysis, branch support was calculated running 1,000 replicates of the SH-like approximate likelihood ratio test and ultrafast bootstrap. November 2018 \| Volume 9 \| Article 2525 Sequence Analysis: Phylogeny and Bioinformatics To characterize the TLR repertoire of B. lanceolatum, we performed a search using the BbtTLR1 sequence (GenBank: DQ400125.2) and an unpublished transcriptome of November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 3 Toll-Like Receptor in B. lanceolatum Ji et al. was puriﬁed using an RNeasy micro kit (Qiagen) and DNAse treated according to manufacturer’s instructions and stored at −80◦C. The ﬁrst-strand cDNA was synthesized with SuperScript III ﬁrst-strand synthesis system (Thermo Fisher Scientiﬁc). RT- PCR reactions were performed with primers speciﬁc for each TLR under following conditions: initial denaturation at 94◦C for 5 min, followed by 35 cycles of denaturation at 94◦C for 45 s, annealing at 60◦C for 45 s, and extension at 72◦C for 50 s, and a ﬁnal extension at 72◦C, 7 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. PCR products were separated in 1% agarose gel electrophoresis and stained with GelGreen Nucleic Acid Gel Stain (Biotium). Agarose gel imaging was performed with a GelDoc XR system (Bio-Rad). Six of the PCR products were puriﬁed and sequenced. predicted using LRRﬁnder software (http://www.lrrﬁnder.com/ lrrﬁnder.php). Full-length protein domain was predicted by the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/). The single cysteine cluster TLRs (sccTLRs) and multiple cysteine cluster TLRs (mccTLRs) were characterized according to Leulier and Lemaitre (15). The ﬁrst annotated sequence was selected according to the blastp software in NCBI. The molecular weight of BlTLR was calculated with ProtParam (http://web.expasy.org/protparam/). The sequence of BlTLR was examined for the presence of a signal peptide using SignalP (http://www.cbs.dtu.dk/services/ SignalP/). N-linked glycosylation site was predicted with NetNGly 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/). Multiple sequence alignment of BlTLR and ﬁsh TLR22 was performed by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/ clustalo/). RNA Isolation, cDNA Synthesis and RT-PCR Total RNA was extracted from the whole animal using TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The homogenization was performed with a Polytron homogenizer (Kinemetica). The quality of the RNA was assessed with a Bioanalyzer (Agilent Technologies) and the concentration was measured with a Nanodrop (Thermo scientiﬁc). The RNA November 2018 \| Volume 9 \| Article 2525 November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 4 Ji et al. Toll-Like Receptor in B. lanceolatum RT-qPCR Analysis RT-qPCR was carried out to analyze the relative transcription level of BlTLR after LPS and Poly I:C treatments. The analysis was performed in the CFX384 Touch Real-Time PCR Detection System (Bio-Rad) using the iTaq universal SYBR green supermix kit (Bio-Rad) following the manufacturer’s protocol. The RT- qPCR primers (Table 1) were designed to detect the transcription level of BlTLR. GAPDH gene was used as a reference gene. 10−1 and 10−2-fold cDNA dilutions were used for BlTLR and GAPDH gene expression analysis, respectively. Each PCR mixture consisted of 5 µl of SYBR green supermix, 0.5 µM of primers, 2.5 µl of diluted cDNA, and 1.5 µl sigma water in a ﬁnal volume of 10 µl. All samples were run in triplicate using the following steps: initial denaturation at 95◦C for 3 min, 39 cycles of 95◦C for 10 s and 60◦C for 30 s, and ﬁnally, 95◦C for 10 s, increase every 0.5◦C for 5 s from 65 to 95◦C. The relative transcription levels were calculated using the 2−11CT method (43). All the data were analyzed using GraphPad software and signiﬁcant diﬀerences were analyzed by one-way analysis of variance (ANOVA) using the value of 1Ct (normalize each technical repeat’s gene-speciﬁc Ct value by subtracting from it the reference gene Ct value) (44). Cell Culture, Transient Transfection and Stable Cell Lines Cell Culture, Transient Transfection and Stable Cell Lines LPS and Poly I:C Treatment in vivo Adult amphioxi were treated with either 10 µg/ml bacterial lipopolysaccharide (LPS) from Escherichia coli O111:B4 strain (Sigma-Aldrich) or 10 µg/ml Poly I:C, a synthetic analog of dsRNA viruses (Invivogen) by bath immersion. The stocks of LPS and Poly I:C solution were prepared in PBS (Sigma-Aldrich) and diluted to the indicated working concentrations with sterile seawater. Seawater sterilization was performed with 0.22 µm sterile ﬁlter. PBS prepared in seawater (1% v/v) was used as a control. Three, 6, 12 and 24 h after immersion, 3 animals from each group were sampled separately. The animals were frozen in liquid nitrogen immediately and stored in −80◦Cuntil use. Total RNA was prepared from the whole animal and the ﬁrst-strand cDNA was synthesized for RT-qPCR analysis. HEK293 cells were grown in complete medium: DMEM (Life Technologies, 31885) supplemented with 10% (v/v) FBS (Gibco) and 1% (v/v) penicillin and streptomycin (Gibco) at 37◦C and 5% CO2. Plasmids were transiently transfected in HEK293 cells using linear polyethylenimine (PEI, CliniScience) at a ratio of 3:1 (µg PEI: µg plasmid). HEK293 cell lines stably expressing BlTLRHA and chimeric BlTLR were generated by Geneticin selection (Invitrogen, G418). In brief, 24 h after transient transfection, the culture medium was substituted with selective medium containing 1 mg/ml G418. Selective medium was refreshed every 2–3 days until the G418-resistant foci could be identiﬁed and all non-transfected cells (control) were dead (around 2 weeks). The colonies were picked and expanded in selective culture medium containing 1 mg/ml G418 for the following 2 weeks. Then, HEK293 stable cell lines were isolated via GFP-positive cell sorting (FACSJazz) in order to enrich the stable cell line. Finally, the HEK293 stable cells lines were cultured in DMEM complete medium at 37◦C and 5% CO2. Flow Cytometry y y To assess the transient transfection eﬃciency of plasmid BlTLRHA in HEK293 cells, ﬂow cytometry was performed using a FACS Canto (Becton Dickinson, USA). In brief, HEK293 cells were seeded on 6-wells plate (Thermo Scientiﬁc) at 50% density. The cells were transfected with empty vector (pIRES2-EGFP) and BlTLRHA plasmid using PEI as described above. Non-transfected cells were used as negative control. Cells were detached using TrypLE (Gibco) and re-suspended in PBS for cytometry analysis at 24, 48 and 72 h after transfection. The cytometer was set to detect the GFP signal and a total 10,000 events were recorded. The raw data were analyzed with Flowing software (Finland) and GraphPad software. Flow cytometry was also used to assess the percentage of transfected cells when setting up the stable cells lines BlTLRHA and chimeric BlTLR. Frontiers in Immunology \| www.frontiersin.org RESULTS The TLR Family in B. lanceolatum A search for TIR and LRR domains was performed and proteins with both domains were selected as candidates. Then, these candidates were manually curated and a list of putative TLRs was obtained (Supplementary Data 2). Despite there are TLR-related molecules lacking extracellular LRR domains reported in some species of Hydra and coral (15), we only considered those sequences with at least one LRR domain, one TM domain and one TIR domain to obtain our ﬁnal list of true TLR candidates. Using this rule, we obtained 30 TLRs. In order to understand the evolution of TLR of B. lanceolatum, we performed a phylogenetic analysis with representative vertebrate and invertebrate TLR sequences. Other authors had used either the full-length protein or the TIR domain to study the TLR evolution (32, 46–48). Therefore, we used full-length protein to perform the phylogenetic analysis when the sequences were complete, or TIR domain when there were incomplete or truncated sequences. The phylogenetic analysis of B. ﬂoridae, B. belcheri and B. lanceolatum using TIR domain sequences showed that there are two major clusters of TLRs (mccTLRs and sccTLRs) in Branchiostoma. However, we obtained a single clade with almost all the Branchiostoma sequences, clustered with vertebrate TLR3, 5 and 7 families (Supplementary Figure 1). This approach did not allow the identiﬁcation of inter-taxa relationships between vertebrate and Branchiostoma TLR families. Roach et al. predicted that a strong selective pressure for speciﬁc PAMPs recognition maintains a largely unchanged repertoire of TLR recognition in vertebrates (16). Thus, we did phylogenetic analysis using the highly reﬁned full-length TLR sequences of B. lanceolatum to better understand the evolutionary relationships with vertebrate TLRs. The phylogenetic analysis showed that the vertebrate TLRs were grouped into six clusters (TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11 families) with high branch support within their own clusters conﬁrming the reliability of the tree (Figure 1 and Supplementary Figure 2). Twenty B. lanceolatum sequences formed a strongly supported clade distinct from the mccTLR sequences and grouped with the TLR11 family. One TLR (Bl19922) is not clustered with any TLRs, probably because it is an N-terminal truncated sequence. Moreover, six B. lanceolatum TLRs, which were identiﬁed as mccTLR (invertebrate type) were clustered separately from the main vertebrate branch (Figure 1 and Supplementary Table 4). Plasmids HEK293 cells were transiently transfected with empty vector (pIRES2-EGFP) and BlTLRHA plasmid as described above. Cells were lysed in 200 µl cell lysis buﬀer (250 mM sacarose, 150 mM Tris, 5 mM EDTA, 125 mM DTT, 5% SDS, 2.5% bromophenol blue and 7.5% β-mercaptoethanol in water) and detached on ice using a cell scraper (BD Falcon) at 24, 48 and 72 h after transfection. The lysed cells were subjected to sonication for 10 s and centrifugation. After heating at 100◦C for 5 min, the cell extracts were loaded into 10% SDS-PAGE and then transferred to PVDF membranes (EMD Millipore) using a Mini-protean Tetra (Bio-Rad). After 1 h blocking in 5% (w/v) BSA (Sigma-Aldrich) in TBST (50 mM Tris, 150 mM NaCl and 0.1% Tween 20), membranes were incubated with 1 µg/µl mouse anti-HA primary antibody (Covance, MMS-101P) overnight at 4◦C, followed by incubation with a secondary HRP-conjugated antibody for 1 h at room temperature (RT). Proteins were visualized with a GelDoc system (Bio-Rad) by adding the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientiﬁc). To study the subcellular localization of BlTLR in HEK293 cell, the coding sequence was cloned into pIRES2-EGFP vector (BD Biosciences Clontech, 6029-1) with two HA-tags (YPYDVPDYA) at 3′ end (named BlTLRHA) using XhoI and EcoRI as restriction sites. For testing the speciﬁc ligand binding of BlTLR, the ectodomain and transmembrane domain (amino acids 1-774) of BlTLR fused with human TLR2 cytoplasmic region (amino acids 611-784; NCBI: NP_001305716.1) was cloned into pIRES2- EGFP vector (named chimeric BlTLR) between SacII and EcoRI restriction sites. The eukaryotic expression vector pIRES2-EGFP was purchased from BD Biosciences. The NF-κB-dependent luciferase reporter vector (pNFκB) and the Renilla luciferase vector (pRenilla) were provided by Dr. José Miguel Lizcano. All the plasmids were conﬁrmed by sequencing and agarose gel electrophoresis digested with the corresponding restriction enzymes. All the plasmids were puriﬁed at large scale using NucleoBond Maxi endotoxin-free plasmid isolation kit (Fisher Scientiﬁc) and stored at −20◦C until use. November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 5 Toll-Like Receptor in B. lanceolatum Ji et al. Immunoﬂuorescence and Confocal Microscopy 100 µl of luciferase assay reagent II (LAR II). After quantifying the ﬁreﬂy luminescence, the reaction was quenched. The Renilla luciferase reaction was initiated by adding 100 µl Stop & Glo Reagent to the same well and the Renilla luminescent signal was detected. The luciferase activity was expressed as the ratio of NF-κB-dependent ﬁreﬂy luciferase activity divided by Renilla luciferase activity. py HEK293 cells were seeded (50% density) on 24 × 24 mm cover glasses (Labbox) coated with Poly-D-lysine hydrobromide (Sigma-Aldrich). The BlTLRHA plasmid was transiently transfected as described above. Cells were washed 3 times with DMEM at 48 h after transfection. For non-permeabilization, cells were blocked with 2% BSA in DMEM for 10 min at 37◦C, and then incubated with mouse anti-HA primary antibody (1/500 diluted in DMEM) for 1 h at 37◦C. Cells were washed 3 times with DMEM and ﬁxed with 4% paraformaldehyde (PFA, Sigma- Aldrich) for 15 min at RT. After PBS washing, for transient transfection, ﬁxed cells were incubated with anti-mouse Alexa Fluor 555 secondary antibody (Invitrogen) at 1:1,000 dilution for 2 h at RT; for stable transfection, cells were incubated with 5 µg/ml wheat germ agglutinin (WGA) conjugated with Alexa Fluor 647 for 10 min at RT before applying the secondary antibody at 1:1,000 dilution for 2 h at RT. For permeabilization, cells were washed with DMEM for 3 times and ﬁxed with 4% PFA for 15 min at RT. After 3 washes with PBS, for transient transfection, cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 15 min at RT; for stable transfection, cells were incubated with 5 µg/ml WGA for 10 min at RT and then permeabilized with 0.1% Tween (Sigma-Aldrich) for 10 min at RT or the freeze and thaw method according to Mardones and González (45). After that, cells were blocked with 2% BSA in PBS for 1 h at RT, incubated with mouse anti-HA primary antibody (1/1,000 dilution) overnight at 4◦C, followed by incubation with secondary anti-mouse AlexaFluor 555 antibody (Invitrogen) at 1:1,000 dilution for 2 h at RT. For both methods, cover glasses with cells were placed on SuperFrost Plus slides (Thermo scientiﬁc) covered with Fluoroshield with DAPI mounting medium (Sigma-Aldrich). Confocal imaging was performed using a Leica SP5 confocal microscope with a 63 × oil objective. The images were analyzed with Fiji software (46). Frontiers in Immunology \| www.frontiersin.org Ligand Stimulation and NF-κB Luciferase Reporter Assay variegatus, representative vertebrates and D. melanogaster Toll were used. Three TLR sequences (Bl10262, Bl22164 and Bl08928c) with incomplete TIR domain were removed from the analysis. Sequences were aligned with MAFFT choosing L-INS-i method and the alignments were trimmed using TrimAL with “Automated 1” mode. The best evolutionary model was established by ModelFinder according to BIC. The branch labels (numbers) are SH-aLRT support (%)/ultrafast bootstrap support (%) at the tree nodes. The tree was generated in FigTree. Dm Toll, Bl mccTLRs, Bl sccTLRs, Sk TLR, Lv TLR and 6 vertebrate TLR families (highlighted in different colors) are shown. TLR22 clade is shown with a red background. The detailed tree with all node supports can be found in Supplementary Figure 2. FIGURE 1 \| Phylogenetic analysis of B. lanceolatum TLRs. The phylogenetic tree was constructed using maximum-likelihood (IQ-TREE) with the full-length protein sequences. TLR sequences of B. lanceolatum, S. kowalevskii, L. variegatus, representative vertebrates and D. melanogaster Toll were used. Three TLR sequences (Bl10262, Bl22164 and Bl08928c) with incomplete TIR domain were removed from the analysis. Sequences were aligned with MAFFT choosing L-INS-i method and the alignments were trimmed using TrimAL with “Automated 1” mode. The best evolutionary model was established by ModelFinder according to BIC. The branch labels (numbers) are SH-aLRT support (%)/ultrafast bootstrap support (%) at the tree nodes. The tree was generated in FigTree. Dm Toll, Bl mccTLRs, Bl sccTLRs, Sk TLR, Lv TLR and 6 vertebrate TLR families (highlighted in different colors) are shown. TLR22 clade is shown with a red background. The detailed tree with all node supports can be found in Supplementary Figure 2. Ligand Stimulation and NF-κB Luciferase Reporter Assay p y Human TLR1-9 agonist kit (tlrl-kit1hw) and murine TLR13 agonist (tlrl-orn19) were purchased from Invivogen. HEK293 stable cell lines were used to minimize the deviation among diﬀerent experiments. The stable cell lines were transfected with 0.5 µg/ml pNFκB and 0.05 µg/ml pRenilla (0.5 ml per well) using PEI. Renilla was used as internal control to normalize the diﬀerences in the reporter due to diﬀerent transfection eﬃciencies. Twenty-four hours after transfection, cells were treated with indicated concentrations of ligands (Supplementary Table 3) for 16 h. As a positive control, 20 ng/ml human TNFα (Sigma-Aldrich) was used. The experiment was performed in triplicate. Luciferase activity assay was performed with the Dual-luciferase reporter assay system (Promega) using the Victor3 (PerkinElmer) according to the manufacturer’s instructions. Brieﬂy, after removing the growth medium from the well, cells were washed with PBS (2X). One hundred µl of passive lysis buﬀer (PLB) were added to each well. Then, the NF- κB-dependent ﬁreﬂy luciferase reporter was measured by adding The transcription of the 30 TLRs in B. lanceolatum was conﬁrmed by RT-PCR analysis in adult animals. Each primer pair was designed based on the nucleotide sequences reconstructed from transcript sequences of B. lanceolatum. We found gene November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 6 Ji et al. Toll-Like Receptor in B. lanceolatum FIGURE 1 \| Phylogenetic analysis of B. lanceolatum TLRs. The phylogenetic tree was constructed using maximum-likelihood (IQ-TREE) with the full-length protein sequences. TLR sequences of B. lanceolatum, S. kowalevskii, L. variegatus, representative vertebrates and D. melanogaster Toll were used. Three TLR sequences (Bl10262, Bl22164 and Bl08928c) with incomplete TIR domain were removed from the analysis. Sequences were aligned with MAFFT choosing L-INS-i method and the alignments were trimmed using TrimAL with “Automated 1” mode. The best evolutionary model was established by ModelFinder according to BIC. The branch labels (numbers) are SH-aLRT support (%)/ultrafast bootstrap support (%) at the tree nodes. The tree was generated in FigTree. Dm Toll, Bl mccTLRs, Bl sccTLRs, Sk TLR, Lv TLR and 6 vertebrate TLR families (highlighted in different colors) are shown. TLR22 clade is shown with a red background. The detailed tree with all node supports can be found in Supplementary Figure 2. FIGURE 1 \| Phylogenetic analysis of B. lanceolatum TLRs. The phylogenetic tree was constructed using maximum-likelihood (IQ-TREE) with the full-length protein sequences. TLR sequences of B. lanceolatum, S. kowalevskii, L. Identiﬁcation and Characterization of a Novel BlTLR transcription in basal conditions for all the 30 TLRs. The TLRs with gene ID of BlTLR, Bl48785, Bl18798b, Bl08928b and Bl30396 showed a weak transcription while others were strongly expressed (Figure 2). Five of the genes were sequenced using speciﬁc primers conﬁrming the identity of these genes (data not shown). We focused on the amphioxus TLR11 family described in section The TLR Family in B. lanceolatum and speciﬁcally in a B. lanceolatum TLR sequence (BlTLR) because it was highly similar to the published bbtTLR1 (GenBank: DQ400125.2). This B. belcheri gene was annotated as TLR1 based on phylogenetic and functional data (34). Nonetheless, our phylogenetic analysis pointed out that BlTLR was a clear TLR11 family member. TLR11 family includes several teleost speciﬁc members (e.g., TLR19 or TLR22) that are not present in mammalian genomes and it is of great interest to know whether they are present in a more basal organism. To begin, we cloned the full-length of this novel BlTLR (GenBank: MG437061) and its 5′ and 3′- UTRs were obtained based on three orthologous found in the Branchiostoma genus. The length of the novel BlTLR cDNA is 3,772 bp, containing a 227 bp long 5′UTR, a 2,913 bp ORF (which encodes a putative 970 amino acid-long protein), and a 616 bp long 3′UTR with a putative polyadenylation signal (AATAAA) 17 nucleotides upstream of the poly(A) tail (Supplementary Figure 3). SMART domain analysis predicted To better understand the Branchiostoma TLR evolution, we compared the domain structure of B. lanceolatum, B. belcheri and B. ﬂoridae. Therefore, we identiﬁed a total number of 30 TLRs in B. lanceolatum, 22 TLRs in B. ﬂoridae and 37 TLRs in B. belcheri (Supplementary Tables 4–6) according to the common TLR pattern. We also discriminated sccTLR and mccTLR in these three species according to the domain structure and phylogenetic analysis (Supplementary Figure 1). There are 3 mccTLRs in B. ﬂoridae, 5 mccTLRs in B. belcheri and 6 mccTLRs in B. lanceolatum. In addition, the mccTLRs found in the three Branchiostoma species consistently blast with invertebrate type TLRs (Supplementary Tables 4–6). We also studied the number of LRR from each TLR using LRRﬁnder software. The results showed that the LRR number of TLRs in the three species ranges from 1 to 25. November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 7 Ji et al. Toll-Like Receptor in B. FIGURE 3 \| Expression of BlTLR gene after LPS or Poly I:C treatment. Animals previously immersed in 10 µg/ml LPS or 10 µg/ml Poly I:C, were collected at 3, 6, 12 and 24 h. The untreated animals were used as a control and assigned a value of 1 in the histogram. GAPDH was used as a reference gene. The bars indicate mean expression of 3 individual animals ± S.D. Signiﬁcant differences of mean values were analyzed according to one-way ANOVA followed by Tukey’s test. Expression Analysis of BLTLR After LPS and Poly I:C Treatment or Poly I:C-treated groups, indicating that 10 µg/ml LPS or Poly I:C administered by immersion within this time frame could not signiﬁcantly induce up- or down-regulation of the BlTLR gene in adult amphioxus. y We performed RT-qPCR to investigate the expression proﬁle of the BlTLR in response to PAMP administration. This approach is often used to identify which family a putative TLR belongs to. Two representative PAMPs of bacterial and viral infection (LPS and Poly I:C, respectively) were used to challenge amphioxus in vivo. Amphioxi were immersed in 10 µg/ml LPS or 10 µg/ml Poly I:C to mimic the natural infection route. The gene transcription of BlTLR was analyzed by RT-qPCR in a time course at 3, 6, 12 and 24 h post-immersion (Figure 3). However, no signiﬁcant diﬀerences in gene expression were observed in any of the LPS Identiﬁcation and Characterization of a Novel BlTLR lanceolatum FIGURE 2 \| Expression of TLR genes in B. lanceolatum. The cDNA used in all ampliﬁcations was prepared from whole animals. RT-PCR reactions were accomplished using equal number of cycles, the PCR products were loaded equally on two 1% agarose gels and GAPDH was used as a reference gene. Images were taken with the same exposure time using a Geldoc. FIGURE 2 \| Expression of TLR genes in B. lanceolatum. The cDNA used in all ampliﬁcations was prepared from whole animals. RT-PCR reactions were accomplished using equal number of cycles, the PCR products were loaded equally on two 1% agarose gels and GAPDH was used as a reference gene. Images were taken with the same exposure time using a Geldoc. that the BlTLR protein has the following domains: a C-terminal TIR domain (from residue 800 to 947), a transmembrane (TM) domain (from residue 752 to 774), a N-terminal signal peptide (ﬁrst 27 residues), 21 tandem extracellular leucine-rich repeats (LRRs), a leucine rich repeat C-terminal domain (LRRCT) and a LRR N-terminal domain (LRRNT). The domain diagram of BlTLR was made with IBS software (49) and shown in Supplementary Figure 4. The LRRs are ﬂanked by one LRRCT and one LRRNT domain. The BlTLR has only one LRRCT like most of the TLRs found in deuterostomes (sccTLRs). The highly conserved consensus sequence (LxxLxLxxNxL) of each LRR was identiﬁed with the LRRﬁnder (Supplementary Figures 3, 4). Ten potential N-linked glycosylation sites were predicted by NetNGly 1.0: N101-N114-N154-N163-N276-N375-N393-N522-N573-N632. The deduced molecular weight of BlTLR protein is 111.3 kDa and the full-length protein showed 78.8% identity with the bbtTLR1 of B. belcheri. Three conserved boxes were identiﬁed in TIR domain of BlTLR (Supplementary Figure 3). Box 1 and 2 are involved in binding downstream signaling molecules while box 3 is involved in the localization of the receptor through interactions with cytoskeletal elements (50). Importantly, a key residue in box 2 (Proline 681 in human TLR2 sequence) involved in MyD88 signaling was substituted by Ala in the BlTLR sequence (51). Frontiers in Immunology \| www.frontiersin.org deduced molecular weight of BlTLR protein is 111.3 kDa and the full-length protein showed 78.8% identity with the bbtTLR1 of B. belcheri. Three conserved boxes were identiﬁed in TIR domain of BlTLR (Supplementary Figure 3). Box 1 and 2 are involved in binding downstream signaling molecules while box 3 is involved in the localization of the receptor through interactions with cytoskeletal elements (50). Importantly, a key residue in box 2 (Proline 681 in human TLR2 sequence) involved in MyD88 signaling was substituted by Ala in the BlTLR sequence (51). Subcellular Localization of BlTLR in HEK293 Cells In order to conﬁrm that the up-regulation of luciferase activity is due to the Poly I:C recognition by the chimeric BlTLR but not by endogenous TLRs, we performed the luciferase assay using chimeric BlTLR stable cells and HEK293 cells without chimeric BlTLR. The NF-κB luciferase activity was up-regulated in chimeric BlTLR stable cells with respect to HEK293 cells treated with Poly I:C (LMW and HMW; Figure 6). To explore the subcellular localization of BlTLR, we overexpressed the HA-tagged BlTLR in HEK293 cells and we visualized the localization using immunoﬂuorescence and confocal microscopy. We did not observe the HA-tagged BlTLR in both transient and stable transfected cells when the cells were not permeabilized (Figures 5B,D). Non-transfected cells were used as a control (Figure 5A). This result indicates that, ﬁrst, BlTLR might be an intracellular protein; second, BlTLR might localize on the plasma membrane but could not be detected in non-permeabilized cells due to the HA-tag location at the C- terminal. To further understand the localization of BlTLR, we performed the assay with a plasma membrane marker (WGA) and diﬀerent permeabilization methods. Interestingly, when the cells were permeabilized using diﬀerent permeabilization methods (from weak to strong), we found that BlTLR was mainly localized on the plasma membrane in both transient and stable transfected cells (Figures 5C,E,F). Our results showed that the novel BlTLR localized at the plasma membrane and responded to Poly I:C. These characteristics are only compatible with TLR22, thus we postulated that the novel receptor is a TLR22-like receptor. The alignment of BlTLR with 12 teleost TLR22 sequences showed that BlTLR had 27.8–30.8% of identity with ﬁsh TLR22 (Supplementary Table 7). Subcellular Localization of BlTLR in HEK293 Cells We used HEK293 cells because these cells could be eﬃciently transfected and they have been extensively used for the study of TLR subcellular localization. Cells were transiently transfected with empty vector and the vector expressing the full-length BlTLR. Flow cytometry analysis showed that cells November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org 8 Toll-Like Receptor in B. lanceolatum Ji et al. were successfully transfected at 24, 48 and 72 h and the transfection eﬃciency at 48 and 72 h (both around 60%) was higher than at 24 h (around 30%) post-transfection (Figure 4A). Western blot analysis conﬁrmed that the BlTLR protein was properly expressed in HEK293 cells, and it was not degraded by intracellular proteases. The BlTLR protein was detected at 24, 48 and 72 h post-transfection (Figure 4B). The transcription levels were much higher at 48 and 72 h than at 24 h which agrees with the cytometry results. The molecular weight of BlTLR protein was around 135 kDa which is slightly bigger than the theoretical one (111.27 kDa). This may be due to post-translational modiﬁcations such as glycosylation, phosphorylation, ubiquitination, ubiquitin-like modiﬁcations or S-nitrosylation among others. NF-κB reporter assay allows functional discrimination between TLRs. To shed light on the role of novel BlTLR in PAMPs recognition, a HEK293 cell line stably expressing BlTLR was generated. However, the BlTLR stable cells could not activate the NF-κB promoter stimulated by any of the tested PAMPs (data not shown). To further study the receptor activity, we design a chimeric receptor fusing the ectodomain of BlTLR with the TIR domain of human TLR2 and we generated a stable cell line. This approach has been used before to ensure a correct downstream signaling avoiding the diﬀerences in the set of adaptors and accessory proteins between vertebrates and non- vertebrates (34, 52). The chimeric BlTLR stable cells responded to Poly I:C (LMW and HMW) which usually binds to TLR3 or TLR22. Conversely, other ligands, including Pam2CSK4 for TLR1/2, HKLM for TLR2, LPS for TLR4, ﬂagellin for TLR5, FSL- 1 for TLR2/6, imiquimod for TLR7, ssRNA for TLR8, ODN2006 for TLR9, ORN Sa19 for TLR13 (mouse) failed to induce NF-κB transactivation (Figure 6). Human recombinant TNFα was used as a positive control since it is a well-known NF-κB activator. BlTLR Could Respond to Poly I:C in HEK293 Cells To further explore the phylogenetic relationship of BlTLR, BbtTLR1 and vertebrate TLRs, phylogenetic trees were constructed based on full-length protein sequence using maximum-likelihood analysis (Supplementary Figure 5). As Mammalian TLRs can transactivate the transcription factor NF- κB in response to ligand binding. Usually, each TLR has a restricted PAMPs preference (Supplementary Table 3) and the FIGURE 4 \| BlTLR expression in HEK293 cells. (A) HEK293 cells transfected with an empty vector (pIRES2-EGFP) and a vector expressing BlTLR were analyzed at 24, 48 and 72 h post-transfection by ﬂow cytometry. Non-transfected cells (NC) were used as a control. Transfection efﬁciency was evaluated as the percentage of GFP positive cells. (B) Non-transfected cells, cells transfected with the empty vector and the vector expressing BlTLR with HA tag were analyzed at 24, 48 and 72 h post-transfection by western blot. Protein molecular weight standards (Niborlab) are shown on the right side. FIGURE 4 \| BlTLR expression in HEK293 cells. (A) HEK293 cells transfected with an empty vector (pIRES2-EGFP) and a vector expressing BlTLR were analyzed at 24, 48 and 72 h post-transfection by ﬂow cytometry. Non-transfected cells (NC) were used as a control. Transfection efﬁciency was evaluated as the percentage of GFP positive cells. (B) Non-transfected cells, cells transfected with the empty vector and the vector expressing BlTLR with HA tag were analyzed at 24, 48 and 72 h post-transfection by western blot. Protein molecular weight standards (Niborlab) are shown on the right side. November 2018 \| Volume 9 \| Article 2525 9 Frontiers in Immunology \| www.frontiersin.org Ji et al. Toll-Like Receptor in B. lanceolatum GURE 5 \| Subcellular localization of BlTLR in HEK293 cells. Confocal images showing HEK293 cells transiently transfected (A–C) or stably transfected (D–F) TLR. (A) Not transfected cells; (B) Cells transfected with BlTLR and non-permeabilized; (C) Cells transfected with BlTLR and permeabilized with 0.2% Triton X BlTLR stable cells not permeabilized; (E) BlTLR stable cells permeabilized using freeze and thaw protocol; (F) BlTLR stable cells permeabilized with 0.1% een-20. Nuclei are stained with DAPI (in blue). Transfected cells were GFP labeled (in green). BlTLR was detected with anti-HA antibody and AF555-conjugat FIGURE 5 \| Subcellular localization of BlTLR in HEK293 cells. Confocal images showing HEK293 cells transiently transfected (A–C) or stably transfected (D–F) with BlTLR. (A) Not transfected cells; (B) Cells transfected with BlTLR and non-permeabilized; (C) Cells transfected with BlTLR and permeabilized with 0.2% Triton X-100. BlTLR Could Respond to Poly I:C in HEK293 Cells Non-treated cells (NC) and cells treated with human TNFα (20 ng/ml) were used as negative and positive controls, respectively. The luciferase activity was expressed as the ratio of NF-κB-dependent ﬁreﬂy luciferase activity divided by Renilla luciferase activity. Bars represented mean ± S.D. Signiﬁcant differences of mean values were analyzed according to one-way ANOVA followed by Tukey’s test. P < 0.05; P < 0.01; *P < 0.0001. expected, D. melanogaster Tolls clustered independently and all the vertebrate TLRs clustered into six clades. Furthermore, BlTLR clustered in the same TLR11 family clade together with BbtTLR1. The SH-aLRT support (%) and ultrafast bootstrap support (%) are 94.6 and 94 (Supplementary Figure 5). This result indicates that BlTLR is very likely to be a member of TLR11 family and could be identiﬁed as TLR11, 12, 13, 19, 20, 21 22, or 23. Overall, all the results strongly support the identiﬁcation of the novel receptor that carries the TLR22 function (BlTLR22-like). 8 and 20 (16); but in cephalochordates, B. ﬂoridae has an expansion of 48 TLRs according to Huang et al. (33). This expansion of TLRs in invertebrate deuterostomes remains to be understood by a comprehensive and thorough study of TLRs evolution. Amphioxus is a good model to study the invertebrate- chordate to vertebrate transition and the evolution of vertebrates. Therefore, studying TLR functions in such organism could improve our understanding of the ancestral innate immune system of vertebrates. In this study, we identiﬁed 30 TLRs in B. lanceolatum, 22 TLRs in B. ﬂoridae and 37 TLRs in B. belcheri according to the basic TLR structure: “LRR+TM+TIR.” Diﬀerences in the total number of B. ﬂoridae TLRs between Huang et al. and our data probably reﬂects discrepancies in the consensus of what is the basic structure of TLRs. Our stringent rule includes only those putative receptors with a TIR domain, a transmembrane domain and at least one LRR domain, known as true TLRs (15). Our available transcriptomic data maybe do not include all the possible TLRs. Probably the total number of TLRs in the 3 species of lancelet should be similar. Among them, we identiﬁed 6 mccTLRs in B. lanceolatum, 3 mccTLRs in B. ﬂoridae and 5 mccTLRs in B. belcheri. This ﬁnding is diﬀerent from the observation by Huang et al. BlTLR Could Respond to Poly I:C in HEK293 Cells concerning amphioxus TLR family: it has a high rate of domain combination acquisition and therefore a high number of TLRs (prediction of 36 sccTLRs and 12 mccTLRs) (33). Importantly, Bányai and Patthy provided evidence to dispute that the rate of protein innovation is exceptionally high in lancelets. They surmised these high rates are BlTLR Could Respond to Poly I:C in HEK293 Cells (D) BlTLR stable cells not permeabilized; (E) BlTLR stable cells permeabilized using freeze and thaw protocol; (F) BlTLR stable cells permeabilized with 0.1% Tween-20. Nuclei are stained with DAPI (in blue). Transfected cells were GFP labeled (in green). BlTLR was detected with anti-HA antibody and AF555-conjugated anti-mouse IgG (in red). Plasma membrane was stained with WGA AF647 conjugated (in purple). Figures were analyzed with Fiji software. FIGURE 5 \| Subcellular localization of BlTLR in HEK293 cells. Confocal images showing HEK293 cells transiently transfected (A–C) or stably transfected (D–F) with BlTLR (A) Not transfected cells; (B) Cells transfected with BlTLR and non permeabilized; (C) Cells transfected with BlTLR and permeabilized with 0 2% Triton X 10 FIGURE 5 \| Subcellular localization of BlTLR in HEK293 cells. Confocal images showing HEK293 cells transiently transfected (A–C) or stably transfected (D–F) with BlTLR. (A) Not transfected cells; (B) Cells transfected with BlTLR and non-permeabilized; (C) Cells transfected with BlTLR and permeabilized with 0.2% Triton X-100. (D) BlTLR stable cells not permeabilized; (E) BlTLR stable cells permeabilized using freeze and thaw protocol; (F) BlTLR stable cells permeabilized with 0.1% Tween-20. Nuclei are stained with DAPI (in blue). Transfected cells were GFP labeled (in green). BlTLR was detected with anti-HA antibody and AF555-conjugated anti-mouse IgG (in red). Plasma membrane was stained with WGA AF647 conjugated (in purple). Figures were analyzed with Fiji software. November 2018 \| Volume 9 \| Article 2525 10 Frontiers in Immunology \| www.frontiersin.org Ji et al. Toll-Like Receptor in B. lanceolatum FIGURE 6 \| HEK293 cells expressing chimeric BlTLR induce the activation of NF-κB in response to Poly I:C. HEK293 chimeric BlTLR stable cells were treated with 11 different ligands (gray columns). Non-transfected HEK293 cells were treated with ﬁve potential ligands (black columns). Non-treated cells (NC) and cells treated with human TNFα (20 ng/ml) were used as negative and positive controls, respectively. The luciferase activity was expressed as the ratio of NF-κB-dependent ﬁreﬂy luciferase activity divided by Renilla luciferase activity. Bars represented mean ± S.D. Signiﬁcant differences of mean values were analyzed according to one-way ANOVA followed by Tukey’s test. P < 0.05; P < 0.01; **P < 0.0001. FIGURE 6 \| HEK293 cells expressing chimeric BlTLR induce the activation of NF-κB in response to Poly I:C. HEK293 chimeric BlTLR stable cells were treated with 11 different ligands (gray columns). Non-transfected HEK293 cells were treated with ﬁve potential ligands (black columns). Frontiers in Immunology \| www.frontiersin.org DISCUSSION p y In mammals, TLRs can be divided into two main groups according to localization: on the cell surface or in intracellular compartments (60). Among human TLRs, the ones located at the plasma membrane (TLR1, 2, 4, 5 and 6) recognize microbial pathogenic components of the cell wall, while the others (TLR3, 7, 8 and 9) located intracellularly in endosomes or lysosome recognizing nucleic acids (4). However, the above ligand recognition pattern in non-mammalian organisms may be not always as in mammals. For instance, mouse TLR13 recognizes a conserved 23S ribosomal RNA (rRNA) from bacteria in the endolysosomal compartment (11). In teleost, TLR13 of M. croaker could respond to Poly I:C both in vivo and in vitro and is localized in the cytoplasm of HeLa cells (19). Fugu TLR22 recognizes long-sized dsRNA on the cell surface whereas TLR3 resides in the endoplasmic reticulum and recognizes relatively short-sized dsRNA (20). TLR22 of grass carp (C. idella) recognizes Poly I:C stimulation in CIK (C. idella kidney) cell line and is localized on the cell membrane (21). In our study, immunoﬂuorescence and confocal microscopy showed that BlTLR22-like is mainly localized on the plasma membrane. In mammals, TLRs can recognize speciﬁc PAMPs with high levels of sensitivity (61). To test B1TLR22-like ligand speciﬁcity, we performed diﬀerent assays with commercially available mammalian TLRs ligands, using NF-κB activity as a reporter. We could not observe signiﬁcant diﬀerences of NF- κB activation in HEK293 cells expressing BlTLR22-like. There are diﬀerent possible explanations but apart from problems with protein expression levels, intracellular degradation or incorrect The phylogenetic analysis of BlTLR22-like protein sequence and representative vertebrate TLR protein sequences revealed that BlTLR22-like clusters with the vertebrate TLR11 family. Interestingly, the phylogenetic analysis of B. ﬂoridae TIR domain and vertebrate TLRs has indicated that 33 variable-type TLRs show a paraphyletic relationship with the vertebrate TLR11 lineage (33). The TLR11 family is represented in humans only by a pseudogene and the major divisions of the TLR11 family are clearly very ancient (16). Moreover, BlTLR22-like has a single domain structure of the ectodomain which should be classiﬁed into TLR13 subfamily (Supplementary Table 8) according to the ectodomain architecture analysis of vertebrate TLRs (17). DISCUSSION TLRs play crucial roles in the innate immune system by recognizing PAMPs from pathogens in vertebrates. In addition, TLRs have multiple functions ranging from developmental signaling to cell adhesion in protostomes (48). The study of TLRs may help to understand the role of TLR-mediated responses which could increase our range of strategies to treat infectious diseases and manipulate immune responses by drug intervention (53). From the evolutionary point of view, TLRs are conserved across invertebrates to vertebrates and absent from non-animal phyla (plants and fungi). However, there are vast structural and functional divergences in TLRs between invertebrates and vertebrates (15). In vertebrates, humans and mice have 10 and 12 TLRs, respectively and at least 16 TLRs have been identiﬁed in teleost; in urochordates, Ciona intestinalis has only two TLRs (54) whereas Ciona savignyi has between November 2018 \| Volume 9 \| Article 2525 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 11 Toll-Like Receptor in B. lanceolatum Ji et al. traﬃcking, the two most likely reasons could be: (1) BlTLR22-like could not directly recognize PAMPs and the recognition process might require the assistance of other proteins that are speciﬁc for amphioxus and are not present in a mammalian system. For instance, D. melanogaster Tolls do not bind any PAMPs directly (62) and mammalian TLR4 cannot recognize LPS without the assistance of MD2 and CD14 (63) or; (2) BlTLR22-like has a TIR domain that interacts with a species speciﬁc adaptor protein not present in mammalian cells. This hypothesis could be supported by the fact that P681 (human TLR2), extremely important to activate MyD88 signaling pathways in mammals (51), was not present in BlTLR22-like neither in BbtTLR1 (Supplementary Figure 3). Thus, we could hypothesize that the absence of this Pro in the TLR22 sequence (Ala in BlTLR22- like) explains why the TIR domain of BlTLR22-like cannot activate MyD88 dependent signaling pathway in HEK293 unless we combine the ectodomain of TLR22 with the human TLR2 TIR domain. To test this hypothesis, we designed a chimeric protein containing the ectodomain and transmembrane domain of BlTLR22-like fused to the human TLR2 TIR domain and we tested whether it could respond to ligand stimulation when stably transfected in HEK293 cells. Indeed, the cells expressing chimeric BlTLR22-like activated signiﬁcantly the NF-κB reporter in response to both LMW and HMW Poly I:C. The magnitude of the stimulation is similar to other published data. Frontiers in Immunology \| www.frontiersin.org DISCUSSION For instance, Ji et al. characterized the activation of IFN and NF- κB pathways by a teleost TLR19, and they found similar fold changes (around 2-fold change) as in our data (2.12 ± 0.1- fold change Poly I:C HMW and 1.95 ± 0.09-fold change Poly I:C LMW) (64). Other authors also have obtained similar fold- changes in the NF-κB reporter assay (65, 66). On the other hand, Voogdt et al. showed an extremely high activation of the NF-κB signaling pathway after ﬂagellin stimulation but the main diﬀerence with our approach is that they used cells stably expressing NF-κB reporter (67). Poly I:C is a speciﬁc ligand of vertebrate TLR3 including many ﬁsh species (20, 23, 65, 68), of M. croaker TLR13 (19) and of diﬀerent ﬁsh TLR22 (20–22, 24, 69). likely due to gene prediction errors (55). This might be the reason why there are less TLRs found in our study than the genomic prediction. Interestingly, if we remove 3 mccTLR sequences in B. ﬂoridae from our list, the total number of TLRs would be the same as reported by Tassia et al. which identiﬁed 19 TLRs (56). Moreover, the RT-PCR analysis showed that all the 30 TLRs of B. lanceolatum were truly expressed in adult animals. Our work shows that amphioxus and vertebrates share a conserved TLR framework in terms of protein structure. On the other hand, amphioxus TLRs maintain some features of invertebrates, such as the mccTLRs which are mainly found in protostomes (15). The function of remaining TLRs in PAMPs recognition remains unclear and needs further investigation. We cloned the full-length sequence of BlTLR22-like from Mediterranean amphioxus (B. lanceolatum). The full-length protein showed the highest identity (78.8%) with bbtTLR1 of B. belcheri that was annotated by the authors as a TLR1 based on the expression analysis after PAMPs injection in vivo (34) but the authors did not study the subcellular localization or the direct ligand speciﬁcity. The domain analysis of BlTLR22-like protein sequence showed that it has a complete vertebrate-like ectodomain including a LRRCT, 21 LRRs and a LRRNT. The ectodomain forms a horseshoe structure to bind the speciﬁc PAMPs including the LRRCT that is responsible for dimerization which is necessary for complete ligand binding (57–59). The full-length protein sequences of BlTLR22 are highly similar to the TLR22 of many ﬁsh species, suggesting that they may have similar ligand recognition, intracellular signal transduction pathway mechanisms and localization. AUTHOR CONTRIBUTIONS JJ performed all the experiments and phylogenetic analysis. DR-V and AB helped to design the phylogenetic analysis. EN- P, CH-Ú and JG-F provided the TLR gene candidates from the transcriptome of B. lanceolatum and assisted with the phylogenetic analysis. JL provided the reporter assay plasmids and helped in the reporter assay design. HE provided the amphioxus and partial sequence of BlTLR from the genome scaﬀold of B. lanceolatum. NR and JJ designed the experiments. JJ and NR prepared all the tables and ﬁgures and wrote the manuscript. All authors contributed to the correction of the ﬁnal manuscript. Supplementary Figure 4 \| Predicted domain architecture of BlTLR protein. The domain structure was predicted using the SMART program. Signal peptide (SP), leucine-rich repeat N-terminal domain (LRRNT), leucine-rich repeat (LRR), leucine rich repeat C-terminal domain (LRRCT), Transmembrane domain (TM) and Toll/interleukin-1 receptor (TIR) domain are indicated in ﬁgure. Figure was prepared with IBS software. DISCUSSION Taken together with its plasma membrane localization and functional analysis, we could further conﬁrm the annotation of this TLR as an ortholog of vertebrate TLR11s, carrying a TLR22-like function and probably share a common ancestor with the ﬁsh speciﬁc TLR22. Overall, we provide evidence suggesting that TLR22 function may be an ancient and evolutionarily conserved antiviral response which emerged in Chordates. In mammals, TLRs can recognize speciﬁc PAMPs with high levels of sensitivity (61). To test B1TLR22-like ligand speciﬁcity, we performed diﬀerent assays with commercially available mammalian TLRs ligands, using NF-κB activity as a reporter. We could not observe signiﬁcant diﬀerences of NF- κB activation in HEK293 cells expressing BlTLR22-like. There are diﬀerent possible explanations but apart from problems with protein expression levels, intracellular degradation or incorrect November 2018 \| Volume 9 \| Article 2525 12 Toll-Like Receptor in B. lanceolatum Ji et al. Ji et al. ACKNOWLEDGMENTS Supplementary Figure 5 \| Phylogenetic analysis of BlTLR. The phylogenetic tree was constructed by maximum-likelihood method (IQ-TREE) using full-length protein sequences. BlTLR, BbtTLR1 and representative vertebrate TLR sequences were used in the analysis. D. melanogaster Toll was used as an outgroup to root the tree. Sequences were aligned with MAFFT choosing L-INS-i method and the alignments were trimmed using TrimAL with “Automated 1” mode. The best evolutionary model was established by ModelFinder according to BIC. One-thousand replicates of the SH-aLRT support and ultrafast bootstrap support are represented as percentages at the tree nodes. The tree was generated in FigTree. Outgroup and six vertebrate TLR families (by colors) are shown in ﬁgure. BlTLR is indicated by a red arrow. This work was supported by grants from the Spanish Ministry of Science, European Commission and AGAUR to NR (AGL2015- 65129-R MINECO/FEDER and 2014SGR-345 AGAUR). JJ was supported by a Ph.D. fellowship from the China Scholarship Council (201306300075), NR was supported by the Ramón y Cajal program (RYC-2010-06210, 2010, MINECO). AB and DR-V thanks to the grants BFU2012-34398, BFU2015- 69717-P (MINECO/FEDER), SAF2014-52624-REDT, Ramón y Cajal Fellowship RYC-2011-08391, Career Integration Grant 304111 (EU), CERCA Programme/Generalitat de Catalunya and AGAUR 2014-SGR-297 and 2017-SGR-1776. HE was supported by the ANR (ANR16-CE12-0008-01). We thank N. Barba (Servei de Microscòpia) and Dr. M. Costa (Servei de Cultius Cel·lulars, Producció d’Anticossos i Citometria) from Universitat Autònoma de Barcelona for their technical assistance. Supplementary Table 1 \| Primers used for RT-PCR analysis. Supplementary Table 1 \| Primers used for RT-PCR analysis. Supplementary Table 2 \| Vertebrate and invertebrate protein sequences used in the phylogenetic analysis. Supplementary Table 3 \| TLR ligands used in this study. Supplementary Table 4 \| TLRs in B. lanceolatum. Supplementary Table 5 \| TLRs in B. ﬂoridae. Supplementary Table 6 \| TLRs in B. belcheri. DATA AVAILABILITY STATEMENTS maximum-likelihood method (IQ-TREE) using TIR domain sequences of B. ﬂoridae, B. belcheri, B. lanceolatum, S. kowalevskii and representative vertebrate TLRs. D. melanogaster Toll sequence was used as an outgroup to root the tree. Sequences were aligned by MAFFT with G-INS-i method and the best evolutionary model was established by ModelFinder according to BIC. Branch support was calculated running 1,000 replicates of the SH-aLRT and ultrafast bootstrap and they are represented as percentages at the tree nodes. The tree was generated in FigTree. Outgroup, mccTLRs and six vertebrate TLR families (by colors) were shown in the ﬁgure. BlTLR is indicated by a red arrow. The datasets for this manuscript are not publicly available yet because the paper in which all these data are presented is in the ﬁnal review process. Once the paper is published all the transcriptomic and genome data will be freely available, but until then requests to access the datasets should be directed to Dr. Jordi Garcia-Fernàndez (jordigarcia@ub.edu) and Dr. Hector Escrivà (hescriva@obs-banyuls.fr). Supplementary Figure 2 \| Complete phylogenetic analysis of B. lanceolatum TLRs. The phylogenetic tree was constructed by IQ-TREE using full-length protein sequences. This tree is a more detailed version of the tree shown in Figure 1. All the values of SH-aLRT support and ultrafast bootstrap support are shown at the tree nodes. Outgroup, mccTLRs and 6 vertebrate TLR families (highlighted in different colors) are shown. The red arrow indicates BlTLR. Additional information about the sequences can be found in Supplementary Table 2, Supplementary Datas 1, 2. Datas 1, 2. Supplementary Figure 3 \| Nucleotide and deduced amino acid sequences of BlTLR. Predicted transcription start site (TSS) is marked with a curved arrow. TATA box is boxed with a rectangle. The putative STAT5 and APIB transcription factor binding sites have a thick underline. The start codon (ATG), the stop codon (TAA) and the polyadenylation signal sequence (AATAAA) are in bold. The predicted signal peptide and the transmembrane region are underlined. The potential N-linked glycosylation sites are underlined and in bold. LRRCT domain predicted by LRRﬁnder is double underlined. The TIR domain predicted by SMART is underlined and highlighted in gray. 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https://openalex.org/W4283792364	https://nora.nerc.ac.uk/id/eprint/533627/1/N533627JA.pdf	English	null	Fifty years of reduction in sulphur deposition drives recovery in soil pH and plant communities	bioRxiv (Cold Spring Harbor Laboratory)	2,022	cc-by	13,530	Received: 5 July 2022 \| Accepted: 14 October 2022 DOI: 10.1111/1365-2745.14039 Received: 5 July 2022 \| Accepted: 14 October 2022 DOI: 10.1111/1365-2745.14039 R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Fifty years of reduction in sulphur deposition drives recovery in soil pH and plant communities Fiona M. Seaton1 \| David A. Robinson2 \| Don Monteith1 \| Inma Lebron2 \| Paul Bürkner3 \| Sam Tomlinson1 \| Bridget A. Emmett2 \| Simon M. Smart1 1UK Centre for Ecology & Hydrology, Lancaster Environment Centre, Lancaster, UK 2UK Centre for Ecology & Hydrology, Environment Centre Wales, Bangor, UK 3Cluster of Excellence SimTech, University of Stuttgart, Stuttgart, Germany Correspondence Fiona M. Seaton Email: fseaton@ceh.ac.uk Funding information Natural Environment Research Council, Grant/Award Number: NE/R016429/1; Department for the Environment Food and Rural Affairs Handling Editor: Qiuying Tian 1UK Centre for Ecology & Hydrology, Lancaster Environment Centre, Lancaster, UK 2UK Centre for Ecology & Hydrology, Environment Centre Wales, Bangor, UK 3Cluster of Excellence SimTech, University of Stuttgart, Stuttgart, Germany Correspondence Fiona M. Seaton Email: fseaton@ceh.ac.uk Funding information Natural Environment Research Council, Grant/Award Number: NE/R016429/1; Department for the Environment Food and Rural Affairs Handling Editor: Qiuying Tian This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Journal of Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society. 464 \| wileyonlinelibrary.com/journal/jec This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited 1 \| INTRODUCTION Increasing temperature has been shown to influence plant and soil functional response to acid deposition in rainfall, in some cases leading to greater effects—for example in- creased susceptibility of plants to air pollution at higher tempera- tures due to increased stomatal opening—and in other cases leading to reduced effects—for example soil respiration increase with tem- perature being reduced by acid rain (Bytnerowicz et al., 2007; Chen et al., 2021). Changes in precipitation will influence both deposition of atmospheric pollutants and soil acidity directly through changes in soil moisture (Hole & Engardt, 2008; Marwanto et al., 2018; Yu et al., 2020). As soil moisture increases, soil pH also increases, due to increased anaerobic conditions that lead to a microbially medi- ated increase in proton consumption during denitrification reactions (Dobbie & Smith, 2001; Zárate-Valdez et al., 2006). Recent analysis of soil pH trends across England and Wales suggested that changes in management practices, and particularly the cessation of liming due to reduced subsidies, may have confounded recent soil pH recovery (Rawlins et al., 2017; Seaton et al., 2021). This shows that soils may in future either stabilise at the current pH, or potentially begin to acid- ify again with corresponding effects upon plant communities and ecosystem health and reversal of ecosystem recovery. On the other hand, recent proposals to increase carbon sequestration in soils by incorporating basalt or alternative rocks into cropland would lead to alkalinisation of the soil (Beerling et al., 2018). Understanding the interrelationship between changes in management, climate, soil pH and vegetation is necessary to better predict the consequences of these technologies upon biotic communities across realistic combi- nations of ecosystem and land-use. Decreasing soil acidity should influence plant community com- position towards species adapted to less acidic, more nutrient rich environments (Peppler-Lisbach et al., 2020; Stevens, Duprè, et al., 2011). Decreasing aluminium solubility and toxicity with in- creasing pH is likely to increase soil microbial activity and facilitate colonisation of acid-sensitive plant species (Jones et al., 2019; Zhao & Shen, 2018). In addition, increased macronutrient availability, as- sociated with an increase in soil pH (Zhao & Shen, 2018) and reduc- tion in ionic aluminium, could theoretically unlock the eutrophying potential of accumulated N, particularly in areas most exposed to N deposition historically (Schneider et al., 2018; Smart et al., 2014; Stevens, Manning, et al., 2011). However, it should be noted that some specific elements (e.g. 1 \| INTRODUCTION attributed to changes in management intensity and N deposition but could also be related to changes in acidity (Diekmann & Dupré, 1997; Duprè et al., 2010; Smart et al., 2003). 1 Atmospheric deposition of sulphur (S) and nitrogen (N) compounds has led to the widespread acidification of natural ecosystems and resulting loss of biodiversity and changes in ecosystem functions (Bytnerowicz et al., 2007; Kirk et al., 2010; Maskell et al., 2010). In Europe and North America, S deposition dramatically declined in the late 20th and early 21st century due to switches away from fossil fuels and more effective monitoring and control of S emission sources. In China more recent declines in emissions are only now beginning to result in decreases in precipitation acidity while in other countries such as India emissions are still increasing (Grennfelt et al., 2020; Smith et al., 2011; Zhang et al., 2020). This raises the possibility of examining ecological recovery within areas now ex- posed to a long period of reduced S deposition in order to predict and manage change in areas where S deposition is still high. Within the UK, there was a 96% reduction in sulphur dioxide (SO2) emis- sions from 1970 to 2019, concurrent with a 57% decline in emis- sions of oxides of nitrogen (NOx). Ammonia (NH3) emissions have remained relatively stable, showing only a 13% decrease from 1980– 2019 (NAEI (National Atmospheric Emissions Inventory), 2019). The late 20th century reduction in S deposition to natural ecosystems has been linked to decreases in soil and water acidity (Hughes et al., 2012; Kirk et al., 2010; Reynolds et al., 2013), while N deposi- tion has been associated with both eutrophication and acidification effects upon plant communities (Bytnerowicz et al., 2007; Schweiger & Beierkuhnlein, 2017; Stevens et al., 2010; Tipping et al., 2021). Changes in the atmospheric deposition of acidity and nutri- ent N have been occurring at a time of increasing pressures from change in climate and land use, and the influence of interactions between these drivers on natural ecosystems have been identified (Bytnerowicz et al., 2007). Abstract 1. Sulphur deposition through rainfall has led to species loss and ecosystem deg- radation globally, and across Europe huge reductions in sulphur emissions since the 1970s were expected to promote the recovery of acidified ecosystems. However, the rate and ecological impact of recovery from acidification in ter- restrial ecosystems is still unclear as is the influence of management and climate, as to date there has been no long-term spatially extensive evaluation of these changes. 1. Sulphur deposition through rainfall has led to species loss and ecosystem deg- radation globally, and across Europe huge reductions in sulphur emissions since the 1970s were expected to promote the recovery of acidified ecosystems. However, the rate and ecological impact of recovery from acidification in ter- restrial ecosystems is still unclear as is the influence of management and climate, as to date there has been no long-term spatially extensive evaluation of these changes. 2. Here, we present data from thousands of sites across Great Britain (pH range 3.3–8.7) surveyed repeatedly from 1978–2019 and assess change in soil pH and plant acidity preference (Ellenberg R) in response to atmospheric deposition of sulphur and nitrogen. We analyse change in grasslands managed for pasture, referred to as high-intensity habitats, and compare to seminatural habitats com- prising rough grassland, broadleaved woodland, bog and heathland, referred to as low-intensity habitats. 3. Soil pH increased from 1978 to 2007 but then decreased between 2007 and 2019, resulting in a net increase of ~0.2 pH units in low-intensity habitats but no change in high-intensity habitats. The community average Ellenberg R increased in seminatural habitats by ~0.2 units but remained stable in intensive grasslands. 4. In seminatural habitats, but not intensive grasslands, these changes in plant community composition were associated with the soil pH changes which were in turn linked to decreasing sulphur deposition and differences in rainfall. 5. Nitrogen deposition, which was relatively stable over the survey period, showed no additional effect upon soil acidity once sulphur deposition was accounted for. 6. Synthesis: Our results provide conclusive evidence that reductions in acid emis- sions are stimulating the gradual recovery of chronically acidified terrestrial eco- systems at a whole-country scale, while also suggesting this recovery is being compromised by changing climate and land management. Journal of Ecology. 2023;111:464–478. SEATON et al. Journal of Ecology 465 1 \| INTRODUCTION Ca2+, Mg2+, K+, and Na+) decrease under acidic conditions due to increased leaching and therefore recovery of availability of those nutrients would require additional inputs of these elements and not just soil pH recovery. Here, we use data from the UKCEH Countryside Survey of Great Britain to evaluate trends in soil pH and plant community acidity preference (mean Ellenberg R) and examine their connec- tion to atmospheric S and N deposition. The Countryside Survey (CS) comprises a representative sample of co-located vegetation and soil data from the British landscape. It was first carried out in 1978 and since then in 1990, 1998, 2007 and most recently a partial resurvey starting in 2019 (Wood et al., 2017). This long-term, large scale monitoring enables us to better evaluate change in seminatu- ral ecosystems in response to a wide range of concurrent biotic and abiotic pressures and over timescales relevant to plant community assembly and related ecological processes. Due to the extensive quality assurance protocols that are part of this survey we are also Nitrogen deposition not only acts as a eutrophying agent but may also add to the acid load, depending on how it is processed within catchments. Over the late 20th century, plant communities across Europe were observed to be shifting towards communities more adapted to higher nutrient conditions, which has largely been SEATON et al. Journal of Ecology 466 FI G U R E 1 Map of survey square locations to the nearest 10 km FI G U R E 1 Map of survey square locations to the nearest 10 km square. able to incorporate quantitative assessment of measurement error in the soils and vegetation data into our statistical modelling to more robustly detect change over time and attribute it to hypothesised drivers. We selected sites that were within seminatural habitats (low-intensity management) or intensively managed grassland (high- intensity management) habitats and estimated change over time in soil pH and vegetation community acidity preference, represented by mean Ellenberg R. 2.1 \| Field survey low intensity management land. The high intensity management land comprised improved and neutral grassland where the summed cover of agricultural forage species, that is Lolium perenne, Lolium multi- florum and Trifolium repens, is over 25%. The low intensity manage- ment land included the rest of the neutral grassland, acid grassland, broadleaved woodland, dwarf shrub heath, bog, bracken, fen, marsh, swamp and calcareous grassland. Coniferous woodland, arable, coastal and urban habitats were not included as we expected their Ellenberg scores to show limited relation to the drivers analysed as their plant communities are largely driven by human interference or dominated by saline influence. In total, within our management cat- egories there are 6665 measurements (i.e. a plot recording from a specific year) from 2717 plots surveyed for vegetation across 5 years and 588 squares, with 3797 measurements having co-located soil measurements (no soil samples were taken in 1990). A total of 2383 measurements that had both plant and soils data were categorised as low-intensity management. The data presented here is from the UKCEH Countryside Survey, a repeated programme of field surveys with locations spread across the GB countryside (Figure 1, Carey et al., 2008). We include data from 1978, 1990, 1998 and 2007 as well as results from the recent resurveys of the previously surveyed sites. The recently surveyed sites were mostly visited in 2019; however, a small number in Wales were surveyed in 2016 (Emmett & the GMEP team, 2017). Within each 1 km square there are multiple vegetation plots, including five square plots measuring 14.14 by 14.14 m. These plots are surveyed for plant composition in a nested structure, with soil samples taken to 15 cm depth from a central position on the corner of the inner 2 by 2 m nest within the plot. Within the 2019 data, a subset of plots had only the inner 2 by 2 m square surveyed for vegetation (221 plots out of 559 plots). All vegetation data are available at the NERC Environmental Information Data Centre (EIDC): see the data avail- ability statement. Broad habitat types were assigned to each plot by the surveyors according to JNCC guidance (Jackson, 2000). These broad habitats were grouped into high intensity management and Soil pH was measured in deionised water, and additionally in 2007 and 2019 soil pH was also measured in a CaCl2 solution. 1 \| INTRODUCTION We hypothesised that, over the last five de- cades of major reductions in acid deposition, soil pH and mean Ellenberg R values would have increased from their low levels to- wards nearer- neutral values due to recovery from acidification, but that high intensity management sites would show less change due to decreasing acid deposition occurring at the same time as a reduction in liming which had artificially increased soil pH in the mid-20th cen- tury. We also hypothesised that the increased sensitivity of cover- weighted estimates of Ellenberg R to environmental change would be counteracted by increased measurement error of cover estimates dependent on plot size. We then tested whether changes in soil pH were associated with decreases in atmospheric S deposition and with cumulative N deposition at each site reflecting legacy effects of acidifying N deposition in parallel with a eutrophying effect (Rowe et al., 2020). The relative influences of S deposition and N deposi- tion upon pH change were evaluated within a Bayesian framework, also including the difference in field season rainfall between survey years to incorporate effects of changes in climate as represented by changing precipitation due to the known large influence of soil mois- ture upon soil acidity. We also hypothesised that the changes in soil pH resulted changes in mean Ellenberg R and hence plant species composition, and tested this using a multivariate Bayesian model. //besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.14039 by Ukri C/O Uk Shared Business NATURAL ENVIRONMENT RSCH COUNCIL, Wiley Online Library on [26/04/2023]. See the Terms and Condi inelibrary.wiley.com/doi/10.1111/1365-2745.14039 by Ukri C/O Uk Shared Business NATURAL ENVIRONME FI G U R E 1 Map of survey square locations to the nearest 10 km square. 2.3 All statistical modelling presented here was undertaken in the brms package (v2.15.0) in R (v4.0.3), an interface to Stan (v2.21.2) (Bürkner, 2017; Carpenter et al., 2017). Ellenberg R and soil pH were both separately modelled as a response to an interaction be- tween year (categorical) and the management intensity, with square identity as a group effect and a first order autoregressive process by year (sequentially numbered) and grouped by plot ID. The num- ber of individual measurements in each model varied, with 6660 for the 4 m2 unweighted Ellenberg R over 2717 plots, 6530 for the 4 m2 cover weighted Ellenberg R over 2705 plots, and 6502 for the 200 m2 Ellenberg R measurements over 2703 individual plots. For the soils’ data, there were 3836 measurements of pH in water over 2323 plots, of which 2247 measurements were from the 21st cen- tury and had corresponding pH in CaCl2 measurements. Pairwise contrasts between years were obtained using the emmeans pack- age (v1.5.5–1, Lenth, 2021) and plotted using the tidybayes package (v2.3.1, Kay, 2020). Within every plot, Ellenberg R scores were calculated from the known species scores, with either the unweighted average or an average weighted by proportion of cover. Ellenberg R is an ordi- nal score assigned to plant species that ranges from one to nine and indicates the preferred acidity of that plant species, which can be averaged over the plant community. It is part of a suite of Ellenberg scores originally developed for Central Europe and adapted for Great Britain by Hill et al. (2000). This procedure was repeated for the full 14.14 by 14.14 m plots and the inner 2 by 2 m nest. An estimate of the error in these plant community vari- ables was taken from the plots resurveyed as part of the Quality Assurance procedures in the survey years from 1990 onwards, the 1978 error is assumed to be the same as the 1990 error (see Supplementary Table S4 for all of the error estimates). In order to extract a standard deviation for use in the statistical modelling of the change in Ellenberg R the difference between the Ellenberg R score from the original and repeat survey was fitted to a normal distribution using the MASS package (Venables & Ripley, 2002). 2.3 To estimate the potential impacts of this shift in plant commu- nity upon plant service provision the Ellenberg R scores of each plant species were compared to known values of service provision, including nectar-yielding species (Baude et al., 2016), agricultural forage grasses and crop wild relatives (Jarvis et al., 2015), and food plants for lowland birds and butterfly larvae (Smart et al., 2000). All data manipulation and statistical analysis was undertaken in R version 4.0.3 (R Core Team, 2020), plots were created using gg- plot2 (Wickham, 2009). To investigate the relationship between changing Ellenberg R and soil pH, they were modelled in a multivariate Bayesian model with measurement error in the response variables being included. The change in Ellenberg R was modelled as a function of the change in soil pH, with an interaction with management intensity also in- cluded. The change in soil pH was modelled as a function of the change in sulphur deposition in the eight-year period before the second survey, the cumulative N deposition in the eight-year period before the second survey and the difference in presurvey rainfall between the two time periods, to ensure standardisation of model coefficients all of these variables were scaled to have mean 0 and standard deviation of 1. The original means and standard deviations of these variables were − 5.3 and 4.5 for S deposition, 134 and 72 for N deposition, and 8 and 30 for field season rainfall. All response vari- ables were assumed to follow a Gaussian likelihood, and had square identity as a random effect, as well as an autoregressive component that accounted for time period where change occurred and had plot identity as a grouping factor. See Supplementary Methods for ex- ample for the code used to fit these models. We also explored the possibility of a nonlinear relationship between S and N deposition and soil pH change but found it was not appropriate for our data, see Supplementary Methods for a discussion of this. Residuals from each model were plotted against broad habitat to check if there were systematic differences between habitat types (data not shown, no differences were found). The sample size varied per model due to differing numbers of small and large vegetation plots, being 1309 for the mean Ellenberg scores from the largest plots, and 1336 and 1458 for the weighted and unweighted mean Ellenberg scores from the 4 m2 subplots respectively. 2.3 Models were compared with 10-fold cross-validation using the loo package (v2.4.1, Vehtari et al., 2020). 2.1 \| Field survey For a full description of the soil methods see Emmett et al. (2008). All soil SEATON et al. Journal of Ecology 467 difference between the field season rainfalls was calculated for each year comparison. physicochemical data is available online at the NERC EIDC; see the data availability statement. The measurement error of soil pH was calculated from randomly selected samples that were remeasured in 2007 and 2019. This procedure did not occur in 1978 and 1998; however, due to a slight difference in the soil pH measurement pro- cedure in 1998, there was a process of remeasuring samples in both 1998 and 2007 to compare the methods (Black et al., 2002; Emmett et al., 2010). The error from these remeasurements was used to cre- ate an expected error of the difference between 1978 and 1998, and 1998 and 2007 respectively. difference between the field season rainfalls was calculated for each year comparison. 3.1 \| Trends in soil pH and Ellenberg R Over the 40-year survey period, soil pH increased in both high and low-intensity management habitats up until 2007, as reported previ- ously from this data (Reynolds et al., 2013), but decreased in 2019 compared to 2007 (Figure 2a). Soil pH levels in 2019 were on aver- age no different to the pH levels of 1978 and 1998 (Table 1)—note that no soils data was collected within the 1990 survey. However, the difference in acidity between 2007 and 2019 was much less ap- parent when pH was measured in a 0.01 M CaCl2 solution, a method applied in the 21st century surveys only. Those data provided no evidence for a drop in mean pH from 2007 to 2019 in low-intensity management habitats, and a smaller drop in pH in high-intensity habitats (Table 1, Supplementary Figure S1). Over the survey pe- riod plant communities under low-intensity management showed sustained increases in mean Ellenberg R, while high-intensity management land showed no overall trend in mean Ellenberg R (Figure 2b, Supplementary Figure S2). In order to evaluate the in- fluence of the mean Ellenberg R calculation method on the trend estimate, mean Ellenberg R was calculated with and without cover weighting, and with or without constraining to species enumerated in the innermost nest of the plot. Measurement error was highest in the cover-weighted and smaller plot-based measurements. All four mean Ellenberg R metrics showed a continued increase in low intensity habitats, while remaining stable in high-intensity habitats Overall, soil pH increased the most in the sites that showed the largest decreases in S deposition and the greatest cumulative N deposition (Figure 3). These two variables were negatively cor- related such that the sites with the biggest decreases in S deposition also showed the largest cumulative N deposition (Pearson correla- tion −0.65). The estimated relationships between pH change and the two variables were broadly similar for the two management intensi- ties. Changes in mean Ellenberg R were slightly negatively correlated FI G U R E 2 The change in soil pH and mean Ellenberg R (from the 4 m2 plot and unweighted average) as modelled in response to FI G U R E 2 The change in soil pH and mean Ellenberg R (from the 4 m2 plot and unweighted average) as modelled in response to management regime and year, represented by the median estimate and ribbons for the 50%, 80% and 95% tiles. 3 \| RESULTS (Supplementary Figure S2, Table S1). This indicates the robustness of these trends to both measurement method and the variation in sample size, as more 4 m2 plots were surveyed due to changes in survey protocol across the years. The measured changes in soil pH and mean Ellenberg R occurred during a period of continuous de- cline in S deposition (Figure 2c) and span a period during which N deposition initially increased before declining again (Figure 2d) but against a backdrop of at least 200 years of elevated N deposition (Fowler et al., 2005). Rainfall during the survey period also varied across the different years, with the highest levels of rainfall in 2007 and the lowest in 1990 (Supplementary Figure S3). The increase in mean Ellenberg R in low-intensity habitats can be related to declines in plant species with low Ellenberg values, such as Calluna vulgaris, Erica tetralix and Trichophorum cespitosum (Table 2). In our dataset, higher mean Ellenberg R scores were associated with a greater di- versity of plants and a higher number of species associated with multiple ecosystem functions, including nectar-yielding species, agricultural forage grasses, crop wild relatives, and food plants for lowland birds and butterfly larvae (Supplementary Figure S4). 2.2 \| Atmospheric data sources Atmospheric N and S deposition was taken from a 1 km grid surface from the FRAME atmospheric chemistry transport model (Tipping et al., 2017; Tomlinson et al., 2021) and is available at the NERC EIDC, see data availability statement. Cumulative N deposition for the 8 years previous to survey was calculated for each square, as was the difference in S deposition over the 8 years previous to survey. This time period for statistic calculation was chosen due to the limited temporal range of the 1 km FRAME atmospheric model outputs. Monthly rainfall data was taken from the HadUK-Grid 1 km gridded climate observations provided by the Met Office (Met Office et al., 2020). Monthly rainfall over the month of sampling and the 3 months previous was averaged for every plot surveyed. The SEATON et al. Journal of Ecology 468 3.1 \| Trends in soil pH and Ellenberg R For low intensity habitat, only the magnitude was marginally higher, −0.14 and 0.08. change for a change in soil pH of 1 unit (Supplementary Table S3). change for a change in soil pH of 1 unit (Supplementary Table S3). change for a change in soil pH of 1 unit (Supplementary Table S3). Soil pH change was still slightly negatively associated with the change in S deposition in low intensity habitat once changes in field season rainfall and N deposition were accounted for. However, the effect was smaller for high intensity management areas and the 95% interval on the posterior parameter distribution included zero (Figure 5, Supplementary Figure S6). Soil pH change was pos- itively associated with the difference in presurvey rainfall between survey years in all four models, with an increase of around 0.1 pH units for every 30 mm increase in rainfall across the field season (95% quantile 0.04–0.14, Supplementary Table S2). The N deposi- tion effect was reduced to around zero once the S deposition effect and rainfall changes were accounted for (Figure 5, Supplementary Figure S6, Table S2), with a positive correlation between the N and S deposition effects showing that when the model estimated a strong positive effect of N deposition this only occurred with a corre- sponding decrease in the estimated effect of S deposition (Pearson correlation ~0.75 in all four models). Adding direct linkages from N and S deposition to mean Ellenberg R resulted in no improvement in model fit, with no evidence of a residual effect of N and S deposi- tion upon mean Ellenberg R change once pH change is accounted for (Supplementary Figure S7). However, within the unweighted mean Ellenberg R score for the whole 200 m2 plot, in theory the most sen- sitive to change in rare species, there was some evidence for a direct negative effect of S deposition on mean Ellenberg R change in low intensity management habitats only (Supplementary Figure S7). 3.2 \| Soil pH change drives Ellenberg R change We found a positive relationship between soil pH and mean Ellenberg R change, with an estimated increase in mean Ellenberg R of ~0.15 units per unit increase in soil pH (~30% of the interquar- tile range in mean Ellenberg R, Figure 4, Supplementary Figure S6, Table S2). This was consistent across both management intensities and all four mean Ellenberg R metrics (Supplementary Figure S6). Temporal autocorrelation was estimated to be negative for both pH and Ellenberg R, indicating a “regression to the mean” effect and therefore justifying accounting for correlation of observations over time. The hierarchical 1 km square effect was greater in the soil pH model than in the mean Ellenberg R model, and there was no evidence of correlation between the square effects in the two models (Supplementary Table S2, Figure S5). The weighted meas- urements of mean Ellenberg R appeared slightly more responsive to change in soil pH in the high intensity management habitats but this effect was small, while absent in the low intensity management habitat (Supplementary Figure S6). There was no effect of habitat type upon the model residuals, indicating no identifiable difference in the response of mean Ellenberg R to pH change, or pH change to atmospheric deposition, between woodland, grassland and heath- land. If we had not taken into account measurement error in soil pH and mean Ellenberg R this relationship would have remained positive 3.1 \| Trends in soil pH and Ellenberg R Also shown are the changes in average S and N deposition across all of the plots in the two management categories, represented by a mean line and ribbons for the mean ± standard deviation. Note, 1 km resolution S and N deposition estimates are not available between 1970 and 1990. FI G U R E 2 The change in soil pH and mean Ellenberg R (from the 4 m2 plot and unweighted average) as modelled in response to management regime and year, represented by the median estimate and ribbons for the 50%, 80% and 95% tiles. Also shown are the changes in average S and N deposition across all of the plots in the two management categories, represented by a mean line and ribbons for the mean ± standard deviation. Note, 1 km resolution S and N deposition estimates are not available between 1970 and 1990. SEATON et al. Journal of Ecology 469 TA B LE 1 Modelled difference between years for both pH in deionised water and pH in CaCl2 (most recent period only). The estimated difference and the lower and upper limits of the 95% highest posterior density (HPD) interval are given for each year contrast. Differences where the 95% HPD do not cross zero are in bold Contrast pH (DIW) pH (CaCl2) Estimate Lower HPD Upper HPD Estimate Lower HPD Upper HPD High intensity management 1998–1978 0.203 0.110 0.294 — — — 2007–1978 0.504 0.417 0.598 — — — 2019–1978 0.092 −0.063 0.231 — — — 2007–1998 0.302 0.206 0.388 — — — 2019–1998 −0.112 −0.262 0.037 — — — 2019–2007 −0.412 0.548 0.278 −0.141 −0.272 −0.007 Low intensity management 1998–1978 0.350 0.279 0.425 — — — 2007–1978 0.418 0.345 0.496 — — — 2019–1978 0.177 0.066 0.281 — — — 2007–1998 0.068 0.001 0.137 — — — 2019–1998 −0.173 −0.276 −0.065 — — — 2019–2007 −0.242 −0.337 −0.149 0.018 −0.069 0.107 but at a smaller effect size of around 0.1 units of mean Ellenberg R but at a smaller effect size of around 0.1 units of mean Ellenberg R with S deposition and slightly positively correlated with cumulative N deposition across all four mean Ellenberg R measurement meth- ods; however, the magnitude of this was small, with the greatest Pearson correlations being −0.10 and 0.04 for S and N deposition, respectively, with the unweighted score from the larger plot. 4 \| DISCUSSION Plant communities in seminatural habitats have shown progressive recovery from acidification over the past 40 years, demonstrating 470 SEATON et al. Journal of Ecology 2 The proportion of plots each species appears in per year and management category, limited to plant species that appear in at least 35% of plots in at least one management/year ation and have a known Ellenberg R score s Ellenberg R High intensity management Low intensity management 1978 (n = 355) 1990 (n = 610) 1998 (n = 664) 2007 (n = 720) 2019 (n = 133) 1978 (n = 503) 1990 (n = 1014) 1998 (n = 1225) 2007 (n = 1221) 2019 (n = 281) is stolonifera 7 0.293 0.451 0.405 0.436 0.263 0.101 0.201 0.269 0.236 0.171 m arvense 7 0.321 0.333 0.363 0.364 0.12 0.097 0.122 0.125 0.143 0.103 s glomerata 7 0.53 0.498 0.423 0.356 0.173 0.149 0.137 0.146 0.142 0.096 m pratense sens.lat. 7 0.482 0.43 0.405 0.275 0.195 0.068 0.052 0.061 0.043 0.018 obtusifolius 7 0.346 0.41 0.449 0.482 0.293 0.054 0.066 0.082 0.086 0.043 erennis 6 0.431 0.349 0.309 0.225 0.128 0.127 0.083 0.073 0.045 0.028 lanatus 6 0.575 0.564 0.604 0.708 0.353 0.348 0.357 0.376 0.4 0.263 perenne 6 0.901 0.974 0.947 0.932 0.421 0.151 0.17 0.171 0.152 0.103 nua 6 0.535 0.444 0.473 0.342 0.12 0.135 0.107 0.103 0.066 0.05 vialis 6 0.437 0.308 0.384 0.375 0.293 0.111 0.107 0.153 0.136 0.132 culus repens 6 0.659 0.59 0.697 0.739 0.338 0.225 0.233 0.219 0.221 0.135 a media 6 0.355 0.325 0.324 0.278 0.12 0.056 0.059 0.056 0.044 0.028 m repens 6 0.825 0.856 0.839 0.849 0.353 0.258 0.241 0.232 0.231 0.149 um fontanum 5 0.544 0.515 0.539 0.649 0.346 0.264 0.235 0.257 0.234 0.16 is capillaris 4 0.372 0.338 0.426 0.478 0.188 0.423 0.45 0.427 0.404 0.206 xanthum odoratum 4 0.13 0.154 0.178 0.21 0.15 0.455 0.41 0.43 0.398 0.249 a ovina agg. 4 \| DISCUSSION 4 0.062 0.031 0.023 0.019 0.015 0.356 0.309 0.309 0.249 0.142 saxatile 3 0.014 0.018 0.029 0.011 0.03 0.336 0.367 0.37 0.352 0.164 a caerulea 3 0 0.003 0.005 0.007 0 0.433 0.417 0.387 0.377 0.167 stricta 3 0.011 0.003 0.009 0.013 0.008 0.404 0.371 0.344 0.312 0.132 lla erecta 3 0.037 0.021 0.027 0.024 0.023 0.604 0.575 0.522 0.509 0.253 a vulgaris 2 0 0.002 0.002 0.001 0 0.596 0.536 0.495 0.471 0.21 ampsia flexuosa 2 0.008 0.005 0.005 0.004 0 0.35 0.361 0.351 0.331 0.132 etralix 2 0 0 0 0 0 0.394 0.344 0.302 0.296 0.139 squarrosus 2 0.003 0 0.005 0.006 0 0.386 0.374 0.34 0.314 0.139 SEATON et al. Journal of Ecology 471 FI G U R E 3 Change in pH between survey periods is negatively related to the change in S deposition (left) and positively related to cumulative N deposition (right). Both deposition statistics are from the 8 year period prior to the second survey. The points are the actual data, with each point representing a site/time period combination, and the lines plus ribbons are the estimated mean and the 2.5% and 97.5% quantiles of that mean. FI G U R E 3 Change in pH between survey periods is negatively related to the change in S deposition (left) and positively related to cumulative N deposition (right). Both deposition statistics are from the 8 year period prior to the second survey. The points are the actual data, with each point representing a site/time period combination, and the lines plus ribbons are the estimated mean and the 2.5% and 97.5% quantiles of that mean. FI G U R E 4 Soil pH change is positively related to mean Ellenberg R change within both high and low intensity management habitats. Data and model shown here are the mean Ellenberg R measured within the 4 m2 plot and unweighted by cover. The points are the actual data and the lines plus ribbons are the estimated mean and the 2.5% and 97.5% quantiles of that mean. parts of the ecosystem, intensive land management effects and higher pH starting conditions have to an extent eclipsed atmos- pheric deposition effects. However, this does not mean that these habitats have not responded to changes in soil acidity over time as we still found a clear link between soil pH change and Ellenberg R change in intensive grasslands. 4 \| DISCUSSION It appears that the lack of evidence for a direct link between plant community response and decreasing S deposition in intensive pasture is likely due to soil pH changes not being as strongly linked to S deposition changes in these systems. Increases in soil pH across GB over the late 20th and first de- cade of the 21st century appear to have stalled, or in some case reversed, in the past decade. We can be confident of the decreas- ing pH in high-intensity management areas, which was found in both soil pH in water and in CaCl2; however, low-intensity man- agement habitats showed no change in soil pH in CaCl2 versus a decline in soil pH in water. Our data can be compared to a previ- ous study of soil pH from farm survey data, which indicated that soil pH has recently decreased in the north-west of England and Wales (Rawlins et al., 2017). The increasing acidity in land man- aged for pasture is potentially related to the decline in liming of agricultural soils across the UK, which is now below the level re- quired to maintain soil pH in the productive zone (Goulding, 2016). Increasing liming or alternatively applying basalt to crop and pas- ture land could therefore increase pH again, and in the latter op- tion, also provide a valuable service in carbon capture (Beerling et al., 2018). However, liming of agricultural soils in the UK dropped in the 1990s and has remained at a fairly constant level since 2000 (Holland et al., 2018; The British Survey of Fertiliser Practice, 2020), indicating that there may be other factors in- volved in the difference between 2007 and 2019 soil pH such as changes in fertiliser use. There is also the possibility that climate change may be impacting the recovery from acid deposition, as both changing temperature and precipitation have been found to FI G U R E 4 Soil pH change is positively related to mean Ellenberg R change within both high and low intensity management habitats. Data and model shown here are the mean Ellenberg R measured within the 4 m2 plot and unweighted by cover. The points are the actual data and the lines plus ribbons are the estimated mean and the 2.5% and 97.5% quantiles of that mean. 4 \| DISCUSSION both the promising reality of recovery from pollution impacts within seminatural ecosystems as well as the longer time scales required to see the positive ecological impacts of legislative change. Our results clearly demonstrate that air pollution control policy can lead to eco- logical recovery even though reductions in S deposition may have been initiated by economic, rather than ecological, considerations (RoTAP, 2012). As expected, the pattern of recovery from acidifi- cation was not apparent in the more highly managed pasture habi- tats which showed relatively stable acidity preference over the past 40 years. This indicates that in both the above- and below-ground 472 SEATON et al. Journal of Ecology FI G U R E 5 Graphical representation of the multivariate model results (mean Ellenberg R score from the unweighted 4 m2 plot) with results from high intensity management land on the left and low intensity management land on the right, where each arrow represents a specific model parameter and is labelled with the median parameter estimate (2.5%/97.5% quantiles). The rainfall effect was assumed to be constant in the two habitat categories but is shown separately for graphical clarity. Blue arrows represent positive median relationships and yellow negative, note that the 95% interval contains zero for the N deposition arrows and the S deposition arrows in high intensity habitats. FI G U R E 5 Graphical representation of the multivariate model results (mean Ellenberg R score from the unweighted 4 m2 plot) with results from high intensity management land on the left and low intensity management land on the right, where each arrow represents a specific model parameter and is labelled with the median parameter estimate (2.5%/97.5% quantiles). The rainfall effect was assumed to be constant in the two habitat categories but is shown separately for graphical clarity. Blue arrows represent positive median relationships and yellow negative, note that the 95% interval contains zero for the N deposition arrows and the S deposition arrows in high intensity habitats. the 2007 season was on average wetter, which may explain some of the increase in soil pH. Integrating rainfall measurements into our multivariate modelling allowed us to demonstrate how both S deposition and rainfall together influenced the soil pH measured over these time periods illustrating individual and additive roles for each driver. 4 \| DISCUSSION This also implies that ongoing impacts of reduced atmospheric deposition are likely to be modified by a backdrop of ongoing changes in precipitation again emphasising the im- portance of accounting for interactions between global change drivers when attributing signals of ecosystem change (Perring et al., 2018). impact soil pH and interact with S deposition; moreover, we also found an effect of rainfall upon pH change (Adamson et al., 2001; Chen et al., 2021; Li et al., 2019). Increasing rainfall could affect pH through increasing frequency of soil saturation events, lead- ing to anaerobic conditions and thus increasing importance of proton consumption processes which promote N2O emissions and increasing pH (Davidson et al., 2000; Dobbie & Smith, 2001; Russow et al., 2000). The effect of rainfall upon soil pH will vary with soil texture, with heavier soils spending more time under sat- urated conditions after rainfall—the majority of our soils are loamy in texture, with clayey loam more common to the south and sandy loam more common to the north (Lawley, 2012). However, the ex- tent of the difference between the predicted pH changes from the two different measurement methods—in water versus in CaCl2— raises the issue of whether our measured pH change optimally represents changes in “plant-visible” soil acidity. Soil pH measure- ments made in water are known to be more affected by salt solu- tion and therefore soil moisture effects (Hester & Shelton, 1933; Kissel et al., 2009). Measurement of soil pH in CaCl2 should be more robust to changes in soil moisture, and more closely relates to the ionic strength of the near-root and near-microbe surfaces, and so would be more suited for tracking changes over time in soil acidity relevant to plant communities and soil functions (Custos et al., 2020; Schofield & Taylor, 1955). Unfortunately, we do not have pH in CaCl2 measurements for the entire soil series as this was first measured in 2007. Instead, we must consider carefully the role of soil moisture in driving the patterns of increasing pH in the early part of the time series. While field season rainfall across the UK in 1978, 1998 and 2019 field seasons was broadly similar, Our analysis has shown that the major drivers of soil pH change over the past 40 years are decreasing S deposition and variation in rainfall. 4 \| DISCUSSION This particular for- mulation of the Ellenberg R score is more sensitive to rarer species and is also likely to be the least spatially coupled to the single point measurement of soil pH as it gives equal weight to plant species that are found in small numbers farther from the soil sampling location within the 200 m2 plot. As soil pH experiences high levels of spa- tial variation at a local scale (Ball & Williams, 1968), it may be that this link between S deposition and Ellenberg R change appears to be separate from soil pH change only due to this decoupling of the Ellenberg R and soil measurements. Surprisingly, we found only a limited interaction between management intensity and the Ellenberg R ~ pH relationship, despite having found divergent trends in soil pH and Ellenberg R change over time in high-intensity management hab- itats. The stability of Ellenberg R from 2007 to 2019, while soil pH decreased in high-intensity habitats could be related to lag effects or indicative of less close coupling of soil and vegetation change under intense management. However there was no indication of less close coupling of soil and vegetation change in the multivariate model results which showed a continued relationship between soil pH and Ellenberg R change in high intensity habitats. This was also unexpected due to the curvilinear relationship between Ellenberg R and soil pH, such that above pH 6 there tends to be a flattening of the Ellenberg R relationship with soil pH (Diekmann, 2003). Potential explanations for the persistence of the relationship between soil pH and Ellenberg R change in high-intensity management habitats in- clude the co-occurrence of soil pH and plant community interven- tions in highly managed areas, such that as the soil is limed there is also introduction of high Ellenberg R plant species within seed mixes. It should also be noted that there was a lower response of soil pH change to S deposition within these high-intensity management habitats, indicating that acidity changes in both soil pH and plant communities are less affected by atmospheric deposition. Therefore, despite there being no overall trend in Ellenberg R in high-intensity pasture over the time period studied we can still conclude that the high-intensity pasture sites that did undergo a change in Ellenberg R were on average sites that showed equivalent change in soil pH. 4 \| DISCUSSION Rainfall was found to be strongly positively related to soil pH change such that a wetter field season had higher soil pH on average, consistent with our knowledge of soil chemistry and pre- vious studies on the effect of seasonality upon soil pH (Hester & Shelton, 1933; Kissel et al., 2009; Marwanto et al., 2018). Inclusion of this rainfall term also enables us to have greater confidence that the S deposition effect we have found is representative of a direct effect once changes in climate and therefore soil moisture are accounted for. While we found a strong effect of the decreases in S deposition upon soil pH, we found no clear evidence of an additional eutrophy- ing effect of cumulative N deposition on plant species composition where soil pH had increased. It is possible that the eutrophication effect on Ellenberg R may be only slightly positive or nonlinear and thus not detected by our analysis; however, the noisiness of our data and the difficulties we encountered in fitting nonlinear models (see Supplementary Methods) indicate that this dataset would not be SEATON et al. Journal of Ecology 473 able to distinguish the subtle nonlinear relationships implied by this hypothesis from ecological noise. within our modelling framework, which represented changes in at- mospheric fertilisation of plant communities. Previously it has been proposed that decreasing acidity in aquatic systems may unlock an eutrophication effect such that the impacts of N deposition are greater as S deposition decreases (Schneider et al., 2018). This might also be expected to occur in land ecosystems as the impacts of alu- minium toxicity decrease once pH is raised and macronutrient avail- ability maximised above 5–6 (Jones et al., 2019; Stevens et al., 2010; Zhao & Shen, 2018). However, we found no evidence of effects of N deposition upon either soil pH or directly upon Ellenberg R change. While the range of our estimated N deposition is large enough to include some possibility of an effect, on balance the likelihood is that Ellenberg R change is driven by soil acidity change rather than changes in N fertilisation. This could partly be due to the measured N deposition in the 8 years prior to the survey not being represen- tative of either historical N deposition, or the N accumulated within the soil. 4 \| DISCUSSION It is possible that phosphorus fertilisation or limitation could be linked to changes in Ellenberg R as phosphorus is known to dif- ferentially influence plant species of varying Ellenberg N scores (Löfgren et al., 2020). Use of phosphorus fertilisers has declined in the UK and there is some evidence of a decline in available phos- phorus in Scottish soils from the 1980s to the present, which would affect both the fields directly fertilised and also leaching to the sur- rounding landscape (Lilly et al., 2020). However, analysis of changes from the 1990s to the present within data collected from large-scale surveys and to support farm management has not found a change in available phosphorus in UK soils (Edwards et al., 2016; Rawlins et al., 2017; Seaton et al., 2021). Residual variation in Ellenberg R change over these decades could be explained by the local-scale changes in nutrient availability, biotic interactions and land use that we were unable to consider in this broad-scale analysis. Our multivariate modelling demonstrates the close linkages be- tween soil pH and plant species compositional change, tracked by the Ellenberg R indicator, with atmospheric deposition effects on the plant communities mediated by the changes in soil condition. The potential exception to this full mediation was the direct respon- siveness of Ellenberg R to S deposition when the R score is measured as an unweighted average of the larger plot size. This particular for- mulation of the Ellenberg R score is more sensitive to rarer species and is also likely to be the least spatially coupled to the single point measurement of soil pH as it gives equal weight to plant species that are found in small numbers farther from the soil sampling location within the 200 m2 plot. As soil pH experiences high levels of spa- tial variation at a local scale (Ball & Williams, 1968), it may be that this link between S deposition and Ellenberg R change appears to be separate from soil pH change only due to this decoupling of the Ellenberg R and soil measurements. Surprisingly, we found only a limited interaction between management intensity and the Ellenberg R ~ pH relationship, despite having found divergent trends in soil pH and Ellenberg R change over time in high-intensity management hab- itats. 4 \| DISCUSSION The stability of Ellenberg R from 2007 to 2019, while soil pH decreased in high-intensity habitats could be related to lag effects or indicative of less close coupling of soil and vegetation change under intense management. However there was no indication of less close coupling of soil and vegetation change in the multivariate model results which showed a continued relationship between soil pH and Ellenberg R change in high intensity habitats. This was also unexpected due to the curvilinear relationship between Ellenberg R and soil pH, such that above pH 6 there tends to be a flattening of the Ellenberg R relationship with soil pH (Diekmann, 2003). Potential explanations for the persistence of the relationship between soil pH and Ellenberg R change in high-intensity management habitats in- clude the co-occurrence of soil pH and plant community interven- tions in highly managed areas, such that as the soil is limed there is also introduction of high Ellenberg R plant species within seed mixes. It should also be noted that there was a lower response of soil pH change to S deposition within these high-intensity management habitats, indicating that acidity changes in both soil pH and plant communities are less affected by atmospheric deposition. Therefore, despite there being no overall trend in Ellenberg R in high-intensity pasture over the time period studied we can still conclude that the high-intensity pasture sites that did undergo a change in Ellenberg R were on average sites that showed equivalent change in soil pH. While we identified a clear response by Ellenberg R to soil acid- ity, as originally intended by Ellenberg's scheme, species' Ellenberg R scores also tend to correlate with Ellenberg N (fertility) scores in a nonlinear fashion (Diekmann, 2003; Diekmann & Dupré, 1997). Our multivariate modelling demonstrates the close linkages be- tween soil pH and plant species compositional change, tracked by the Ellenberg R indicator, with atmospheric deposition effects on the plant communities mediated by the changes in soil condition. The potential exception to this full mediation was the direct respon- siveness of Ellenberg R to S deposition when the R score is measured as an unweighted average of the larger plot size. 4 \| DISCUSSION The inclusion of measurement error within our modelling frame- work allowed for identification of a stronger relationship between soil pH change and Ellenberg R change than would have been found otherwise. Measurement error affects every part of the sampling to analysis pipeline, and it has frequently been found to be exten- sive and pervasive in ecological and soil science (Desaules, 2012; Morrison, 2016). Within our data, there were greater levels of mea- surement error within the Ellenberg R estimates that were weighted by plant cover, and in the smaller plots. Taking all of these errors into account resulted in robust relationships between Ellenberg R change both over time and in relation to pH change across all four measurement methods of the plant community. While we might ex- pect the Ellenberg R scores weighted by plant cover to be more re- sponsive to environmental change, it may be that this is balanced out by the increased error of these measurements (Diekmann, 2003), which could explain previous results showing no greater correlation of cover weighted Ellenberg scores with environmental variables (Carpenter & Goodenough, 2014; Käfer & Witte, 2004). Inclusion of measurement error within our analysis did limit the analysis we could perform due to the limited quality assurance data from the early survey years; however, we consider this to be well worth it for the increased realism of our results. While we identified a clear response by Ellenberg R to soil acid- ity, as originally intended by Ellenberg's scheme, species' Ellenberg R scores also tend to correlate with Ellenberg N (fertility) scores in a nonlinear fashion (Diekmann, 2003; Diekmann & Dupré, 1997). Therefore, as the plants that prefer acidic environments also prefer low-fertility environments, the changing Ellenberg R may be reflect- ing the dual drivers of change of decreasing acid rain and increasing fertilisation (Maskell et al., 2010; Peppler-Lisbach et al., 2020). We attempted to explore this possibility by inclusion of N deposition 474 Journal of Ecology SEATON et al. peak and decline being greater in intensively managed grasslands. Plant communities have responded to this changing soil pH, with an overall increase in mean Ellenberg R in seminatural habitats. We have linked these ecosystem changes to changing seasonal rainfall and decreasing sulphur deposition, with no evidence of an additional effect of nitrogen deposition. DATA AVAILABILITY STATEMENT All vegetation, soils and atmospheric deposition data is avail- able for download from the NERC Environmental Information Data Centre. Vegetation data is available at: https://doi. org/10.5285/67bbfabb-d981-4ced-b7e7-225205de9c96 (Barr, Bunce, Smart, & Whittaker, 2014); https://doi.org/10.5285/26e79792-5ffc- 4116-9ac7-72193dd7f191 (Barr, Bunce, Gillespie, Hallam, et al., 2014); https://doi.org/10.5285/07896bb2-7078-468c-b56d-fb8b4 1d47065 (Barr, Bunce, Gillespie, Howard, et al., 2014); https://doi. org/10.5285/85c71959-0f7c-4f04-b3a7-152673107a85 (Bunce et al., 2014); https://doi.org/10.5285/fd6ae272-aeb5-4573-8e8a-7ccfae64f506 4 \| DISCUSSION Our findings indicate that ecosystem recovery from acidification is affected by changing precipitation patterns and intensity of land management, showing the importance of understanding the interplay of climate, land management and at- mospheric pollution when predicting and managing ecosystem re- covery from anthropogenic stressors. Our results indicate that positive effects on plant biodiversity and ecosystem service provision are likely to have resulted from the causal chain linking reduced S deposition to change in our index of plant acidity preferences. As this mean acidity index increases, con- ditions becomes more favourable to a larger pool of species. This also results in passive selection for larger subsets of species valued because of the support they provide to ecosystem services and functions (Supplementary Figure S4). These groups include plants that underpin positive services such as crop wild relatives (Jarvis et al., 2015), nectar-yielding species (Baude et al., 2016), agricultural forage grasses and food plants for lowland birds and butterfly larvae (Smart et al., 2000). However, there are other plant groups less ben- eficial to human activities such as injurious weeds that are also more common at higher Ellenberg R scores (Maskell et al., 2020). Overall, these changes in plant service provision indicate how changing at- mospheric deposition not only influences soil acidity and thus plant composition but also acts through these changes in plant communi- ties to influence the health of the whole ecosystem, including bird and pollinator communities. AUTHOR CONTRIBUTIONS Simon M. Smart conceived this project, with Fiona M. Seaton and Simon M. Smart designed the analyses with input from David A. Robinson, Don Monteith, Inma Lebron, and Bridget A. Emmett. David A. Robinson and Inma Lebron provided the soils data, Simon M. Smart the plant data and Sam Tomlinson the atmospheric deposi- tion data. Fiona M. Seaton carried out the statistical analysis with assistance from Paul Bürkner. Fiona M. Seaton wrote the first draft of the manuscript, with subsequent revisions by Simon M. Smart, Don Monteith, David A. Robinson, Sam Tomlinson, Paul Bürkner and Inma Lebron. All authors read and approved the final draft of this manuscript. The close linkages between the plant community and the soil chemical status we have found here demonstrate the range of po- tential future impacts of a variety of environmental stressors. Soil pH is an emergent property of the soil system that is responsive to a variety of factors as well as influencing a wide range of both microbially and chemically driven soil functions (Neina, 2019). The range of environmental stressors that ecosystems are currently ex- periencing can affect some elements of the soil and plant system more than others, for example soil fungal communities are known to be particularly responsive to synergistic effects of multiple stressors (Rillig et al., 2019). These changes in microbial communities could then feedback to soil pH changes with knock on effects and posi- tive feedback loops influencing soil and plant health. Moreover, it is clear that changes in soil pH driven by anthropogenic changes in atmospheric deposition and land management will have concomitant impact on soil and general ecosystem health. Of particular concern is the potential for changes to occur on different timeframes, as seen here within the changes in the plant community over time relative to changes in soil pH. There have also been observed changes in the soil pH ~ carbon relationship over time in GB soils (Thomas et al., 2020). This raises the possibility of a change in the mechanisms that drive soil and ecosystem functions and increasing disconnect between differing ecosystem components, such that future change will be- come more difficult to predict and mitigate (Hawkes & Keitt, 2015; Smith et al., 2009). The authors declare no competing interests. The authors declare no competing interests. ACKNOWLEDGEMENTS This work was funded by the Natural Environment Research Council (part of UK Research and Innovation) under the UK-SCAPE Programme delivering National Capability (Grant Reference NE/ R016429/1). The Countryside Survey was funded by a consortium of funders led by the Natural Environment Research Council and the Department for the Environment Food and Rural Affairs: the full list available at the Countryside Survey web-page (countrysidesurvey. org.uk). PEER REVIEW The peer review history for this article is available at https://publo ns.com/publon/10.1111/1365-2745.14039. REFERENCES Adamson, J. K., Scott, W. A., Rowland, A. P., & Beard, G. R. (2001). Ionic concentrations in a blanket peat bog in northern England and correlations with deposition and climate variables. European Journal of Soil Science, 52(1), 69–79. https://doi.org/10.1046/ j.1365-2389.2001.t01-1-00350.x Carpenter, B., Gelman, A., Hoffman, M. D., Lee, D., Goodrich, B., Betancourt, M., Brubaker, M. 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https://openalex.org/W4251423338	https://www.researchsquare.com/article/rs-939651/latest.pdf	English	null	Effects Of Spent Engine Oil On Soil Characteristics And Selected Phytochemicals In Amaranthus Hybridus	Research Square (Research Square)	2,021	cc-by	6,332	Effects Of Spent Engine Oil On Soil Characteristics And Selected Phytochemicals In Amaranthus Hybridus Arathy Arunan Swapna All Saints' College Rajani Vijayammal (  rajanijayasankar@gmail.com ) All Saints' College, TVPM, Kerala KSMDB College: Kumbalathu Sankupillai Memorial Devaswom Board College Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/11 Page 1/11 Abstract Spent engine oil is hazardous to the environment. Indiscriminate disposal of spent engine oil drain from engines after servicing has been found to affect the environment. An experiment was carried out to determine the effect of spent engine oil pollution on soil characteristics and the ability of Amaranthushybridusto thrive in the soil supplemented with varying concentrations of spent engine oil ranging from 50- 300 mL. Soil pH was slightly increased due to spent engine oil pollution. Nitrogen, phosphorus and potassium were reduced in the polluted soils and the soil organic carbon was increased. Soil samples polluted with spent engine oil showed increased bulk and particle densities and also decreased water holding capacity and porosity. The spent engine oil pollution affected the phytochemicals and resulted in the increased concentration of anti- nutrient tannin and decreased concentrations of nutrients like alkaloids, flavonoids, etc. The results of this study suggest that spent engine oil at any concentration seriously affects the soil properties and phytochemical analysis showed the inhibitory effects of spent engine oil on Amaranthushybridus. Materials The soil samples were collected from a domestic area at Anayara, Thiruvananthapuram district, Kerala. Soil samples were collected in 6 polythene bags. Better growing seeds of Amaranthushybridus(plate 1) used for the study were purchased from Agro bazar. Spent engine oil was obtained from an auto-mechanic workshop at Thiruvananthapuram. I. Plate 1: Amaranthushybridus Introduction Oil pollution is a serious threat to the environment and the ecosystem as a whole. Oil spill include any spill of crude oil or oil distilled products such as gasoline, diesel fuels, jet fuels, kerosene, Stoddard solvents, hydraulic oils, lubricating oils, etc., that can pollute the land, air and water environments. The soil system was considered to be the most important long term repository for these toxic compounds mainly PAHs and it also considered to be a steady indicator of the state of environmental pollution. Oil pollution changes the pH of the soil and thereby affects the physical, chemical and biological properties of the soil. Pore spaces in the soil might be clogged which could reduce soil aeration and water infiltration. It increases the bulk density subsequently affecting plant growth. Oil pollution also reduces and restricts the soil permeability. Oil pollution in soil may leads to the development of essential and non- essential elements in soil and the eventual translocation in plant tissues. This study was carried out with the sole objective to provide information on the effect of oil pollution on soil properties and the growth response of Amaranthushybriduscrops to spent engine oil with the view to establish minimum concentration of this contaminant could pose hazardous effects on its growth. Amaranthushybriduscrops to spent engine oil with the view to establish minimum concentration of this contaminant could pose hazardous effects on its growth. Treatment of soil samples Seedlings used for the study were raised from Amaranthus seeds and were planted in a grow bag. Seedlings were allowed to grow on the grow bags for 14 days to enable them attain a height range of 8-12 cm. 5 grow bags filled with soil samples were indicated as A, B, C, D and E for easy identification. The set A was polluted by soaking with 50 ml of Page 2/11 spent engine oil, sample B by 100 ml, sample C by 200 ml and finally sample D by 300 ml of spent engine oil. Treatment of the soil samples were carried out by manual mixing of soil with known volume of oil. The mixing was gradually done to obtain a thorough and even mixing. Soaked soil samples were left for 24 hours after which seedlings are planted in the bags. The set E served as the control which is devoid of spent engine oil. Seedlings of equal height (11 cm) were selected and transplanted into the grow bags in the order of A, B, C, D and E. Two seedlings were planted in a bag representing each treatment. Water was added to all samples daily. The rate of growth, seedling height and number of leaves were observed at intervals. spent engine oil, sample B by 100 ml, sample C by 200 ml and finally sample D by 300 ml of spent engine oil. Treatment of the soil samples were carried out by manual mixing of soil with known volume of oil. The mixing was gradually done to obtain a thorough and even mixing. Soaked soil samples were left for 24 hours after which seedlings are planted in the bags. The set E served as the control which is devoid of spent engine oil. Seedlings of equal height (11 cm) were selected and transplanted into the grow bags in the order of A, B, C, D and E. Two seedlings were planted in a bag representing each treatment. Water was added to all samples daily. The rate of growth, seedling height and number of leaves were observed at intervals. Analysis of soil parameters Analysis of soil parameters a. Estimation of Soil pH (S D Sawarkar 2012) b. Estimation of Electrical Conductivity (S D Sawarkar 2012) c. Estimation of Soil Organic Carbon (S D Sawarkar 2012) d. Estimation of Nitrogen (Bremner 1965) e. Estimation of Phosphorus (Bray and Kurtz 1945) f. Estimation of Potassium (Ghoshet al., 1983) g. Estimation of Water Holding Capacity (Viji and Prasanna P. Rajesh 2012) h. Estimation of Bulk Density, Particle Density and Porosity (Viji and Prasanna P. Rajesh 2012) a. Estimation of Soil pH (S D Sawarkar 2012) Analysis of Phytochemicals Analysis of Phytochemicals a. Estimation of Flavonoids (Zshishenet al., 1999) b. Estimation of Total Phenols (Chun et al., 2003) c. Estimation of Alkaloids (JoyammaVarkey andSeema S Nair, 2019) d. Estimation of Tannin (Selvakumaret al., 2019) e. Estimation of Phytic acid (Camire and Clydesdale, 2006) f. Estimation of Saponin(Makkar et al., 2007) 3.1.3. Soil Organic Carbon Organic carbon was higher in the soil sample D (2.07%) and it was relatively lower in control soil sample E (1.67%) (Table1). Amount of organic carbon in spent engine oil treated soil samples A, B, C and D were 1.71%, 1.95%, 1.98%, and 2.07% respectively (Table 1). According to Ekundayoet al. (1989), organic carbon content in the soil can be altered by spent engine oil pollution. Similarly, Okonokhua B O, et al., (2007) found that, the organic C (3.68) of the contaminated soils increased compared to the control (2.07). The organic carbon content was highest at the point of discharge of the spent engine oil (2–2.9%) having a mean value of 2.5%, the surrounding soil and control had the same mean OC value of 1.2% was observed in the study conducted by Otobong B. Iren and Victoria F. Ediene (2017). Nitrogen and organic carbon increased markedly with an increase in the oil treatment concentration for all the crops were found in a study conducted by Uquetan U I et al., in 2017. Similar results are also obtained in the study of Nwite J N and Alu M O (2015). The percentage of organic carbon (%OC) across the treatments of spent engine oil was significantly (P<0.05) higher than control. 3.1.4. Soil Nitrogen Nitrogen was found to be higher in the control soil sample E (Table 1). As the treatment concentration increased, the nitrogen content in the soil samples was decreased. The amount of nitrogen in the soil samples A, B, C, and D were 191, 184, 176 and 165 kg/ha respectively. The nitrogen content in the control soil sample was 203 kg/ha (Table 1). Similar results are found in the results of Kayodeet al. (2009). They observed that, there was reduced nitrogen in soil treated with spent lubricant oil. Many authors prove that pollution by petroleum products may cause a reduction in nitrogen levels in plants (Wyszkowski and Wyszkowska2005). Soils contaminated with petroleum substances causes changed nitrogen–carbon ratio mainly due to the presence of hydrocarbons. This contributes to the inhibition of many nitrogen involving reactions in the soil (regarding both mineral and organic forms of nitrogen), as well as to a reduction in the intensity of ammonification and nitrification (Adam and Duncan 2003; Kucharski and Jastrzębska2005) which may explain the reduced levels of nitrogen in the soil. 3.1.2. Soil Electrical conductivity Electrical Conductivity was higher in the control soil sample E (0.41 mhos/cm) and it was lower in soil sample D (0.21 mhos/cm) (Table 1). Electrical Conductivity of spent engine oil treated soil samples A, B, C, and D were found to be 0.26, 0.26, 0.24, and 0.21 respectively. The application of spent engine oil reduced the availability of exchangeable cations. This could be due to the presence of enhanced nutrients such as available Phosphorus. Kayodeet al., in 2009 and Uhegbuet al., in 2012 reported that spent engine oil pollution in the soils reduced the concentration of soil exchangeable cations. 3.1.1. Soil pH In the analysis of physico-chemical parameters of soil, the pH was found to be higher in soil sample D and it was lower in control soil sample E (Table 1). The soil became slightly alkaline in the sample D. The pH of the soil samples treated with 50, 100, 200 and 300 ml spent engine oil was 6.3, 6.5, 6.9 and 7.3 respectively (Table 1). This was agreed with the findings of Odjegba V J and Atebe J O (2007). They found that, soil pH was increased, when amended with used engine oil. The control (unpolluted) soil had a pH value of 7.1 and oil polluted soil had pH values ranging from 7.1 - 7.5. Also Nwite J N and Alu M O (2015) found that, pH was generally higher than the control and increased with increase in spent engine oil application across treatments. These findings may contradictory with the results of Oyem and Page 3/11 Page 3/11 Isama Lawrence (2013). That is spent engine oil pollution decrease the soil pH. Some other researchers found no variations on the soil pH by the addition of spent engine oil. There was no significant difference (P>0.05) in the soil pH between the control and the contaminated soil in the study by Okonokhua B O et al., (2007).The optimum soil pH for the growth of plants was between 6 and 7. At this pH, the seeds need good fertile soil and adequate moisture to germinate. High or low pH cause deficiencies in essential nutrients that plants need to grow and causes stunted growth and yellowing of leaves. 3.1.5. Soil Phosphorus Phosphorus content was highest in the control soil sample E (Table 1). Phosphorus content in the spent engine oil treated soil samples A, B, C and D were 86, 74, 70 and 64 kg/ha respectively. Phosphorus was reduced in the oil contaminated soils compared to the control and this agrees with the findings of Ogboghodoet al., (2004) and Okonokhua B O et al., (2007). In the study of Okonokhua B O et al., (2007), the phosphorus in the control was 5.76 Page 4/11 Page 4/11 ppm and it progressively decreased into 4.85 ppm in the treated soil samples. In a study of Uquetan U I et al., (2017) observed that the available phosphorus content decreased from 93.40mg/kg in the control to 50.24mg/kg in treatment concentration of 800ml/20kg and further decreased to 41.0mg/kg as the treatment concentration increased. Similarly, Nwite J N and Alu M O (2015) found that, available phosphorus of control was significantly (P<0.05) higher than those of spent engine oil application across the treatments. Available phosphorus decreased with increase in spent engine oil application with the one applied at 1.0 l/poly bag giving the least value of 10.20 mg kg-1. The available phosphorus of control was 83% higher than the one applied at 1.0 l/poly bag. 3.1.7. Soil Water Holding Capacity Water holding capacity of the soil was higher in the soil sample E (43.84%) and it is lower in the soil sample D (31.46%) (Table1). The water holding capacity of soil samples A, B, C and D are 41.10%, 38.70%, 31.53%, and 31.46% respectively. This observation was agreed with the study of Rasaiahet al., (1990). They observed a decreased soil water holding capacity in soils polluted with spent hydrocarbon oil. Spent lubricant oil pollution in the soil causes an increased bulk density, decreased water holding capacity and aeration propensity (Kayodeet al., 2009). Agbogidi and Enujeke (2012) reported that plots with spent oil contamination had reduced water infiltration and percolation. Reduced hydraulic conductivity results in a low “soil water transmission”. It also leads to less water availability for plant roots to access for photosynthetic processes. 3.1.8. Soil Bulk Density Bulk density was higher in the soil sample D (1.40g/cc) (Table 1) compared to the control soil sample. Bulk density of the soil samples A, B, C and D, treated with spent engine oil are 0.98, 1.17, 1.24, and 1.40g/cc respectively. The bulk density of control soil sample was 1.00g/cc. This result was agreed with the study done by Ewetola E Abosede (2013) observed that crude oil pollution increased bulk density and reduced total porosity of the soil. When comparing the control soil with polluted soil, the bulk density was increased by 7.1% and also the total porosity was reduced by 8.5%. This may result as blockage of pores spaces with the pollutant. Similar observations are also found in the study of Oyem and Isama Lawrence (2013). In this, values of the average bulk density obtained from Orgonoko and Arunton areas were1.31g/cm3 and 1.27g/cm3 respectively. But the oil impacted sample has the value of 4.16 g/cm3. Bulk density was higher in the soil sample D (1.40g/cc) (Table 1) compared to the control soil sample. Bulk density of the soil samples A, B, C and D, treated with spent engine oil are 0.98, 1.17, 1.24, and 1.40g/cc respectively. The bulk density of control soil sample was 1.00g/cc. This result was agreed with the study done by Ewetola E Abosede (2013) observed that crude oil pollution increased bulk density and reduced total porosity of the soil. When comparing the control soil with polluted soil, the bulk density was increased by 7.1% and also the total porosity was reduced by 8.5%. This may result as blockage of pores spaces with the pollutant. Similar observations are also found in the study of Oyem and Isama Lawrence (2013). In this, values of the average bulk density obtained from Orgonoko and Arunton areas were1.31g/cm3 and 1.27g/cm3 respectively. But the oil impacted sample has the value of 4.16 g/cm3. Similarly Kayodeet al., in 2009, observed an increased bulk density in soils polluted with spent lubricant oil. Effect of bulk density of the soil by spent engine oil pollution is evident in the study of Nwite J N and Alu M O (2015) that, bulk density of spent engine oil treated soil was significantly (P<0.05) higher than the control. The significant increase in bulk density of spent engine oil treated soil could be attributed to compaction resulting from oil contamination as well as reduced porosity. 3.1.6. Soil Potassium Potassium was higher in the soil sample E (220 kg/ha) and it was lower in the soil sample D (103 kg/ha) (Table 1). Potassium content in spent engine oil treated soil samples A, B, C and D are 152, 143, 121 and 103 kg/ha respectively. Similar observations are also found in the study of Nwite J N and Alu M O in 2015. Exchangeable cations of potassium were generally higher in control than in different levels of spent engine oil application. The exchangeable cations of K and Mg were decreased with increasing spent engine oil pollution in the soil. 3.1.9. Soil Particle Density Particle density was higher in the soil sample D (Table 1). The particle density of the soil samples A, B, C and D are 1.79, 1.96, 2.01 and 2.03 g/cc respectively. Particle density of the control was 1.62 g/cc, which was less than the particle densities of soil samples A, B, C and D. Increased bulk density of spent engine oil treated soil also affect the Page 5/11 Page 5/11 particle density of the soil. Compaction of soil results in increased particle density and also caused reduction in total porosity as the pore spaces could have been clogged by dispersed soil particles (Nwite J N and Alu M O, 2015). 3.2.1. Estimation of Alkaloids Alkaloid content in the leaf samples was high in the control E compared to the A, B, C, and D samples (Table 2). Alkaloids in the leaf samples A, B, C and D are 2.45, 2.18, 1.83, and 0.98 mg/100gm respectively. This observation was agreed with the results of OnyegemOkerentaet al., (2002). In their study, the alkaloid content in the plant material was significantly decreased due to the spent engine oil pollution. In a study conducted by Emurotu and Marvelous Olubunmi (2019), the bean plant grown in crude oil contaminated soil only alkaloid was present while the rest of phytochemicals (phenol, flavonoids) were absent. 3.1.10. Soil Porosity The porosity of soil sample was higher in the control soil sample E (44.13%). The porosity of spent engine oil treated soil samples A, B, C and D are 39.63%, 37.07%, 32.36%, and 17.09% respectively. Porosity was very less in the soil sample D (Table 1). This result was agreed with the study of Nwite J N and Alu M O (2015). There was an inverse relationship between bulk density and total porosity of the soil. Spent engine oil pollution increased the total porosity of the soils. This was mainly because of the compaction of the soils. In the polluted soils the pore spaces have been clogged by dispersed soil particles. Table 1 Analysis of soil parameters Soil Sample pH EC mhos/cm Org. C (%) N (kg/h) P (kg/ha) K (kg/ha) WHC (%) BD (g/cc) PD (g/cc) Porosity (%) A (50 mL) 6.3 0.26 1.71 191 86 152 41.10 0.98 1.79 39.63 B (100mL) 6.5 0.26 1.95 184 74 143 38.70 1.17 1.96 37.07 C (200mL) 6.9 0.24 1.98 176 70 121 31.53 1.24 2.01 32.36 D (300mL) 7.3 0.21 2.07 165 64 103 31.46 1.40 2.03 17.09 E (control) 6.2 0.41 1.67 203 92 220 43.84 1.00 1.62 44.13 3.2. Effect on selected phytochemicals of Amaranthushybridus 3 2 1 Estimation of Alkaloids 3.2. Effect on selected phytochemicals of Amaranthushybridus 3 2 1 E ti ti f Alk l id 3.2. Effect on selected phytochemicals of Amaranthushybridus 3.2.1. Estimation of Alkaloids 3.2.2. Estimation of Flavonoids Flavonoid content in the control sample was 0.82 mg/100gm and it was higher than that of the samples A, B, C and D. In a study conducted by Emurotu and Marvelous Olubunmi (2019), the bean plant grown in crude oil contaminated soil only alkaloid was present while the rest of phytochemicals were absent. Total phenol and flavonoid contents Page 6/11 Page 6/11 showed a decline of 57.6% and 41.3% respectively in the Amaranthuscruentus samples exposed to PAH pollution (Tandey R et al., 2020). showed a decline of 57.6% and 41.3% respectively in the Amaranthuscruentus samples exposed to PAH pollution (Tandey R et al., 2020). 3.2.4. Estimation of Tannin The tannin concentration was higher in the plant grown in highly polluted soil sample D (0.91mg/100gm). The tannin content was lower in the control sample E (Table 4). Tannin in the leaf samples A, B, C, and D was 0.51, 0.58, 0.65, and 0.91mg/100gm respectively. In food legumes, tannin that occurs naturally reacts with complexes of tannin- protein that form with protein which result to inactivation of some enzymes and protein insolubility as reported by Reddy N.R. et al (1985). 3.2.3. Estimation of Saponin The content of saponin in the leaf sample was higher in the control E (Table 2). Saponin content in leaf samples of the plants grown in the soil samples A, B, C, and D were 1.21, 1.03, 0.87 and 0.73 mg/100gm respectively. This result was agreed with the findings of OnyegemOkerentaet al., (2002). The impact of the pollution results in a marked reduction in saponin concentration in A. esculentus as the level of pollution in the soil increased respectively. The responsive reduction in the saponin content could be attributed to the higher levels of phytotoxic damage to the plant that might have been caused by spent engine oil pollution. 3.2.5. Estimation of Total Phenol The total phenol content in the leaf samples was higher in the control E (0.35mg/100gm) and it was lower in the leaf sample D (Table 2). The total phenol content in the leaf samples A, B, C, and D were 0.26, 0.19, 0.13, and 0.09 mg/100gm respectively. In a study conducted by Emurotu and Marvelous Olubunmi (2019), the bean plant grown in crude oil contaminated soil only alkaloid was present while the rest of phytochemicals were absent. Total phenol and flavonoid contents showed a decline of 57.6% and 41.3% respectively in the Amaranthuscruentus samples exposed to PAH pollution (Tandey R et al., 2020). 3.2.6. Estimation of Phytic Acid The phytic acid concentration was highest in the control sample E (Table 2). The phytic acid concentration in the leaf samples A, B, C, and D are 1.05, 0.87, 0.73 and 0.63 mg/100 gm respectively. Phytic acid concentration represents 50–85 % of total phosphorous in plants (Reddy et al., 1985). Page 7/11 Table 2 Phytochemical Screening of Amaranthushybridus Leaf Samples/ Phytochemicals Alkaloid (mg/100gm) Flavonoid (mg/100gm) Saponin (mg/100gm) Tannin (mg/100gm) Total Phenol (mg/100gm) Phytic acid (mg/100gm) A (50 mL) 2.45 0.71 1.21 0.51 0.26 1.05 B (100mL) 2.18 0.66 1.03 0.58 0.19 0.87 C (200mL) 1.83 0.62 0.87 0.65 0.13 0.73 D (300mL) 0.98 0.58 0.73 0.91 0.09 0.63 E (control) 3.54 0.82 1.68 0.49 0.35 1.32 Summary And Conclusion Table 2 Summary And Conclusion Oil pollution is a serious threat to the environment and the ecosystems. It can pollute neighbouring soil, surfaces and ground water; it may damage ecosystems and negatively affect the health of plants, animals and human beings. Spent engine oil affects the biological, physical, chemical and microbiological components of the soil and hence, it is affecting the growth, development, productivity and yield of plants. Oil pollution is a serious threat to the environment and the ecosystems. It can pollute neighbouring soil, surfaces and ground water; it may damage ecosystems and negatively affect the health of plants, animals and human beings. Spent engine oil affects the biological, physical, chemical and microbiological components of the soil and hence, it is affecting the growth, development, productivity and yield of plants. Spent engine oil pollution changes the pH of the soil and thereby affects the physical, chemical and biological properties of the soil. Pore spaces in the soil might be clogged which could reduce soil aeration and water infiltration. It increases the bulk density subsequently affecting plant growth, length of the shoot, number of leaves, etc. Here, a preliminary study was done on the effects of spent engine oil at different treatment concentrations on soil characteristics and some selected phytochemicals of Amaranthushybridus. Spent engine oil had a prominent negative impact on the soil characteristics, plant growth and the phytochemical content of Amaranthushybridus. This study revealed that as the treatment concentration increased, the soil characteristics are severely affected by spent engine oil pollution and it progressively reduced the fertility of the soil. 3.2.6. Estimation of Phytic Acid The most critical soil parameters like pH, organic carbon, bulk density, particle density, etc were got increased and electrical conductivity, nitrogen, phosphorus, potassium, water holding capacity, and porosity were decreased as the concentration of spent engine oil in the soil increased. The phytochemicals found in the leaf samples such as alkaloids, flavonoids, saponin, total phenol phytic acid, etc are reduced and the tannin is increased in the treatment concentrations. The present study reveals the effects of spent engine oil soil characteristics and selected phytochemicals in Amaranthushybridus. Amaranthushybridus that survived in contaminated soils became stunted. The discharge of spent engine oil in land should be prohibited because of its adverse effect on growth and development of plants. Consequently, the need to encourage the protection of farmlands and surroundings against the disposal of the oil cannot be overemphasized. Additionally, it increased the accumulation of harmful substances in plants which in turn, seriously impact the economy and human health. However, the intensity of the impact depends on the type of derivative found in the petroleum- derived products. Bioremediation supported by the utilization of microorganisms is an advantageous solution that helps in improving the growth of plants, also helps to reducing the harmful effects of petroleum-derived products. Nevertheless, to assess the full picture of the effects of spent engine oil, we recommend extending the scope of this study to soil microbiota in relation to changes observed in soil properties. Also, the extent of the current study can be widened via performing a detailed investigation the entire nutritional and anti-nutritional profiling and also the physiological measurement by analyzing antioxidants. Declarations Ethics approval and consent to participate- Not applicable Consent for publication- Not applicable Consent for publication- Not applicable Availability of data and materials- Not applicable Availability of data and materials- Not applicable Competing interests- Not applicable Competing interests- Not applicable Funding- Not applicable Authors' contributions- A S performed the review, data collection, a part of analysis and writing of the manuscript; V has contributed interpretation including the editing of manuscript and also the corresponding author; S R performed phytochemical analysis. Page 8/11 Acknowledgements- The authors would like to thank Department of Environmental Sciences, All Saints College, TVPM for the completion of the work. Library support from the college is also acknowledged. Acknowledgements- The authors would like to thank Department of Environmental Sciences, All Saints College, TVPM for the completion of the work. Library support from the college is also acknowledged. Authors' information (optional)- Authors' information (optional)- 1. Arathy Arunan Swapna , Dept of Environmental Sciences, All Saints’ College, Thiruvananthapuram, Kerala 2. Rajani Vijayammal, Assistant Professor, Dept of Environmental Sciences, All Saints’ College, Thiruvananthapuram Kerala 1. Arathy Arunan Swapna , Dept of Environmental Sciences, All Saints’ College, Thiruvananthapuram, Kerala 1. Arathy Arunan Swapna , Dept of Environmental Sciences, All Saints’ College, Thiru 1. Arathy Arunan Swapna , Dept of Environmental Sciences, All Saints’ College, Thiruvananthapuram, Kerala 2. Rajani Vijayammal, Assistant Professor, Dept of Environmental Sciences, All Saints’ College, Thiruvananthapuram, Kerala 2. 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https://openalex.org/W4391546362	https://www.scielo.br/j/rbepid/a/7CdP5dR4H6QsWvJhx4QFTXz/?lang=en&format=pdf	English	null	Factors associated with hospitalizations and deaths of pregnant women from Paraná due to COVID-19: a cross-sectional study	Revista brasileira de epidemiologia	2,024	cc-by	8,133	Factors associated with hospitalizations and deaths of pregnant women from Paraná due to COVID-19: a cross-sectional study Avenida Colombo, 5790, Jardim Universitário, CEP: 87020-900, Maringá (PR), Brasil. E-mail: larissasbergantini@gmail.c CONFLICT OF INTERESTS: nothing to declare HOW TO CITE THIS ARTICLE: Bergantini LS, Ichisato SMT, Salci MA, Birolim MM, Santos MLA, Höring CF, et al. Factors associated with hospitalizations and dea of pregnant women from Paraná due to COVID-19: a cross-sectional study. Rev Bras Epidemiol. 2024; 27: e240005. https://doi.org/10.1590/1980-549720240005 This is an open article distributed under the CC-BY 4.0 license, which allows copying and redistribution of the material in any format and for any purpose as long the original authorship and publication credits are maintained. Received on: 08/11/2023 Reviewed on: 11/14/2023 Accepted on: 11/14/2023 Objective: To analyze the factors associated with hospitalization in the ward and intensive care unit (ICU), and with death from COVID-19 in pregnant women with confirmed cases. Methods: Observational, cross-sectional study, carried out with data from pregnant women with a confirmed case of COVID-19 from the Influenza Epidemiological Surveillance Information System and the Paraná’s state COVID-19 notification system. The association between the independent and dependent variables (hospitalization in the ward and ICU, and death) was investigated using the Poisson regression model with robust variance. Results: 4,719 pregnant women comprised the study population. 9.6 and 5.1% were hospitalized in wards and ICU, respectively. 1.9% died. There was an association between advanced maternal age and hospitalization in wards (PR=1.36; 95%CI 1.10–1.62) and ICU (PR=2.25; 95%CI 1.78– 2.71), and death (PR=3.22; 95%CI 2.30–4.15). An association was found between the third trimester and hospitalization in wards (PR=5.06; 95%CI 2.82–7.30) and ICU (PR=6.03; 95%CI 3.67–8.39) and death (PR=13.56; 95%CI 2.90–24.23). The second trimester was associated with ICU admission (PR=2.67; 95%CI 1.36–3.99). Pregnant women with cardiovascular disease had a higher frequency of hospitalization in wards (PR=2.24; 95%CI 1.43–3.05) and ICU (PR=2.66; 95%CI 1.46–3.87). Obesity was associated with ICU admission (PR=3.79; 95%CI 2.71–4.86) and death (PR=5.62; 95%CI 2.41–8.83). Conclusions: Advanced maternal age, the end of the gestational period and comorbidities were associated with severe COVID-19. Keywords: COVID-19. Cross-sectional studies. Pregnant women. Hospitalization. Intensive care units. Maternal mortality. HOR: Larissa Silva Bergantini. Avenida Colombo, 5790, Jardim Universitário, CEP: 87020-900, Maringá (PR), Brasil. E-mail: larissasbergantini@gmail.com CORRESPONDING AUTHOR: Larissa Silva Bergantini. Avenida Colombo, 5790, Jardim Universitário, CEP: 87020-900, Maringá (PR), Brasil. E-mail HOW TO CITE THIS ARTICLE: Bergantini LS, Ichisato SMT, Salci MA, Birolim MM, Santos MLA, Höring CF, et al. https://doi.org/10.1590/1980-549720240005 Revista Brasileira de Epidemiologia www.scielo.br/rbepid ORIGINAL ARTICLE www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 Revista Brasileira de Epidemiologia Factors associated with hospitalizations and deaths of pregnant women from Paraná due to COVID-19: a cross-sectional study Fatores associados a hospitalizações e óbitos de gestantes paranaenses por COVID-19: estudo transversal Larissa Silva BergantiniI , Sueli Mutsumi Tsukuda IchisatoII , Maria Aparecida SalciII , Marcela Maria BirolimIII , Márcia Lorena Alves dos SantosIV , Carla Franciele HöringIV , Roberta RossaII , Luiz Augusto FacchiniV IUniversidade Estadual de Maringá, Postgraduate Program in Physiological Sciences – Maringá (PR), Brazil. IIUniversidade Estadual de Maringá, Postgraduate Program in Nursing – Maringá (PR), Brazil. IIICentro Universitário Guairacá, Postgraduate Program in Health Promotion – Guarapuava (PR), Brazil. IVUniversidade Estadual de Maringá, Department of Statistics – Maringá (PR), Brazil. VUniversidade Federal de Pelotas, Department of Social Medicine – Pelotas (RS), Brazil. Factors associated with hospitalizations and deaths of pregnant women from Paraná due to COVID-19: a cross-sectional study Fatores associados a hospitalizações e óbitos de gestantes paranaenses por COVID-19: estudo transversal Fatores associados a hospitalizações e óbitos de gestantes paranaenses por COVID-19: estudo transversal Larissa Silva BergantiniI , Sueli Mutsumi Tsukuda IchisatoII , Maria Aparecida SalciII , Marcela Maria BirolimIII , Márcia Lorena Alves dos SantosIV , Carla Franciele HöringIV , Roberta RossaII , Luiz Augusto FacchiniV Larissa Silva BergantiniI , Sueli Mutsumi Tsukuda IchisatoII , Maria Aparecida SalciII , Marcela Maria BirolimIII , Márcia Lorena Alves dos SantosIV , Carla Franciele HöringIV , Roberta RossaII , Luiz Augusto FacchiniV IUniversidade Estadual de Maringá, Postgraduate Program in Physiological Sciences – Maringá (PR), Brazil. IIUniversidade Estadual de Maringá, Postgraduate Program in Nursing – Maringá (PR), Brazil. IIICentro Universitário Guairacá, Postgraduate Program in Health Promotion – Guarapuava (PR), Brazil. IVUniversidade Estadual de Maringá, Department of Statistics – Maringá (PR), Brazil. VUniversidade Federal de Pelotas, Department of Social Medicine – Pelotas (RS), Brazil. VUniversidade Federal de Pelotas, Department of Social Medicine – Pelotas (RS), Brazil. ABSTRACT ABSTRACT Objective: To analyze the factors associated with hospitalization in the ward and intensive care unit (ICU), and with death fro COVID-19 in pregnant women with confirmed cases. Methods: Observational, cross-sectional study, carried out with data fro pregnant women with a confirmed case of COVID-19 from the Influenza Epidemiological Surveillance Information System and t Paraná’s state COVID-19 notification system. The association between the independent and dependent variables (hospitalization the ward and ICU, and death) was investigated using the Poisson regression model with robust variance. Results: 4,719 pregna women comprised the study population. 9.6 and 5.1% were hospitalized in wards and ICU, respectively. 1.9% died. There was association between advanced maternal age and hospitalization in wards (PR=1.36; 95%CI 1.10–1.62) and ICU (PR=2.25; 95%CI 1.7 2.71), and death (PR=3.22; 95%CI 2.30–4.15). An association was found between the third trimester and hospitalization in war (PR=5.06; 95%CI 2.82–7.30) and ICU (PR=6.03; 95%CI 3.67–8.39) and death (PR=13.56; 95%CI 2.90–24.23). The second trimester w associated with ICU admission (PR=2.67; 95%CI 1.36–3.99). Pregnant women with cardiovascular disease had a higher frequency hospitalization in wards (PR=2.24; 95%CI 1.43–3.05) and ICU (PR=2.66; 95%CI 1.46–3.87). Obesity was associated with ICU admissi (PR=3.79; 95%CI 2.71–4.86) and death (PR=5.62; 95%CI 2.41–8.83). Conclusions: Advanced maternal age, the end of the gestation period and comorbidities were associated with severe COVID-19. Keywords: COVID-19. Cross-sectional studies. Pregnant women. Hospitalization. Intensive care units. Maternal mortality. CORRESPONDING AUTHOR: Larissa Silva Bergantini. Study design Observational, cross-sectional research, which used secondary data from two COVID-19 epidemiological sur- veillance databases: SIVEP-Gripe and the specific informa- tion system of the state of Paraná, called Sistema Estadual Notifica COVID-19 (Notifica COVID-19). In relation to women of reproductive age, who were not pregnant, symptomatic pregnant women were at increased risk of developing COVID-19 in its severe form10, and, con- sequently, of admission to an intensive care unit (ICU)10-13, of needing mechanical ventilation10-12, and of death10,13. The first 18 months of the pandemic were defined as the investigation period. Specifically, the study comprised pregnant women notified in SIVEP-Gripe between March 29th, 2020 (date of the first registration of a pregnant wom- an from Paraná in this base) and September 29th, 2021, and those registered in Notifica COVID-19, from March 30th, 2020 (date of the first notification of a pregnant woman from Paraná in the state system) to September 30th, 2021. The date of notification of the case in the aforementioned systems was considered for the inclusion of pregnant women in the study. In Brazil, more than 24 thousand cases of severe acute respiratory syndrome (SARS) due to COVID-19 have been recorded in pregnant and postpartum women, according to data from the Brazilian Obstetric Observatory SARS (Ob- servatório Obstétrico Brasileiro SRAG – OOBr SRAG) updated on August 2nd, 2023. 23.7% of these women required inten- sive care, and 8.4% of them died14. Pregnant women from low- and middle-income coun- tries are more vulnerable to the negative outcomes of COVID-19, such as death15. Added to this, the distribution of the number of maternal deaths due to the pathology in Brazilian states appears to be asymmetric, possibly due to socioeconomic differences resulting in unequal access to health services16,17. This points to the relevance of conduct- ing research with this population in the Brazilian context. The research was described based on the guidelines of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) initiative24. Study background The state of Paraná, in South Brazil, is the most popu- lous in the region, and fifth in the Brazilian rank according to data from the 2022 Demographic Census25. The human development index (HDI) of Paraná is equivalent to 0.769, the seventh highest in the country26. Until August 2023, the state had the fourth highest number of confirmed cases and deaths from COVID-19 in Brazil27. g p p Considering that pregnant women are more susceptible to the worsening of COVID-19, it is crucial to determine the con- ditions related to severe cases of the disease. In this context, the presence of comorbidities can act as a risk factor, as well as advanced maternal age18, defined by the Brazilian Ministry of Health as the pregnancy of women aged 35 years old or older19. The influence of the gestational period on the predispo- sition to severe manifestations of COVID-19 is a controversial point in the literature: there are studies that observed associ- ations between the trimester of pregnancy and the severity of the disease20,21, and others that did not find this relationship18,22. Furthermore, despite advances in scientific knowledge on this topic, analyses developed in Brazil involving preg- nant and postpartum women predominantly investigat- ed the risks of morbidity and mortality in the in-hospital setting, based on data from the Influenza Epidemiological Surveillance Information System (Sistema de Informação Considering that pregnant women are more susceptible to the worsening of COVID-19, it is crucial to determine the con- ditions related to severe cases of the disease. In this context, the presence of comorbidities can act as a risk factor, as well as advanced maternal age18, defined by the Brazilian Ministry of Health as the pregnancy of women aged 35 years old or older19. INTRODUCTION de Vigilância Epidemiológica da Gripe – SIVEP-Gripe), which monitors SARS cases17,23. In March 2020, the World Health Organization recog- nized the Coronavirus disease 2019 (COVID-19) pandemic1, caused by Severe Acute Respiratory Syndrome Coronavi- rus 2 (SARS-CoV-2)2, as a Public Health Emergency of Inter- national Concern. The disease has multisystemic manifes- tations3, and its spectrum varies from asymptomatic, mild and moderate, to severe and fatal stages4. This research aimed to contribute to the understand- ing of the relationship between COVID-19 and pregnancy by exploring the factors associated with severe conditions, considering pregnant women affected at different levels of severity, including non-hospitalized cases. Thus, the ob- jective was to analyze the factors associated with hospital- ization in the ward and ICU, and death from COVID-19 in pregnant women with a confirmed case. Pregnant and postpartum women, who were initially not considered more prone to severe conditions compared to the general population5, constitute a vulnerable group for severe forms of the pathology6. Immunological, anatomical, and physiological changes generate a state of greater sus- ceptibility to respiratory infections in women during the ges- tational period7,8. Furthermore, unfavorable prognoses were observed in pregnant women affected by other coronavirus- es from the family of the etiological agent of COVID-199. Factors associated with hospitalizations and deaths of pregnant women from Paraná due to COVID-19: a cross-sectional study Factors associated with hospitalizations and deaths of pregnant women from Paraná due to COVID-19: a cross-sectional study. Rev Bras Epidemiol. 2024; 27: e240005. https://doi.org/10.1590/1980-549720240005 This is an open article distributed under the CC-BY 4.0 license, which allows copying and redistribution of the material in any format and for any purpose as long as the original authorship and publication credits are maintained. Received on: 08/11/2023 Reviewed on: 11/14/2023 Accepted on: 11/14/2023 Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 1 https://doi.org/10.1590/1980-549720240005 www.scielo.br/rbepid Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 2 Participants The influence of the gestational period on the predispo- sition to severe manifestations of COVID-19 is a controversial point in the literature: there are studies that observed associ- ations between the trimester of pregnancy and the severity of the disease20,21, and others that did not find this relationship18,22. The study population consisted of pregnant women residing in and notified by the state of Paraná, with a con- firmed case of COVID-19, aged between 10 and 49 years, and without reservations regarding the gestational trimes- ter. Women who did not require hospitalization due to the disease were included, in addition to those who required hospitalization, in a ward or ICU, regardless of whether the condition progressed to cure or death. Furthermore, despite advances in scientific knowledge on this topic, analyses developed in Brazil involving preg- nant and postpartum women predominantly investigat- ed the risks of morbidity and mortality in the in-hospital setting, based on data from the Influenza Epidemiological Surveillance Information System (Sistema de Informação Women with incomplete information on the following were excluded from the analysis: gestational age, date of first www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 Data Sources symptoms, outcome (cure or death), and place of hospital- ization (ward or ICU). Asymptomatic hospitalized pregnant women were not included, with the aim of avoiding hospital- izations caused by obstetric reasons, such as childbirth. Information was extracted from SIVEP-Gripe and Notifi- ca COVID-19 databases combined. SIVEP-Gripe is a nation- wide information system that monitors SARS cases, whose notification is mandatory in the country. In 2020, COVID-19 surveillance was incorporated into SIVEP-Gripe28. The processes for defining the study population are il- lustrated in Figure 1. he study period corresponded to the first 18 months of the pandemic, that is, the period from March 2020 to September 2021. gure 1. Flowcharts for sample selection of pregnant women from the SIVEP-Gripe (left) and Notifica C * The study period corresponded to the first 18 months of the pandemic, that is, the period Entries in SIVEP-Gripe until 10/19/2021 (n=202,176) Entries in Notifica COVID-19 until 11/16/2021 (n=5,787,621) Non-target population (n=200,201) Non-target population (n=5,742,467) Notified pregnant women (n=2,005) Notified pregnant women (n=45,154) Pregnant women without a diagnosis of COVID-19 (n=1,031) Pregnant women without a diagnosis of COVID-19 (n=36,520) Pregnant women with a confirmed case (n=974) Pregnant women with a confirmed case (n=8,634) Pregnant women with incomplete data (n=73) Notifications outside the study period (n=82) Pregnant women with incomplete data (n=4,341) Notifications outside the study period (n=156) Pregnant women notified during the study period* (n=819) Pregnant women notified during the study period* (n=4,137) Pregnant women from other states (n=41) Asymptomatic pregnant women (n=85) Pregnant women from other states (n=52) Pregnant women with duplicated names in SIVEP-Gripe (n=59) Pregnant women included (n=693) Pregnant women included (n=4,026) Final population (n=4,719) Entries in Notifica COVID-19 until 11/16/2021 (n=5,787,621) Non-target population (n=5,742,467) Notified pregnant women (n=45,154) Entries in Notifica COVID-19 until 11/16/2021 (n=5,787,621) Entries in SIVEP-Gripe until 10/19/2021 (n=202,176) Entries in SIVEP-Gripe until 10/19/2021 (n=202,176) until 11/16/2021 (n=5,787,621) Pregnant women without a diagnosis of COVID-19 (n=1,031) Pregnant women with a confirmed case (n=974) Pregnant women with a confirmed case (n=8,634) Pregnant women with incomplete data (n=73) Notifications outside the study period (n=82) Pregnant women notified during the study period* (n=4,137) Pregnant women notified during the study period* (n=819) Final population (n=4,719) The study period corresponded to the first 18 months of the pandemic, that is, the period from March 2020 to September 2021. Figure 1. Flowcharts for sample selection of pregnant women from the SIVEP-Gripe (left) and Notifica COVID-19 (right) databases. RESULTS A total of 4,719 pregnant women with COVID-19 made up the study population. Women’s mean age was 27.8 years (SD=6.4; median=27); 25.4% were in the first, 38.1% in the second, and 36.5% in the third trimester of pregnancy; 40.5% were white, but 45.5% were missing this information. The objective was to model the Y/t=rate of pregnant women hospitalized in the ward; Z/t=rate of pregnant women admitted to the ICU; and W/t=rate of pregnant women who died, where t corresponds to the total number of pregnant women in Paraná diagnosed with COVID-19 in the first 18 months of the pandemic. Furthermore, 586 (12.4%) women were asymptomat- ic at the time of notification, and 4,133 (87.6%) had some symptoms. Most common signs and symptoms included coughing (61.9%), sore throat (41.8%), fever (32.2%), dys- pnea (23.1%), symptoms related to the gastrointestinal tract (diarrhea, nausea or vomiting) (11.9%), and oxygen saturation below 95% (7.2%). To construct the analysis, only the explanatory vari- ables common to both databases were selected, namely: age range, subdivided into younger than 35 years (refer- ence category) and 35 years or more; gestational period, categorized as first (reference category), second, and third trimesters; and the following comorbidities: heart disease, diabetes, obesity and others, all dichotomized as yes or no (reference category). Uncompleted (ignored) records for comorbidities were included in the “no” group. Of the total number of infected pregnant women, 4,026 (85.3%) did not require hospitalization and received outpa- tient care; 693 (14.7%) were hospitalized. Considering all those notified, 453 (9.6%) women were treated in the ward and 240 (5.1%) in the ICU. The age range variable was categorized based on the advanced maternal age stratification proposed by the Min- istry of Health19. The race/color factor was excluded from the inferential analysis due to the high number of incom- plete cases. The majority of patients (98.1%) recovered from the dis- ease. 92 (1.9%) deaths from COVID-19 were recorded, 87 among those hospitalized (94.6%), 12 in the ward and 75 in the ICU, and five (5.4%) among pregnant women who were not hospitalized, registered in Notifica COVID-19. Data Sources The study period corresponded to the first 18 months of the pandemic, that is, the period from March 2020 to September 2021. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 3 s for sample selection of pregnant women from the SIVEP Gripe OVID 19 (right) databases https://doi.org/10.1590/1980-549720240005 www.scielo.br/rbepid tion (SD) and absolute and relative frequencies. Inferen- tial statistical analysis was performed using R® software, version 4.1.1. Records of mild and asymptomatic cases of COVID-19 in Brazilian territory were sent to the e-SUS Notifica28 system. In the state of Paraná, however, the State Health Secretar- iat (Secretaria de Estado da Saúde – SESA/PR) implemented Notifica COVID-19, its own system for monitoring mild oc- currences. Subsequently, data from Notifica COVID-19 is passed on to e-SUS Notifica. The Poisson regression model with robust variance was used to investigate possible associations between explan- atory variables and responses based on the estimation of crude and adjusted prevalence ratios (PR) and the respec- tive 95% confidence intervals (95%CI). For this research, data were collected from non-hos- pitalized pregnant women, treated only on an outpatient basis, through the Notifica COVID-19 database. Occur- rences of hospitalization in the ward and ICU were iden- tified using the SIVEP-Gripe database. In order to avoid duplicates, the pregnant women’s names were checked in both databases and, in case of duplicity, only the SIVEP- Gripe data were kept. The variables that presented p-value <0.20 in the crude analysis were maintained in the multiple model. Backward selection was used to determine which variables would remain in the final model, with non-significant variables (p-value>5%) being removed one by one from the final model. The association between variables was considered significant when the 95% confidence interval did not in- clude the value 1.00.i Versions of databases that are not available to the gen- eral public and that contain sensitive data were analyzed. The SIVEP-Gripe database provided to the authors by SESA/ PR covered notifications made until October 19th, 2021, and the Notifica COVID-19 database until November 16th, 2021. The information was collected in December 2021. The Wald test was applied to verify the significance of the estimated coefficients. The quality of fit of the models was evaluated according to deviance and the likelihood ra- tio test (significance level of 5%). Characteristics The research was approved with Certificate of Presenta- tion of Ethical Appreciation (Certificado de Apresentação de Apreciação Ética – CAAE) number 34787020.0.0000.0104/2020, by protocol number 4.165.272/2020. Three outcomes were determined for the analysis: hospitalization in the ward, hospitalization in the ICU, and death due to COVID-19. In the present research, response variables are counts and, therefore, have a discrete quan- titative nature. They can be expressed as: Y=number of pregnant women hospitalized in the ward, Z=number of pregnant women hospitalized in the ICU, and W=number of pregnant women who died. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 4 Statistical methods Furthermore, 32.5% of those hospitalized for COVID-19 had more than eight years of education, however, there was a predominance (56.1%) of incomplete records for Data were subjected to descriptive analysis in the Ex- cel® software to calculate median, mean, standard devia- www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 this variable. Almost all (90.9%) of hospitalized pregnant women lived in the urban area, and 41.1% were part of the Eastern health macro-region, which encompasses Curitiba (Paraná’s capital) and its metropolitan region. There was no association between diabetes, other comor- bidities and the three response variables. Obesity did not re- main in the final model for the response variable hospitaliza- tion in the ward, and heart disease did not remain in the death outcome, as both presented p-value>5% (backward selection). The average length of stay was approximately nine days (SD=11.5; median=5), with 42.6% of those hospitalized not needing to use ventilatory support. Among those assisted in the ward, 58.3% did not receive ventilatory therapy, while the majority (85%) of patients in the ICU used the treat- ment. Considering the 87 hospitalized women who died, eight (9.2%) did not use ventilatory support. These eight patients were hospitalized for an average of nine days, and most were admitted to the hospital in the first half of 2021. The deviance test applied to evaluate the quality of fit of the models obtained p-values>5%, demonstrating the suitability of the proposed models for the three outcomes. The adjusted analysis identified that pregnant women aged 35 or over were almost 1.4 times more likely to be hospitalized in a ward (95%CI 1.10–1.62) compared to pa- tients below this age range. Pregnant women in the third trimester were approxi- mately five (95%CI 2.82–7.30) times more likely to be ad- mitted to the ward compared to those in the first trimester. There was no statistically significant difference between women in the second and first trimesters for this outcome. Furthermore, having heart disease doubled (95%CI 1.43– 3.05) the probability of a pregnant woman with COVID-19 requiring hospitalization in a ward. Of the total number of patients who required hospital- ization, 108 (15.6%) received invasive ventilation. Among those hospitalized who used invasive ventilatory support, 49 (45.4%) survived and 59 (54.6%) died. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 5 Statistical methods In Table 1, the prevalence of hospitalization in the ward and ICU and the proportions of deaths due to COVID-19 are described according to the categories of explanatory variables in relation to the total number of pregnant women reported. The probability of admission to the ICU was increased by approximately two (95%CI 1.78–2.71) times among pregnant women of advanced maternal age. Compared to women in the first trimester, the probability of admission to the ICU for those in the second and third trimesters in- creased by almost three and six times, respectively. Table 2 presents the crude prevalence ratios of the sur- vey response variables according to the explanatory vari- ables. In Table 3, the adjusted prevalence ratios are shown according to the variables that remained in the multiple model for each outcome. Table 1. Prevalence of hospitalization in wards and intensive care units and proportions of deaths in relation to the total population of pregnant women according to the categories of independent variables. Paraná, Brazil, March 2020 to September 2021 Table 1. Prevalence of hospitalization in wards and intensive care units and proportions of deaths in relation to the total population of pregnant women according to the categories of independent variables. Paraná, Brazil, March 2020 to September 2021 Characteristics Outcomes Total notifications (n=4,719) Hospitalization in a ward (n=453) Hospitalization in ICU (n=240) Death (n=92) n Prevalence (%) n Prevalence (%) n Proportion (%) n Age range (years) <35 353 9.01 159 4.06 54 1.38 3,918 ≥35 100 12.48 81 10.11 38 4.74 801 Gestational trimester First 42 3.50 17 1.42 3 0.25 1,199 Second 103 5.72 71 3.94 31 1.72 1,800 Third 308 17.91 152 8.84 58 3.37 1,720 Comorbidity Heart disease No 436 9.36 221 4.75 85 1.83 4,656 Yes 17 26.98 19 30.16 7 11.11 63 Diabetes No 427 9.35 218 4.77 82 1.80 4,568 Yes 26 17.22 22 14.57 10 6.62 151 Obesity No 429 9.42 202 4.44 74 1.62 4,554 Yes 24 14.55 38 23.03 18 10.91 165 Others No 443 9.64 231 5.03 87 1.89 4,597 Yes 10 8.20 9 7.38 5 4.10 122 ICU: intensive care unit. www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 Table 2. Crude prevalence ratios of outcomes according to age group, gestational trimester, and comorbidities. Paraná, Brazil, March 2020 to September 2021. Table 2. Crude prevalence ratios of outcomes according to age group, gestational trimester, and comorbidities. Paraná, Brazil, March 2020 to September 2021. Table 2. Crude prevalence ratios of outcomes according to age group, gestational trimester, and comorbidities. Paraná, Brazil, March 2020 to September 2021. Patients with obesity were more likely to be hospital- ized in the ICU, with a probability practically four (95%CI 2.71–4.86) times higher than those without the comorbidi- ty. Having heart disease increased the probability of need- ing intensive care by around two times (95%CI 1.46–3.87). The probability of death was three (95%CI 2.30–4.15) times higher among pregnant women aged 35 years old or older. Being in the third trimester increased the chance of death from COVID-19 by approximately 13 (95%CI 2.90– 24.23) times, however, there was no statistically significant difference with regard to the second and first trimesters of pregnancy. The adjusted analysis found an association be- tween obesity and death, increasing the probability of the outcome by approximately five (95%CI 2.41–8.83) times. ization in the ward and ICU, and death due to COVID-19. The second trimester was related to the need for intensive care. Having a heart disease increased the probability of admission to the ward and ICU. There was an association between obesity and ICU hospitalization and death. Patients with obesity were more likely to be hospital- ized in the ICU, with a probability practically four (95%CI 2.71–4.86) times higher than those without the comorbidi- ty. Having heart disease increased the probability of need- ing intensive care by around two times (95%CI 1.46–3.87). The probability of death was three (95%CI 2.30–4.15) times higher among pregnant women aged 35 years old or older. Being in the third trimester increased the chance of death from COVID-19 by approximately 13 (95%CI 2.90– 24.23) times, however, there was no statistically significant difference with regard to the second and first trimesters of pregnancy. The adjusted analysis found an association be- tween obesity and death, increasing the probability of the outcome by approximately five (95%CI 2.41–8.83) times. The use of secondary data implies limitations inher- ent to this set of information: there was a high number of records with important elements not filled out, which resulted in purification of the study population and made it impossible to assess the impact of race/color on severe outcomes of COVID-19. Ethnic-racial disparities in relation to the risk of infection and mortality have been reported among pregnant women10, therefore, the non-inclusion of health determinants in this analysis is a limitation. Table 2. Crude prevalence ratios of outcomes according to age group, gestational trimester, and comorbidities. Paraná, Brazil, March 2020 to September 2021. The probable underreporting of asymptomatic cases is a limit- ing factor. This research was carried out in a limited con- text, the state of Paraná, therefore, generalizing the results to other locations with different socioeconomic and health characteristics must be cautious. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 6 Statistical methods Characteristics Outcomes Hospitalization in a ward Hospitalization in ICU Death PRc (95%CI) p* PRc (95%CI) p* PRc (95%CI) p* Age range (years) <35 (reference) 1.00 - 1.00 - 1.00 - ≥35 1.39 (1.10–1.72) 0.003 2.49 (1.90–3.24) <0.001 3.44 (2.26–5.19) <0.001 Gestational trimester First (reference) 1.00 - 1.00 - 1.00 - Second 1.63 (1.15–2.36) 0.007 2.78 (1.68–4.88) <0.001 6.88 (2.46–28.68) <0.001 Third 5.11 (3.75–7.16) <0.001 6.23 (3.89–10.68) <0.001 13.48 (4.99–55.24) <0.001 Comorbidity Heart disease No (reference) 1.00 - 1.00 - 1.00 - Yes 2.88 (1.70–4.52) <0.001 6.35 (3.85–9.87) <0.001 6.09 (2.56–12.22) <0.001 Diabetes No (reference) 1.00 - 1.00 - 1.00 - Yes 1.84 (1.21–2.68) 0.002 3.05 (1.91–4.62) <0.001 3.69 (1.79–6.77) <0.001 Obesity No (reference) 1.00 - 1.00 - 1.00 - Yes 1.54 (1.00–2.28) 0.038 5.19 (3.62–7.25) <0.001 6.71 (3.89–10.97) <0.001 Others No (reference) 1.00 - 1.00 - 1.00 - Yes 0.85 (0.42–1.50) 0.612 1.47 (0.70–2.69) 0.258 2.17 (0.76–4.81) 0.092 Regarding the Wald Test; ICU: intensive care unit; PRc: Crude prevalence ratio; 95%CI: 95% confidence interval. DISCUSSION Adjusted analysis showed an association between age equal to or over 35 years old, as well as the third trimester of pregnancy and the three severity outcomes: hospital- Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 6 www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 Table 3. Adjusted prevalence ratios of outcomes according to the variables that remained in the final model. Paraná, Brazil, March 2020 to September 2021. Characteristics Outcomes Hospitalization in a ward Hospitalization in ICU Death PRa (95%CI) p† PRa* (95%CI) p† PRa* (95%CI) p† Age range (years) <35 (reference) 1.00 - 1.00 - 1.00 - ≥35 1.36 (1.10–1.62) <0.001 2.25 (1.78–2.71) <0.001 3.22 (2.30–4.15) <0.001 Gestational trimester First (reference) 1.00 - 1.00 - 1.00 - Second 1.63 (0.92–2.34) 0.028 2.67 (1.36–3.99) <0.001 6.74 (0.88–12.61) <0.001 Third 5.06 (2.82–7.30) <0.001 6.03 (3.67–8.39) <0.001 13.56 (2.90–24.23) <0.001 Comorbidity Heart disease No (reference) 1.00 - 1.00 - - - Yes 2.24 (1.43–3.05) <0.001 2.66 (1.46–3.87) <0.001 - - Obesity No (reference) - - 1.00 - 1.00 - Yes - - 3.79 (2.71–4.86) <0.001 5.62 (2.41–8.83) <0.001 *All variables were adjusted for those that remained in the multiple model; †Regarding the Wald Test; ICU: intensive care unit; PRa: Adjusted prevalence ratio; 95% CI: 95% confidence interval. Table 3. Adjusted prevalence ratios of outcomes according to the variables that remained in the final model. Paraná, Brazil, March 2020 to September 2021. Table 3. Adjusted prevalence ratios of outcomes according to the variables that remained in the final model. Paraná, Brazil, March 2020 to September 2021. A study carried out with 240 pregnant women with con- firmed cases of SARS-CoV-2 infection found that hospital- ized patients were more likely to have at least one comor- bidity, such as hypertension, type two diabetes, obesity, among others29. rized the products of 77 studies, did not observe a correla- tion between advanced gestational age and complications arising from the disease18. Another longitudinal analysis found that the gestation- al trimester was not associated with an increased risk of developing moderate to severe and critical forms of the pathology. The authors themselves, however, highlighted that the low representation of pregnant women in the first trimester of pregnancy could restrict the understanding of the interference of this variable22. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 7 DISCUSSION Similarly, advanced maternal age and the existence of any comorbidity, including obesity, chronic lung dis- ease, and hypertension, stood out as risk factors for the moderate to severe and critical condition of COVID-19 in a cohort consisted of 7,950 pregnant women22, in agreement with the present findings. A dose-response effect was found with regard to comorbidities and severe forms of the disease22. On the other hand, a study carried out in Brazil found a predominance of deaths due to COVID-19 in the third tri- mester of pregnancy or in the postpartum period16. Like- wise, the majority of patients hospitalized with the pathol- ogy in an analysis carried out in the United States were in the third trimester29. Two studies, which specifically evaluated ICU admission as a response variable, observed that advanced age and comorbidities increased the risk of the outcome23,30. One of these analyses carried out in Brazil demonstrated that obesity and diabetes were prominent in relation to the risk of ICU admission among comorbidities23. A cohort study developed by North American research- ers found that pregnant women affected by COVID-19 were three times more likely to be admitted to the ICU com- pared to those without the disease, and that the risk was increased by infection during the third trimester31. Two re- view studies also indicated that the last gestational trimes- ter was associated with the severity of the pathology20,21. These results converge with those of this research. Regarding death from COVID-19, the two studies cit- ed23,30 corroborated the results obtained here: advanced maternal age and the presence of comorbidities were relat- ed to mortality from the disease. A second Brazilian survey reinforced that having associated clinical conditions, such as obesity, diabetes, and cardiovascular disease, increased the risk of death17. The anatomic-functional changes observed mainly in the third trimester, such as those in the respiratory system (increased oxygen demand and limitation of lung expansion, for example)32,33, could form the pathophys- Regarding the influence of the gestational period on the prognosis of COVID-19, a systematic review, which summa- www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 iological mechanism that would explain the increased susceptibility of pregnant women affected at the end of their pregnancy34. tributed to the understanding that pregnant women at the end of the gestational period may be more likely to develop severe cases of COVID-19. REFERENCES 1. World Health Organization. WHO Director-General’s opening remarks at the media briefing on COVID-19 — 11 March 2020 [Internet]. 2020 [cited on Aug 8, 2023]. Available at: https://www.who.int/director-general/speeches/detail/who- director-general-s-opening-remarks-at-the-media-briefing- on-covid-19---11-march-2020 2. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020; 5: 536-44. https://doi.org/10.1038/s41564-020-0695-z Furthermore, the so-called indirect effects of the pan- demic on maternal health should also be mentioned38. Measures implemented around the world to prevent, control, and treat COVID-19 have resulted in difficulties in transportation and access to healthcare, discontinuity of maternal care services and shortages of human and ma- terial resources, such as personal protective equipment and medicines39. 3. Gupta A, Madhavan MV, Sehgal K, Nair N, Mahajan S, Sehrawat TS, et al. Extrapulmonary manifestations of COVID- 19. Nat Med. 2020; 26(7): 1017-32. https://doi.org/10.1038/ s41591-020-0968-3 3. Gupta A, Madhavan MV, Sehgal K, Nair N, Mahajan S, Sehrawat TS, et al. Extrapulmonary manifestations of COVID- 19. Nat Med. 2020; 26(7): 1017-32. https://doi.org/10.1038/ s41591-020-0968-3 4. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Guia de vigilância epidemiológica: emergência de saúde pública de importância nacional pela doença pelo coronavírus 2019 [Internet]. Brasília: Ministério da Saúde; 2022 [cited on Aug 8, 2023]. Available at: https://www.gov.br/saude/pt-br/ coronavirus/publicacoes-tecnicas/guias-e-planos/ guia-de-vigilancia-epidemiologica-covid-19/view Responses to the pandemic, particularly in developing countries, may have intensified pre-existing social dispari- ties and exacerbated chronic deficits in health systems37,39. Thus, in Brazil, it is suggested that the significant impact of COVID-19 on maternal health is also linked to precarious obstetric care, access impairment, and social risk17. This research found that around 13% of pregnant wom- en hospitalized in the ICU did not receive ventilatory sup- port; 9.2% of those hospitalized who died did not use the therapy, with the majority of these women being admitted to the hospital in the first half of 2021, between March and June, a period in which there was a significant increase in confirmed cases and deaths due to COVID-19 and, con- sequently, overload of the Brazilian health system40. Five patients who did not survive the disease did not receive hospital care. This highlights that obstacles to accessing intensive care can contribute to the mortality of pregnant and postpartum women17. 5. Chen L, Li Q, Zheng D, Jiang H, Wei Y, Zou L et al. Clinical characteristics of pregnant women with Covid-19 in Wuhan, China. DISCUSSION Furthermore, this analysis em- phasized that pregnant women of advanced maternal age, and equally patients with comorbidities, should receive special attention in the pandemic context. Furthermore, the assertion that pregnancy corresponds to a state of immunosuppression per se is inaccurate34. In the third trimester of pregnancy until delivery, a pro-in- flammatory disposition prevails in the woman’s body. In this sense, it is assumed that infection by SARS-CoV-2 could worsen this process during the final phase of the gestation- al period33. Identifying vulnerable women by the team involved in maternal and child care enables to develop intervention strategies that can prevent or alleviate injuries resulting from COVID-19. Finally, these findings can contribute to the construction of approaches to the continuing education of health professionals, policy formulation, organization of services, tracking of pregnant women, in addition to con- tributing to the development of future studies. The most frequent symptoms among pregnant women with COVID-19 investigated in this research were coughing, sore throat, fever, and dyspnea, similar to those identified by other authors23,35. As previously mentioned, in developing countries, the impact of COVID-19 on maternal health may be discrepant compared to developed nations36. In Brazil, the significant consequences of the disease in pregnant and postpartum women can be explained, primarily, by clinical issues. In the national territory, the main cause of maternal death is hypertensive disorders19, which, combined with COVID-19, can deteriorate the prognosis during the gestational pe- riod. Added to this are the high prevalence of overweight and obesity among Brazilian pregnant women, both pro-in- flammatory states17,37. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 8 REFERENCES N Engl J Med. 2020; 382(25): e100. https://doi. org/10.1056/NEJMc2009226 5. Chen L, Li Q, Zheng D, Jiang H, Wei Y, Zou L et al. Clinical characteristics of pregnant women with Covid-19 in Wuhan, China. N Engl J Med. 2020; 382(25): e100. https://doi. org/10.1056/NEJMc2009226 6. Brasil. Ministério da Saúde. Secretaria de Atenção Primária à Saúde. Departamento de Ações Programáticas e Estratégicas. Manual de recomendações para a assistência à gestante e puérpera frente à pandemia de Covid-19 [Internet]. Brasília: Ministério da Saúde; 2021 [cited on Aug 8, 2023]. Available at: https://bvsms.saude.gov.br/bvs/publicacoes/manual_ assistencia_gestante_puerpera_covid-19_2ed.pdf 6. Brasil. Ministério da Saúde. Secretaria de Atenção Primária à Saúde. 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Rev Bras Epidemiol. 2024; 27: e240005 10 REFERENCES Comorbidity, poverty and social vulnerability as risk factors for mortality in pregnant women with confirmed SARS-CoV-2 infection: analysis of 13 062 positive pregnancies including 176 maternal deaths in Mexico. Ultrasound Obstet Gynecol. 2022; 59(1): 76-82. https://doi.org/10.1002/uog.24797 37. Nakamura-Pereira M, Amorim MMR, Pacagnella RC, Takemoto MLS, Penso FCC, Rezende-Filho J, et al. COVID- 19 and maternal death in Brazil: an invisible tragedy. Rev Bras Ginecol Obstet. 2020; 42(8): 445-7. https://doi. org/10.1055/s-0040-1715138 31. Doyle TJ, Kiros GE, Schmitt-Matzen EN, Propper R, Thompson A, Phillips-Bell GS. Maternal and perinatal outcomes associated with SARS-CoV-2 infection during pregnancy, Florida, 2020– 2021: a retrospective cohort study. Clin Infect Dis. 2022; 75(Suppl 2): S308-S316. https://doi.org/10.1093/cid/ciac441 38. Lucas DN, Bamber JH. Pandemics and maternal health: the indirect effects of COVID-19. Anaesthesia. 2021; 76(Suppl 4): 69-75. https://doi.org/10.1111/anae.15408 32. Wastnedge EAN, Reynolds RM, van Boeckel SR, Stock SJ, Denison FC, Maybin JA, et al. Pregnancy and COVID-19. Physiol Rev. 2021; 101(1): 303-18. https://doi.org/10.1152/physrev.00024.2020 39. Kingsley JP, Vijay PK, Kumaresan J, Sathiakumar N. The changing aspects of motherhood in face of the COVID-19 pandemic in low- and middle-income countries. Matern Child Health J. 2021; 25(1): 15-21. https://doi.org/10.1007/ s10995-020-03044-9 33. Ferrer-Oliveras R, Mendoza M, Capote S, Pratcorona L, Esteve-Valverde E, Cabero-Roura L, et al. Immunological and physiopathological approach of COVID-19 in pregnancy. Arch Gynecol Obstet. 2021; 304(1): 39-57. https://doi.org/10.1007/s00404-021-06061-3 40. Fundação Oswaldo Cruz. Um balanço da pandemia em 2021 em um cenário de incertezas e falta de dados [Internet]. Boletim do Observatório Covid-19; 2021 [cited on Oct 9, 2023]. Available at: https://portal.fiocruz.br/sites/portal.fiocruz.br/ files/documentos/boletim_covid_2021-retrospectiva.pdf 34. Thompson JL, Nguyen LM, Noble KN, Aronoff DM. COVID-19- related disease severity in pregnancy. Am J Reprod Immunol. 2020; 84(5): e13339. https://doi.org/10.1111/aji.13339 Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 10 www.scielo.br/rbepid https://doi.org/10.1590/1980-549720240005 RESUMO RESUMO Objetivo: Analisar os fatores associados à hospitalização em enfermaria e unidade de terapia intensiva (UTI), e ao óbito pela COVID-19 em gestantes com caso confirmado. Métodos: Pesquisa observacional, transversal, realizada com dados de gestantes com caso confirmado para COVID-19 provenientes do Sistema de Informação de Vigilância Epidemiológica da Gripe e do Sistema Estadual Notifica COVID-19, do Paraná. Investigou-se a associação entre variáveis independentes e dependentes (hospitalização em enfermaria e UTI, e óbito) pelo modelo de regressão de Poisson com variância robusta. Resultados: 4.719 gestantes compuseram a população do estudo; 9,6 e 5,1% foram hospitalizadas em enfermaria e UTI, respectivamente; 1,9% evoluíram para óbito. FUNDING: This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel — Brazil (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — Brasil – CAPES) — Financing Code 001. REFERENCES Houve associação entre a idade materna avançada e internação em enfermaria (RP=1,36; IC95% 1,10–1,62) e UTI (RP=2,25; IC95% 1,78–2,71), e óbito (RP=3,22; IC95% 2,30–4,15). Verificou-se associação entre o terceiro trimestre gestacional e hospitalização em enfermaria (RP=5,06; IC95% 2,82–7,30) e UTI (RP=6,03; IC95% 3,67–8,39) e óbito (RP=13,56; IC95% 2,90–24,23). O segundo trimestre associou-se à internação em UTI (RP=2,67; IC95% 1,36–3,99). Gestantes com cardiopatia apresentaram maior frequência de hospitalização em enfermaria (RP=2,24; IC95% 1,43–3,05) e UTI (RP=2,66; IC95% 1,46–3,87). A obesidade foi associada à admissão em UTI (RP=3,79; IC95% 2,71–4,86) e ao óbito (RP=5,62; IC95% 2,41–8,83). Conclusão: A idade materna avançada, o final do período gestacional e comorbidades foram fatores associados a quadros graves de COVID-19. OVID-19. Estudos transversais. Gestantes. Hospitalização. Unidades de terapia intensiva. Mortalidade materna. alavras-chave: COVID-19. Estudos transversais. Gestantes. Hospitalização. Unidades de terapia intensiva. Mor AUTHORS’ CONTRIBUTIONS: Bergantini, L.S.: Project administration, Conceptualization, Writing – original draft, Writing – review & editing, Investigation, Methodology, Validation, Visualization. Ichisato, S.M.T.: Project administration, Conceptualization, Writing – original draft, Writing – review & editing, Methodology, Supervision, Visualization. Salci, M.A.: Conceptualization, Writing – review & editing, Methodology, Visualization. Birolim, M.M.: Writing – review & editing, Methodology, Validation, Visualization. Santos, M.L.A.: Formal analysis, Data curation, Writing – review & editing, Investigation, Methodology, Validation. Höring, C.F.: Formal analysis, Data curation, Writing – review & editing, Investigation, Methodology, Validation, Visualization. Rossa, R.: Methodology, Validation, Visualization. Facchini, L.A.: Conceptualization, Writing – review & editing, Methodology, Supervision, Validation, Visualization. FUNDING: This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel — Brazil (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — Brasil – CAPES) — Financing Code 001. Hospitalizações e óbitos de gestantes paranaenses por COVID-19. Rev Bras Epidemiol. 2024; 27: e240005 11 © 2024 \| Epidemio is a publication of Associação Brasileira de Saúde Coletiva - ABRASCO © 2024 \| Epidemio is a publication of Associação Brasileira de Saúde Coletiva - ABRASCO
https://openalex.org/W4231863859	https://www.qeios.com/read/OHDS6H/pdf	English	null	Vatalanib Succinate	Definitions	2,020	cc-by	102	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Vatalanib Succinate National Cancer Institute National Cancer Institute Qeios ID: OHDS6H · https://doi.org/10.32388/OHDS6H Source National Cancer Institute. Vatalanib Succinate. NCI Thesaurus. Code C74945. The succinate salt of vatalanib, an anilinophthalazine derivative, with antineoplastic activity. Vatalanib binds to and inhibits the protein kinase domain of vascular endothelial growth factor receptors 1 and 2; both receptor tyrosine kinases are involved in angiogenesis. This agent also binds to and inhibits related receptor tyrosine kinases, including platelet-derived growth factor (PDGF) receptor, c-Kit, and c-Fms. Qeios ID: OHDS6H · https://doi.org/10.32388/OHDS6H 1/1
https://openalex.org/W4388831988	https://ojs.academicon.pl/np/article/download/6567/7499	Polish	null	Kościoły i kaplice Chęcin	Nasza Przeszłość	1,982	cc-by-sa	10,980	KS. MARIAN PAULEWICZ KS. MARIAN PAULEWICZ 1. KOŚCIÓŁ SW. STANISŁAWA W STAROCHĘCINACH Pierwsza świątynia parafii została wybudowana jeszcze przed założeniem zamku i miasta, na terenie wsi Chęciny. Początki tego kościoła wiążą się z osobą właściciela wsi Chęciny Mikulona, któ ry występuje w dokumentach historycznych drugiej po&owy XIII w.1. Mikulon będąc zamożnym dygnitarzem księstwa sando mierskiego, wzorem ówczesnych możnowładiców polskich, w swoich dobrach rodzinnych ufundował kościół i nadał mu za patrona, po wszechnie wówczas czczonego, a niedawno kanonizowanego św. Stanisława biskupa i męczennika. Kościół ten, zapewne już od po czątku swego istnienia był kościołem parafialnym. Został on zbu dowany z drewna i przetrwał do końca XVII w.2. Kiedy Władysław Łokietek przejął w swoje ręce wieś Chęciny i dołączył ją do więk szego zespołu wsń książęcych, zmieniła się również rola tego kościo ła. Stał się on wówczas kościołem parafialnym dla rozległego te renu włości książęcych, zaś prawo patronatu przeszło na monar chów Polski. Kiedy zostaje założone miasto Chęciny, pierwotne Chęciny — wieś, przyjmują nazwę Stare Chęciny. Nowo lokowane miasto przez parę dziesiątków lat, mimo iż ma już własny ko ściół, który jest filialnym w stosunku do kościoła św. Stanisława, nadal należy do tej parafii. Kościół parafialny św. Stanisława spełnił podobną rolę wobec kościoła w mieście, jak wieś Chęciny wobec miasta. Wieś ta przekazała swą nazwę zamkowi i póź niejszemu miastu, sama stając się Starymi Chęcinami, zwykłą ci chą wsią, nie odgrywającą żadnego poważniejszego znaczenia. Po dobnie i kościół w tej wsi, pierwsza świątynia chęcińska, był ko ściołem macierzystym, nie tylko kościoła parafialnego św. Bartło mieja w mieście, ale i wszystkich innych świątyń na terenie mia sta Chęcin i parafii. Spełnił .on swoją rolę dziejową, a kiedy cen trum życia przeniosło się na Górę Żarnikową i do miasta, powoli zaczął pustoszeć i chylić się ku upadkowi, a z niim również cała rozległa parafia starochęoińska3. W tym czasie dobra staro stwa chęcińskiego należą do królowej Elżbiety, wdowy po Kazimie rzu Jagiellończyku, jako jej wiano. Królowa ta widząc, że parafia i kościół w Starochęcinach upadają, wystarała się u swego syna kardynała Fryderyka Jagiellończyka, arcybiskupa gnieźnieńskiego Po klęsce, jaka dotknęła Chęciny w czasie najazdu szwedzkiego, kościół św. Stanisława ulegał stopniowo całkowitej ruinie i zni szczeniu. Nowy został wzniesiony na miejscu pierwotnego w latach 1680—1690 z fundacji Stefana Bidizińskłego starosty chęcińskiego, według projektu Tylmana z Gameren, architekta Jana III. Nowy kościół został wykończony przed 1718 r.6. Kościół ten został zbu dowany w stylu barokowym, na planie krzyża greckiego o Czterech krótkich jednoprzęsłowych ramionach. KOŚCIOŁY I KAPLICE CHĘCIN Na terenie parafia Chęciny, powstałej w końcu XIII w., w oią- gu 700 lat jej istnienia było jedenaście świątyń. W liczbie tej sześć kościołów i pięć kaplic. Dwa kościoły były parafialne, dwa klasztorne, jeden szpitalny i jeden górniczy. Każda z pięciu kaplic pełniła inną rolę. Były to kaplice: zamkowa, klasztorna, grobowa, filialna i cmentarna. Spośród tych jedenastu świątyń tylko sześć służy dzisiaj celom sakralnym, dwie przestały istnieć, zaś trzy przebudowane, utraciły charakter budynków sakralnych i służą celom świeckim. Z tych trzech ostatnich jedna jest domem miesz kalnym, druga salą restauracyjną, a trzecia barem kawowym. Dziwne, a zarazem ciekawe dzieje tych świątyń, stanowią przedmiot niniejszej pracy. Budowle sakralne parafii Chęciny według nazwy i tytułu świątyni, czasu powstania, pełnionej aktualnie funkcji r4 łb Nazwa świątyń czas Pełniona funkcja 1. kościół św. Stanisława XIII w. kościół rektoralny 2. .kaplica zamkowa św. 10 tys. m. XIII w. w ruinie 3. kościół św. Bartłomieja XIV w. kościół parafialny 4. kościół Wniebowzięcia N.M.P. XIV w. sala restauracyjna 5. kościół św. Jakuba XIV w. nie istnieje 6. kościół św. Ducha XV w. dom mieszkalny 7. kościół św. Marii Magdaleny XVI w. kościół klasztorny 8. kaplica św. Leonarda XVI w. bar 9. kaplica Trzech Króli XVII w. kaplica k-ła par. 10. kaplica M. B. Anielskiej — Polichno XIX w. kaplica rektoralna 11. kaplica cmentarna XX w. kaplica pogrzebo wa Budowle sakralne parafii Chęciny według nazwy i tytułu świątyni, czasu powstania, pełnionej aktualnie funkcji K O Ś C IO Ł Y I K A P L IC E CH ĘCIN K S. M A R IA N P A U L E W IC Z [3] [2 ] 2 1 2 213 4 J. Ł a s k i , Liber Beneficiorum Archidiecezji Gnieźnieńskiej, wyd. J. Łukowskii, Gmiezno 11880 T. 1 s. 584. 5 Tamże. 6 Katalog zabytków sztuki w Polsce, T. 3, Warszawa 1SÓ7 z. 4 s. 55; A. B a r t a c z k o w a , M. K o z i ń s k a , Chęciny, s. 2; J. C z a r - n e c k i, Chęciny i okolice, Warszawa 1954 s. 53. 7 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 13—22. 8 Lustracja woj. sandomierskiego 1789, Cz. I, wyd. H. Madurowicz — Urbańska, Wrocław—Warszawa— Kraków 1865 s. 172. 9 W. G r z e l i ń s k i , Monografia Chęcin, s. 56. I KOŚCIOŁY i biskupa krakowskiego1, że ten specjalnym dekretem wydanym w 1500 r. połączył obydwie parafie w jedną. Proboszczem jedynej odtąd parafii Chęciny mianował kardynał Jana de Panczelicze4. Odtąd przy kościele św. Stanisława rezyduje wikariusz manualny, spełniający funkcje duszpasterskie w imieniu proboszcza rezydują- oego w mieście przy kościele św. Bartłomieja5. 4 J. Ł a s k i , Liber Beneficiorum Archidiecezji Gnieźnieńskiej, wyd. J. Łukowskii, Gmiezno 11880 T. 1 s. 584. 5 Tamże. 1 F. P i e k o s i ń s k i , Rycerstwo polskie wieków średnich, Kraików 1001). T. 3 s. 177—il78; Kodeks Dyplomatyczny Małopolski wyd. F. Pie- kosińskii, Kraków 1876 T. 1 rur 88 s. 1015—(106, , ( , 2 A. B a r t a c z k o w ą, M. K o z i ń s k a , Chęciny — studium hi- star yc zn o-urb anist y c zn€, Warszawa 1'957 s. 1>—2 (Maszynopis WAP Kielce). Kielce). ’ W. G r z e l i ń s k i , Monografia Chęcin, Kielce 1908 s. 56. 1 F. P i e k o s i ń s k i , Rycerstwo polskie wieków średnich, Kraików 1001). T. 3 s. 177—il78; Kodeks Dyplomatyczny Małopolski wyd. F. Pie- kosińskii, Kraków 1876 T. 1 rur 88 s. 1015—(106, 2 A. B a r t a c z k o w ą, M. K o z i ń s k a , Chęciny — studium hi- star yc zn o-urb anist y c zn€, Warszawa 1'957 s. 1>—2 (Maszynopis WAP Kielce). ’ W. G r z e l i ń s k i , Monografia Chęcin, Kielce 1908 s. 56. 4 J. Ł a s k i , Liber Beneficiorum Archidiecezji Gnieźnieńskiej, wyd. J. Łukowskii, Gmiezno 11880 T. 1 s. 584. 5 Tamże. 6 Katalog zabytków sztuki w Polsce, T. 3, Warszawa 1SÓ7 z. 4 s. 55; A. B a r t a c z k o w a , M. K o z i ń s k a , Chęciny, s. 2; J. C z a r - n e c k i Chęciny i okolice Warszawa 1954 s 53 ) ’ W. G r z e l i ń s k i , Monografia Chęcin, Kielce 1908 s. 56. 1. KOŚCIÓŁ SW. STANISŁAWA W STAROCHĘCINACH Nad częścią centralną kwa dratową, kopuła z latarnią na ośmiobocznym bębnie wspartym na żagdelkach. Bogata barokowa dekoracja stiukowa wykonana zo stała w XVII wieku. Nad arkadą tęczową anioły podtrzymują tar czę z herbem Janina, Stefana Bidzińskiego. Do wystroju kościoła weszły, wykonane wówczas, trzy ołtarze barokowe. Obok kościoła, równocześnie z nim, wzniesiono dwie analogiczne dzwonnice 7. W niedługim czasie, bo już w 1789 r., świątynia ta znalazła się w opłakanym stanie, prawie cała zrujnowana. Lustratorzy króle wscy oglądając ją, we wspomnianym roku, takie wydają o niej świadectwo: „Jest w tej wsi kościółek czyli kaplica publiczna mu rowana w krzyż kawalerski, piękna i ozdobna, całkiem zaniedbana, bo dach na niej zły, okna powybijane. Dwie dzwonnice bez da chów, puste stoją” 8. W aktach chęcińskich jest wzmianka, że w 1787 r. dano pozwolenie na zburzenie zrujnowanego kościoła i wy korzystanie materiału otrzymanego z rozbiórki na budowę szpitala i szkoły. Sytuacja polityczna, jaka wytworzyła się po rozbiorach Polski i zaszłe wówczas zmiany, nie pozwoliły wykonać tych pla nów. W 1844 r. kościół św. Stanisława podźwignął z ruiny i od budował gen. Storożemko, Obdarowany dobrami byłego starostwa chęcińskiego 9. W ostatnich latach, a mianowicie w 1969 r., kościół ten stał się świątynią rektoralną. Zamieszkał przy nim ksiądz rektor, któ ry spełnia w nim wszystkie funkcje duszpasterskie dla mieszkań K S . M A R IA N P A U L E W IC Z [4] 214 K O Ś C IO Ł Y I K A P L IC E CHĘCIN [5] 215 ców: Starorihęcm, Lapowicy, Przymiarek i Podzamcza Chęcińskiego. Dla wiernych ze wspomnianych miejscowości kościół św. Stani sława stanowi ąuasi kościół parafialny. ców: Starorihęcm, Lapowicy, Przymiarek i Podzamcza Chęcińskiego. Dla wiernych ze wspomnianych miejscowości kościół św. Stani sława stanowi ąuasi kościół parafialny. okres stopniowego rozwoju i rozbudowy kościoła. Dziięki rozkwi towi górnictwa kruszcowego w starostwie chęcińskim, miasto prze zywa złote czasy swojego rozwoju. Nic też dziwnego, że w tak rozwijającym się wspaniale mieście, kościół parafialny staje się coraz piękniejszy i bogatszy. Zamożni mieszczanie zarówno kupcy, jak i gómniicy czynią na rzecz tego kościoła liczne fundacje i daro wizny16. W drugiej połowie XVI w. urząd starosty chęcińskiego pełni Stanisław Szafraniec, właściciel Secemina. Porzuca on kato licyzm i przyjmuje kalwinizm, przyczyniając się znacznie do roz szerzenia tego odłamu protestantyzmu w Małopolsce. Wspaniale uposażony i bogaty kościół parafialny w Chęcinach, staje się ła komym kąskiem dla innowierców. 10 M. P a u l e w i c z , Początki górnictwa chęcińskiego i fazy jego rozwoju do początku XVII w. (w:) Biuletyn Informacyjny 1(,Kopa,syny”, Kielce, sierpień 1975 s. 14. 16 J. Ł a s k i, Liber Beneficiorum, s. 584. 17 Lustracja woj. sandomierskiego 1789, Cz. 1 s. 165 18 W. Gr z e 1 i ń s k i, Monografia Chęcin, s. 41. 19 Tamże. 20 Lustracja woj. sandomierskiego 1789, Cz. 1 s. 165. 21 F. M. S o b i e s z e z a ń s k i, Wycieczka archeologiczna w niektóre strony gubemii radomskiej w miesiącu wrześniu 1851 roku. Waonszawa 1852 s. 1,94. 22 Katalog zabytków sztuki w Polsce, T. 3 z. 4 Bi. 13. 1. KOŚCIÓŁ SW. STANISŁAWA W STAROCHĘCINACH Kalwini mając poparcie współ wyznawcy starosty chęcińskiego Szafrańca, w 1568 r. zdobywają i przejmują w posiadanie kościół parafialny w Chęcinach. W czasie swego użytkowania świątyni doprowadzają ten kościół do opłaka nego stanu17. W trzy lata później Szafraniec przekazuje kościół arianom i sam przechodzi, do tego odłamu protestantyzmu pol skiego 18. Kiedy katolicy utracili swój kościół parafialny, ówczesny proboszcz przenosi się do kościoła św. Ducha, który na przeciąg prawie trzydziestu lat staje się kościołem parafialnym dla parafii chęcińskiej 19. Kiedy proboszczem chęcińskim zostaje były sekretarz królewski Mateusz Post, wówczas przy pomocy starosty chęciń skiego Stanisława z Ruszczy Branickiego odzyskuje utracony ko ściół. Zygmunt III na sejmie generalnym w Warszawie, dnia 27 marca 1597 r. zatwierdza wówczas wszystkie dawne przywileje tego kościoła20. Kiedy kościół powrócił w ręce katolików przed stawiał żałosny wlidok. Był całkowicie spustoszony; ołtarze, na czynia, sprzęty, obrazy — zniszczone lub zrabowane. Dopiero po odnowieniu i ponowlneij konsekracji w 1603 r. zaczęto w nim od prawiać nabożeństwa21. Teraz też następuje dlia1 kościoła czas po nownego rozkwitu i zatarcia śladów zniszczeń i dewastacji. W 1628 r. zostaje konsekrowany nowy wczesnoibarokowy główny oł tarz, ozdobiony bramkami, rzeźbami świętych, proroków i anio łów. Następnie świątynia ubogaca się o pięć nowych bocznych ołtarzy, wzniesionych również w stylu barokowym22. 18 W. Gr z e 1 i ń s k i, Monografia Chęcin, s. 41. 19 Tamże. g f ę , , 15 J. D l u g o s z, Liber Beneficiorum Dioecesi Cracoviensis, Kraków 1863, T. 1 s. 466. ’ 13 Informację o wezwaniu kościoła w Trzebnicy zasięgnął autor od duszpasterzy kościoła par. w Trzebnicy na Śląsku. , p 11 J. Ł a s k i, Liber Beneficiorum, s* 584p. 16 J. Ł a s k i, Liber Beneficiorum, s. 584. j j g , 21 F. M. S o b i e s z e z a ń s k i, Wycieczka archeologiczna w niektóre strony gubemii radomskiej w miesiącu wrześniu 1851 roku. Waonszawa 1852 s. 1,94. 10 M. P a u l e w i c z , Początki górnictwa chęcińskiego i fazy jego rozwoju do początku XVII w. (w:) Biuletyn Informacyjny 1(,Kopa,syny”, Kielce, sierpień 1975 s. 14. 11 J. Ł a s k i, Liber Beneficiorum, s* 584p. 12 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 13. 13 Informację o wezwaniu kościoła w Trzebnicy zasięgnął autor od duszpasterzy kościoła par. w Trzebnicy na Śląsku. 14 W. G r z e l i ń s k i . Monografia Chęcin, s. 39—40, 47. 15 J. D l u g o s z, Liber Beneficiorum Dioecesi Cracoviensis, Kraków 1863, T. 1 s. 466. ’ , f , 17 Lustracja woj. sandomierskiego 1789, Cz. 1 s. 165 i i , 22 Katalog zabytków sztuki w Polsce, T. 3 z. 4 Bi. 13. duszpasterzy kościoła par. w Trzebnicy na Śląsku. 14 W. G r z e l i ń s k i . Monografia Chęcin, s. 39—40, 47. 17 Lustracja woj. sandomierskiego 1789, Cz. 1 s. 165 18 W G 1i ń k i M fi Ch i 41 , f , 17 Lustracja woj. sandomierskiego 1789, Cz. Lustracja woj. sandomierskiego 1789, Cz. 1 s 28 M. P a u l e w i c z , E. K o s i k , Kasper Fodyga gwarek chęciń ski (w:) „Informator Towarzystwa Górnictwa, Hutnictwa i Przemysłu Staropolskiego”. Kielce 1974, lipiec s. 5. 24 J. C h ą d z y ń s k i , Historyczno-statystyczne opisy miast staro- żytnych w ziemi sandomierskiej leżących, Warszawa 1855 T. 3 s. 161. 25 Lustracja woj. sandomierskiego 1660— 1664, Cz. I, wyd. H. Opraw ko i K. Schuster, Kraków 1971 s. 68. 26 W. G r z e l i ń s k i , Monografia Chęcin, s. 41— 42. 27 Tamże s. 42. 2. KOŚCIÓŁ SW. BARTŁOMIEJA W CHĘCINACH mieszkańców pozosta ło tylko 86 katolików i 154 Żydów24. Katastrofalny ubytek ludno ści na terenie parafii notują w tym czasie lustratorzy królewscy: „dla niewielkości poddanych zupełnie łąk sprzątać nie mogą” 2S. Po tej klęsce nastały ciężkie czasy nie tylko dla miasta, ale i dla kościoła św. Bartłomieja i Jadwigi. Rrzez 50 lat proboszczowie pa rafii nie mogli usunąć .strat i zniszczeń, jakie po wojnach szwedz kich w nim pozostały. Dopiero na początku XVIII w., kiedy probo szczem tego kościoła zostaje ks. Andrzej Andzelowski, kanonik kamieniecki i mieszczanin chęciński — z całą gorliwością i zapa łem przystępuje do restauracji i renów,acji świątyni i budynków parafialnych. Dopiero po 30 latach pracowitego duszpasterzowamia, mógł on w pełni ocenić to czego dokonał. Było zaś tego dosyć sporo. Odrestaurował wnętrze kościoła, zakrystię zaopatrzył w li czne naczynia srebrne, postawił dom plebański i nowe zabudowa nia gospodarcze. Umierając w 1746 r. sam napisał w testamencie, że kościół w szacie pięknej, jako swą oblubienicę, zostawia: „im meliori et elegantiori statu ut sponsam meam rełinąuo” 26. 3. KOŚCIÓŁ WNIEBOWZIĘCIA NMP. — FRANCISZKANÓW Żaden z kościołów chęcińskich nie 'ma tak dokładnej metryki powstania, jak kościół franciszkanów. Fundatorem i założycielem tego kościoła i istniejącego przy nim klasztoru jest król Kazimierz W ielki. Monarcha ten pragnąc ludności Chęcin zapewnić odpo wiedni poziom życia religijnego, pragnie w mieście tym wybudo wać kościół i osadzić przy nim franciszkanów. O fakcie tym mówi wyraźme petycja króla zwrócona do ówczesnego papieża28. Frag ment tego dokumentu zamieścił papież Urlban V w swej bulli z dnia 5 marca 1367 r. Wynika z niego, że król prosi papieża, aby ten pozwolił w nowo założonych miastach, takich jak Chęciny, Skawina, Opoczno i Szydłów założyć klasztory franciszkańskie. Papież chętnie wyraził zgodę i dzięki temu już w roku następnym oisładają franciszkanie w Chęcinach. Powstaje tu klasztor i kościół z miejscowego kam ienia29. W XV w. kościół ten i klasztor znacznie rozbudowano i powięk szono s0. Przez dwieście lat obiekty te spełniały swoje zadanie, a miejscowi franciszkanie szerzyli kulturę religijną wśród miej skiej ludności parafii. W okresie reformacji kościół i klaszrtor znalazły się w niebezpieczeństwie. Nie wystarczyło protestantom zabranie kościoła parafialnego, ale w trzynaście lat po tym fakcie sięgnęli po kościół klasztorny. W 1581 r. przemocą zagrabili koś ciół i klasztor oraz zajęli dobra zakonne31. Świątynia ta była w rękach innowierców do 1597 r. i została im odebrana w tym sa mym roku co i kościół parafilalny. Zamieniona na zbór pozbawiona została sprzętów, ozdób, naczyń i szat liturgicznych. Kiedy wró ciła ona w ręce zakonu, natychmiast przystąpiono do pracy i za częto usuwać zniszczenia. Już w roku następnym po Objęciu ko ścioła i klasztoru ponownie przez frandisizkanów przybywa z Kra kowa wizytator zakonny O. Donat. Miasto zastaje otoczone murem, który jest w dosyć dobrym stanie. Wewnątrz murów miejskich jest konwent franciszkański. Kościół nosi tytuł św. Franciszka z Asy żu, świątynia ta jest cała murowana, wieża natomiast jest drewnia na. Chodzi tu zapewne o dzwonnicę stojącą luźno obok kościoła. Na niej umieszczone są dzwony. Biidynki Haszom e są drewniane. Na końcu wirydarza jest kaplica św. Leonarda „ad ąuiam est m a- xima devotio hominum”. W kościele jest sześć ołtarzy, wszyst kie odnowione. W kaplicy św. Leonarda jest siódmy ołtarz bartłzo Pod koniec XVIII w. wzrasta znacznie liczlba duchowieństwa przy kościele parafialnym w Chęcinach. Ks. Urban Szostakowicz za zgodą Michała Jerzego księcia Poniatowskiego, arcybiskupa gnieźnieńskiego, ustanowił tu ponownie mansjonarzy, którzy po raz pierwszy zastali tu zorganizowani w XVII w. 2. KOŚCIÓŁ SW. BARTŁOMIEJA W CHĘCINACH Kościół ten został wybudowany w stylu gotyckim. Późniejsze liczne przebudowy nie zdołały tego stylu zatrzeć. Fundatorem ko- ścioła jest założyciel miasta, król Władysław Łokietek. Lokacja miasta najprawdopodobniej miała miejsce w 1315 r.10 i odtąd też miasto zaczęło powstawać „na nowym korzeniu”. Wszystkie dane przemawiają za tym, że razem z miastem został wzniesiony ko ściół, który otrzymał tytuł św. św. Bartłomieja i Jadwigi. Pier wotny niewielki kościół, został zapewne ukończony jeszcze za cza sów króla Władysława Łokietka. Kazimierz Wielki rozbudował go i prace budowlane zakończył w 1350 r. Tę ostatnią datę, zapewne mylnie, podaje w przypisie wydawca Liber Beneficiorum Jana Łas kiego, jako datę rozpoczęcia budowy kościoła11. Ponieważ kościół św. św. Bartłomieja i Jadwigi nie posiada dokumentu erekcyjnego, stąd też dokładna data fundacji kościoła i założenia przy nim pa rafii pozostaje nadal nieznana. Za czasów Kazimierza Wielkiego, a najpóźniej, za rządów jego siostry Elżbiety Łokietkównej, przy kościele tym została założona parafia, tym razem już druga (miej ska) parafia chęcińska 12. Sam tytuł tego kościoła — Bartłomieja i Jadwigi — wskazuje na powiązanie tej świątyni ze Śląskiem. Wydaje się to być oczywistym, gdyż ci, którzy go wznosili przy byli ze Śląska z okolic Trzebnicy jako osadnicy i przekazali taki tytuł nowemu kościołowi, jaki mieli dawniej u siebie na Śląsku13. W kościele tym, jak świadczą źródła parafialne istniało pięć ołta rzy. Były to: ołtarz św. Krzyża, św. Stanisława, św. Mikołaja, św. Trójcy i Wszystkich Świętych. Z ołtarzami tymi związane były fundacje altarii, a następnie prebend 14. Jan Długosz wymieniając dziesięciny pobierane na terenie parafii Chęciny przez kanoników kolegiaty kieleckiej w 1440 r., tak pisze o Chęcinach: „Chęciny miasto mające kościół parafialny murowany w archidiecezji gnieź nieńskiej leżący” 15. Czas od wzniesienia świątyni do 1568 r., to Kościół odrestaurowany i odnowiony uzyskał piękny wygląd, a skarbiec wiele cennych srebrnych naczyń, które przetrwały do K S . M A R IA N P A U L E W IC Z [61 216 [7] K O Ś C IO Ł Y I K A P L IC E CHĘCIN 217 Obecnych czasów. W 1614 r. ukończono budowę w tym kościele Kaplicy Trzech Króli, która posiada wysokie walory artystyczne 23. Druga połowa XVII w. to czasy katastrofalne dla życia i rozwoju miasta Chęcin. Najaizd szwedzki doprowadził do rudny kwitnące miasto, a szalejąca po nim zaraza wyludniła miasto i okolicę. Po tych dwu klęskach w mieście z około 5 tys. 28 Z. K a c z m a r c z y k , Monarchia Kazimierza Wielkiego, organi zacja Kościoła. Poznań 1947 s. 212. 29 Vetera Monumenta Poloniae et Lituaniae, wyd. A. Theiner, T. 1, Rama 1860 dod. 866 s. 642—647. 38 Katalog zabytków sztuki w Polsce T. 3 z. 4 s. 15; K. K a n t a k , Franciszkanie polscy, Kraików 11937 T. li s» U25. 8t Katalog zabytków sztuki w Polsce, T» 3 z. 4 s. 115. 38 Katalog zabytków sztuki w Polsce T. 3 z. 4 s. 15; K. K a n t a k , Franciszkanie polscy, Kraików 11937 T. li s» U25. 28 Z. K a c z m a r c z y k , Monarchia Kazimierza Wielkiego, organi zacja Kościoła. Poznań 1947 s. 212. 29 Vetera Monumenta Poloniae et Lituaniae, wyd. A. Theiner, T. 1, Rama 1860 dod. 866 s. 642—647. 38 Katalog zabytków sztuki w Polsce T. 3 z. 4 s. 15; K. K a n t a k , p y, 8t Katalog zabytków sztuki w Polsce, T» 3 z. 4 s. 115. 28 Z. K a c z m a r c z y k , Monarchia Kazimierza Wielkiego, organi zacja Kościoła. Poznań 1947 s. 212. 29 Vetera Monumenta Poloniae et Lituaniae, wyd. A. Theiner, T. 1, Rama 1860 dod. 866 s. 642—647. 3. KOŚCIÓŁ WNIEBOWZIĘCIA NMP. — FRANCISZKANÓW Napis ten brzmi: „Wie kuista chwała Jaśnie Wielmożnemu Panu z Sidzin Bidzińskiemu, kasztelanowi sandomierskiemu, najwyższemu i najczujniejszemu strażnikowi koronnemu, naówczas chęcińskiemu i skalskiemu sta roście, najszczodrobliwszemu zakonu naszego dobrodziejowi, nie śmiertelne podzięki, którego hojnym nakładem ta część klasztoru z fundamentów została wzniesiona za gwardiaństwa przewielebne go Szymona Boratyńskiego ojca zgromadzenia, który ten pomnik na znak wdzięczności wystawił, iżby wdzięczna czeladfca modliła się o pomyślność i powodzenie dla tak wielkiego dobroczyńcy za jego żywota, za wieczną szczęśliwość po najpóźniejszym zgonie” 39. Tak odbudowany kościół został po raz trzeci poświęcony w 1685 r. Nie danym było zakonnikom cieszyć się jednak zbyt długo swo ją świątynią. W sto trzydzieści dwa lata po ostatnim poświęce niu świątyni został on wraz z klasztorem przejęty przez władze rosyjskie i przeznaczony na więzienie dla całej gubemii kielec kiej 40. W 1851 r. zwiedzał ten kościół F. M. 'Sobieszczański a takie wydał o nim świadectwo: „W roku 1817 mory kościoła i (klaszto ru na więzienie dla całej gubemii zajęte zastały. Teraz tylko jedna kaplica, ciasna, ciemna, zarzucona kościelnymi starymi sprzętami zwiedzaną być może” 41 Stefana Bidzińskiego, miejscowego starosty, odbudowano kościół i klasztor oraz założono przy 'nim ogród. Wdzięczni zakonnicy u- wiecznili ten fakt napisem umieszczonym na płycie marmurowej wmurowanej w ścianę klatki schodowej. Napis ten brzmi: „Wie kuista chwała Jaśnie Wielmożnemu Panu z Sidzin Bidzińskiemu, kasztelanowi sandomierskiemu, najwyższemu i najczujniejszemu strażnikowi koronnemu, naówczas chęcińskiemu i skalskiemu sta roście, najszczodrobliwszemu zakonu naszego dobrodziejowi, nie śmiertelne podzięki, którego hojnym nakładem ta część klasztoru z fundamentów została wzniesiona za gwardiaństwa przewielebne go Szymona Boratyńskiego ojca zgromadzenia, który ten pomnik na znak wdzięczności wystawił, iżby wdzięczna czeladfca modliła się o pomyślność i powodzenie dla tak wielkiego dobroczyńcy za jego żywota, za wieczną szczęśliwość po najpóźniejszym zgonie” 39. Tak odbudowany kościół został po raz trzeci poświęcony w 1685 r. Nie danym było zakonnikom cieszyć się jednak zbyt długo swo ją świątynią. W sto trzydzieści dwa lata po ostatnim poświęce niu świątyni został on wraz z klasztorem przejęty przez władze rosyjskie i przeznaczony na więzienie dla całej gubemii kielec kiej 40. W 1851 r. zwiedzał ten kościół F. M. 'Sobieszczański a takie wydał o nim świadectwo: „W roku 1817 mory kościoła i (klaszto ru na więzienie dla całej gubemii zajęte zastały. Teraz tylko jedna kaplica, ciasna, ciemna, zarzucona kościelnymi starymi sprzętami zwiedzaną być może” 41 Tak oto wyglądały burzliwe dzieje świątyni wzniesionej przez Kazimierza Wielkiego, poświęconej najprzód św. iFTranciszkowi, a następnie Matce Bożej Wniebowziętej. 32 A. K a r w a c k i , Materiały do historii konwentów franciszkań skich prowincji polskiej, T. 9, s. 47—48. Rkps Archiwum Franciszkań skiego w Krakowie. 33 Tamże s. 47. 34 Tamże s. 49. 35 F. M. S o b i e s z c z a ń s k i , Wycieczka archeologiczna, s. 198. 34 A. K a r w a c k i , Materiały, s. 50. 37 W. G r z e l i ń s k i , Monografia Chęcin, s. 64. 38 A. K a r w a c k i , Materiały, s. 46. 81 F. M. S o b i e s z c z a ń s k i , Wycieczka archeologiczna, s. 13. 40 Tamże. Tłumaczenie tekstów łacińskich podano według Sofoie- szczańskiego. 41 Tamże. 3. KOŚCIÓŁ WNIEBOWZIĘCIA NMP. — FRANCISZKANÓW przed najazdem szwedzkim i dał im dobra dawnych starożytnych prebend, zacho wując równocześnie tytuły prebend: Trzech Króli, św. Krzyża, św. Trójcy i Szpitalnej 27. j y p j Po upadku Rzeczypospolitej szlacheckiej z tytułu kościoła usu nięto imię św. Jadwigi, a patronem aż do obecnej chwili pozostał tylko św. Bartłomiej. Zmieniła się też przynależność kościoła i pa rafii do diecezji, weszły one, najprzód do krakowskiej, a następnie kieleckiej diecezji. K S . M A R IA N P A U L E W IC Z [8] [9] 218 K O Ś C IO Ł Y I K A P L IC E CHĘCIN 219 stary (antiquum) 32. Naczynia i szaty liturgiczne są bandizo Skro mne. Jest jedna monstrancja srebrna', pacyfikał srebrny, trzy humerały, dziesięć ornatów, dwie kapy, diwde dalmatyki i to już wszystko 33. W 1612 r. klasztor wizytuje prowincjał Gośki. Wspomi na on, że prezbiterium kościoła ma sklepienie, ale nawa główna nie posiada go jeszcze d nie została Idiotąd 'odbudowana po znisz czeniach dokonanych przez protestantów. Wizytator wspomina, że kościół ma teraz trzy ołtarze poświęcone Matoe Bożej i ma przy jąć tytuł Matki Bożej34. Stanisław z Ruszczy Branitiki, starosta chęciński i miecznik koronny, ibył tym, który pośtwdęcił swoje fundusze na renowację i restaurację kościoła, (kaplicy św. Leonar da i klasztoru. Wdzięczni franciszkanie chęcińscy Wznieśli mu tu taj pomnik marmurowy35. Na pomniku tym umieszczono skrom ny napis upamiętniający zasługi dobrodzieja klasztoru: „Bogoboj nemu dobroczyńcy Jaśnie Wielmożnemu Panu Stanisławowi z Ru szczy Branickiemu miecznikowi koronnemu, staroście 'chęcińskie mu i lelowskiemu, który to 'ogrodzenie z funduszów własnych wzniósł, kości® odnowił i wiele imnydh dobrodziejstw zgroma dzeniu wyświadczył. Roku Pańskiego 1620. Poleca się modłom”. Wizytacja odbyta w 1620 r. przez O. Czerwinę zastała już wszyst ko wspaniale urządzone. Klasztor odbudowany: „Ambitus in qu- adro murafcus”, nakryty drewnianym dachem. Kościół odbudowa ny otrzymał wieżę — sygnaturkę oraz organy o czterech głosach. Przybyło mnóstwo ornatów, humerałów, bielizny kościelnej, sprzę tów liturgicznych oraz trzy obrazy na płótnie: św. Dionizego, św. Salomei i św. Katarzyny38. Stefana Bidzińskiego, miejscowego starosty, odbudowano kościół i klasztor oraz założono przy 'nim ogród. Wdzięczni zakonnicy u- wiecznili ten fakt napisem umieszczonym na płycie marmurowej wmurowanej w ścianę klatki schodowej. g 41 Tamże. 81 F. M. S o b i e s z c z a ń s k i , 40 Tamże. Tłumaczenie tekstów szczańskiego. 41 Tamże. 32 A. K a r w a c k i , Materiały do historii konwentów franciszkań skich prowincji polskiej, T. 9, s. 47—48. Rkps Archiwum Franciszkań skiego w Krakowie. 33 T ż 47 38 A. K a r w a c k i , Materiały, s. 46. 4. KOŚCIÓŁ SW. JAKUBA j p y y p p , Poważną trudność stanowić będzie odtpowiedź na pytanie: Przez kogo i dla kogo ten kościół został wzniesiony? Jedynym kościołem drewnianym wzniesionym na terenie parafii Chęciny był kościół św. Jakuba. Wprawdzie pierwszy kościół chę ciński też był drewniany, ale w XVII w. zastąpiono go nowym murowanym. Ten jednak od początku swego istnienia do końca pozostał tylko drewnianym. Fakt istnienia tego kościoła na tere nie miasta Chęcin był dotąd nie znany. Wzmianka, w niniejszej pracy, o istnieniu tej świątyni jest pierwszą i. jedyną. Ponieważ kościół ten istniał w obrębie posiadłości franciszkanów chęcińskich, stąd też nie był ujmowany w Liber Beneficiorum Łaskiego, gdyż nie stanowił własności diecezjalnej. Informację o istnieniu tego kościoła na terenie klasztoru chęcińskiego przekazał franciszkanin Gośki, prowincjał z Krakowa, wizytujący konwent chęciński w 1612 r. Notatka o tym znajduje się w VIII księdze „Visitatio per- sonalis” {s. 209), znajdującej się w archiwum franciszkańskim w Krakowie. Ojciech CJoski tak o tym kościele mówii: „Preter hanc ecclesiam (kazimierzowskiego murowanego) hic conveńtus aliam ligneam antiąuam siitam in amlhitu, dediCaitam sub titulo S. Ja- cobi, in qua est unium altare S. Jacobi cum imag. lignea anti- qua” 4S. Wydaje się, że odpowiedzi na to pytanie udzieli wzmianka za warta w ordynacji górniczej wydanej przez Jana Płazę z Msty- czowa, żupnika „gór chęcińskich” 44. Ordynacja ta wydana w 1608 r. jest pierwszym w Polsce tak obszernym zestawieniem norm i zwyczajów polskiego prawa górniczego. Została napisana w całości w języku polskim. Zawiera cały szereg zwyczajów, norm i zasad postępowania braci górniczej. Chęciny były tym terenem, gdzie od głębokiego średniowiecza kwitło staropolskie górnictwo krusz cowe 4S. Ta ordynacja wspomina o okolicznościach utraty przez górników prawa do kopania kruszców. Poszczególnym górnikom ma o tym .oznajmić sługa przysięgły prawa górniczego (woźny): ,,u kościoła kędy Gwarkowie odprawiają swoje nabożeństwa, a zwłaszcza w klasztorze Bractwa św. Franciszka, także w mie ście K. J. Moi Chęcinach” 46. Płaza redagując prawo górnicze w jednym z artykułów zanoto wał odwieczany zwyczaj gromadzenia się górników chęcińskich na swoich specjalnych nabożeństwach na terenie klasztoru francisz kańskiego. Prawdopodobnie kościół św. Jakuba był tym kościołem, w którym co niedzielę gromadzili się górnicy chęcińscy na swoje nabożeństwa. Zapewne też nie kto inny jak tylko oni w XIV w. własnymi rękami wznieśli ten kościół. Stąd też nic dziwnego, że w czasie najazdu szwedzkiego, kiedy upada górnictwo kruszcowe i zanika organizacja gwarecka w Chęcinach, kościół św. 3. KOŚCIÓŁ WNIEBOWZIĘCIA NMP. — FRANCISZKANÓW y y W 1628 r. nastąpiło poświęcenie kościoła ii nadano mu nowe wez wanie: Wniebowzięcia Najśw. Maryi Panny. Odtąd też rocznica poświęcenia tego kościoła obchodzona była w piątą niedzielę po Wielkanocy37. Nie upłynęło jeszcze trzydzieści lat od tej chwili, gdy nowe nieszczęście nawiedziło miasto i klasztor, a mianowicie najazd szwedzki. W okresie potopu szwedzkiego, dnia 1 kwietnia 1657 r. w sam dzień Wielkiejnocy Chęciny zostały zajęte przez wojska Jerzego Rakoczego. Zniszczono wówczas kościół klasztor ny i sam klasztor, a trzech zakonników zamordowano38. Hojność dobroczyńców nie pozwoliła jednak popaść w ruinę (kościołowi i klasztorowi franciszkańskiemu w Chęcinach. W 1668 r. kosztem Pod względem architektonicznym budynek kościoła przedstawia dużą wartość. Historyk sztuki tak oto charakteryzuje go: dużą wartość. Historyk sztuki tak oto charakteryzuje go: Dawny kościół pofranciszkański, gotycki pochodzi z XIV w.. Przebudowywany był kilkakrotnie, zwłaszcza w XVII w. Oriento wany, murowany z kamienia łamanego, detale z ciosu, tynkowany. Prezbiterium składa się z trzech nierównej szerokości przęseł. Na wa jest znacznie szersza iod prezbiterium i wyższa, krótka dwu przęsłowa. Sklepienie kolebkowo-krzyżowe zostało wykonane w XVII w. z nakładanymi dekoracyjnymi żebrami, wsparte jest w nawie na filarach przy ścianach. W prezbiterium żebra o układzie sieciowym ze spływami wspartymi na konsolach, w nawie o ukła dzie krzyżowym. Po obu stronach ostrołulkowego otworu tęczowe go pozostałości późnogotyckiej polichromii figuralnej z drugiej po łowy XV w. Z prawej strony sygnatura malarza: „Frater Ange- Wycieczka archeologiczna, s. 13. łacińskich podano według Sofoie- [U] K S . M A R IA N P A U L E W IC Z K O Ś C IO Ł Y I K A P L IC E CHĘCIN [101 2 2 1 2 2 0 le św. Jakuba wzniesiono z fundacji Kazimierza Wielkiego klasztor franciszkanów i kościół klasztorny pod wezwaniem św. Franci szka. lus de Sand.” Zewnątrz ściany oskalpowane. Dachy dwuspadowe. Okna lostoołufcawe w obramieniach 'XIV w. We wschodnim przęśle prezbiterium oknio o charakterze późnogotyckim. Na osi faisady portal 'gotycki, laskofwany wykonany w XIV w. 42 Mpgł też powstać nieco później niż klasztor i kościół klasztor ny, ale wzniesiony został, jako świątynia elitarna, służąca jakiejś specjalnej grupie społecznej czy zawodowej, która w nim się gro madziła, a jeden z franciszkanów pełnił przy nim funkcję kape lana. Ponieważ wszystkie kościoły w mieście były murowane, za mek również był murowany, a kamienia było tu pod dostatkiem — wydaje się, że jeśli kościół ten powstałby w XV czy XVI w. 44 Monumenta Historica Dioeceseos Wladislaviensis, wyd. Z. Chotdyń- ski, Włocławek 1886 t. 9 s. 49. 45 M, P a u l e w i c z , Chęcińska ordynacja górnicza z 1608 r. (w:) Jncformaitor T.P.G.H. i P. S.”, Kielce 1975. marzec, s. 8; Monumen ta Historica Dioeceseos Wladislaińensis, s. 49—‘50. 4li Monumenta Historica Dioeceseos Wladislaviensis, s. 49—50. 42 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 15 43 A. K a r w a c k i , Materiały, s. 49. 3. KOŚCIÓŁ WNIEBOWZIĘCIA NMP. — FRANCISZKANÓW był by na pewno murowany. Musiał więc on być wybudowany w XIV w. i tak jak kościół parafialny otrzymał on za patrona apostoła, Poważną trudność stanowić będzie odtpowiedź na pytanie: Przez Po stu jedenastu latach, więzienie z klasztoru £ kościoła fran ciszkańskiego, zostało przeniesiona na św. Krzyż koło Nowej Słu pi, zaś gmachy po więzieniu chęcińskim zamieniono na magazyny, mieszkania, saile szkolne itip. W .ostatnich latach po gruntownej przebudowie, zamieniono cały obiekt na hotel turystyczny, a bu dynek kościoła na salę restauracyjną. 51 Archiwum Akt Główmyidh. Wairszawa. ASK. 56, C. 6, K. 8 v. 52 J. Ł a s k i , Liber Beneficiorum, s. 587. 53 Archiwum Diecezjalne, Kielce, Sygn. PC 3/2, K. 236. (ADK) 54 W. G r z e l i ń s k i . Monografia Chęcin, s. 54. 55 ADK, Sygn- PC 3/2, K. 236. 4. KOŚCIÓŁ SW. JAKUBA Ducha staje się na okres 30 lat kościołem parafialnym dla parafii chęcińskiej. W tym cza sie mieszkają przy nim proboszczowie parafii chęcińskiej. Tu od prawiają nabożeństwa i tu koncentruje się życie parafii, a na cmentarzu obok tego kościoła chowają zmarłych. Mateusz Post od biera kościół protestantom. Kościół św. św. Bartłomieja i Jadwigi odrestaurowany, zostaje poświęcony i tutaj przenosi się teraz ży cie parafialne. Zostaje też wydany nowy akt erekcji tego ko ścioła 55. Opuszczony w takich -oto okolicznościach kościół św. Du cha opustoszał i zaczął powoli popadać w ruinę. W 1612 r. archi diakon kurzelowski przypomina, że proboszcz chęciński, który w czasie posiadania w swym ręku kościoła św. Ducha pobudował przy nim domy, ma z nich płacić czynsz do kościoła św. Ducha. 47 W. G r z e l i ń s k i , Monografia Chęcin, s. 53. 43 J. Ł a s k i, Liber Beneficiorum, s. 587. 49 J. K ł o c z o w s k i , Wspólnoty chrześcijańskie, Kraków 1964 s. 242. 50 J. Ł a s k i, Liber Beneficiorum, s. 587. 4. KOŚCIÓŁ SW. JAKUBA Jakuba ulega zniszczeniu i zanika o nim pamięć wśród mieszkańców mia sta. q Kościół ten dla prowincjała wizytującego klasztor wydaje się być świątynią starożytną, a więc istniejącą bardzo dawno. Rów nocześnie, według jego świadectwa, jest to kościół bardzo prosty bez (bocznych -ołtarzy, posiadający tylko jedyny ołtarz i poza tym żadnych specjalnych ozdób. Mimo skromnego wystroju i zapewne niewielkiego rozmiaru — prowincjał wizytujący ■— nie nazywa go jednak kaplicą. Patronem tęgo kościoła nie jesit żaden święty zwią zany z zakonem franciszkańskim, ale jeden z apostołów. Wszystko to zdaje się przemawiać aa tym, że kościół ten nie był ndgdy spe cjalnie związany z klasztorem chęcińskim. Mógł on powstać je szcze przed fundacją klasztoru i właśnie przy drewnianym koście- [13] K O Ś C IO Ł Y I K A P L IC E CHĘCIN 223 [12] K S . M A R IA N P A U L E W IC Z 222 też Ludwika Węgierskiego. W inwentarzu dóbr miasta Ghędn z 1508 r. występuje również probostwo szpitalne51. Szczegółowe da ne dotyczące tego probostwa podaje Liber Beneficiorum Łaskiego. Mówi on, że w mieście Chęciny jest szpital z kaplicą pod tytułem Wniebowzięcia N.M.P., położony na cmentarzu szpitalnym. Na tym że cmentarzu jest inny dom, w którym mieszka proboszcz szpital ny, jest również łaźnia i dom dla podróżnych, dosyć obszerny i przestronny. Między domem proboszcza, a miurem miejskim jest ogród nowo urządiziony. Szpital i proboszcz posiadają razem dwa łany ziemi52. Wiek XVI to okres wspaniałego rozwoju i rozkwi tu Chęcin. Niewielka kaplica szpitalna wzniesiona w odległych czasach, była już za skromna dla tego -ośrodka miejskiego. To też w połowie XVI w. -przystąpiono do budowy dużej świątyni szpitalnej. Wydaje się, że wykorzystano stary gmach poprzedniej świątyni i na jej zrębach rozbudowano nową. Nowowzniesiony ko ściół był już duży. Zerwano też z dawną tradycją i według ów czesnych zwyczajów nowo wzniesionej świątyni szpitalnej nadano nowy tytuł: św. Ducha. Został on konsekrowany i oddany do użytku. Akt erekcji pochodzi z 1569 r. i do połowy XIX w. prze chowywany był w aktach parafii Chęciny53. Po rozbudowie katpli- cy szpitalnej na kościół, zaszły w Chęcinach istotne zmiany. Stani sław Szafraniec, starosta chęciński, oddał kościół parafialny św. Bartłomieja innowiercom54. Wówczas po ugodzie proboszcza z burmistrzem i radą miejską, probositwo szpitalne zostaje złączo ne z kościołem św. Marii Magdaleny, który to kościół został wzniesiony z fundacji władz miejskich i znajdował się w odle głości 200 m. f 49 J. K ł o c z o w s k i , Wspólnoty chrześcijańskie, Kraków 1964 s. 242. 4. KOŚCIÓŁ SW. JAKUBA od szpitalnego, ale w obrębie murów miejskich. Po dokonaniu tej zmiany kościół św. Ducha staje się na okres 30 lat kościołem parafialnym dla parafii chęcińskiej. W tym cza sie mieszkają przy nim proboszczowie parafii chęcińskiej. Tu od prawiają nabożeństwa i tu koncentruje się życie parafii, a na cmentarzu obok tego kościoła chowają zmarłych. Mateusz Post od biera kościół protestantom. Kościół św. św. Bartłomieja i Jadwigi odrestaurowany, zostaje poświęcony i tutaj przenosi się teraz ży cie parafialne. Zostaje też wydany nowy akt erekcji tego ko ścioła 55. Opuszczony w takich -oto okolicznościach kościół św. Du cha opustoszał i zaczął powoli popadać w ruinę. W 1612 r. archi diakon kurzelowski przypomina, że proboszcz chęciński, który w czasie posiadania w swym ręku kościoła św. Ducha pobudował przy nim domy, ma z nich płacić czynsz do kościoła św. Ducha. też Ludwika Węgierskiego. W inwentarzu dóbr miasta Ghędn z 1508 r. występuje również probostwo szpitalne51. Szczegółowe da ne dotyczące tego probostwa podaje Liber Beneficiorum Łaskiego. Mówi on, że w mieście Chęciny jest szpital z kaplicą pod tytułem Wniebowzięcia N.M.P., położony na cmentarzu szpitalnym. Na tym że cmentarzu jest inny dom, w którym mieszka proboszcz szpital ny, jest również łaźnia i dom dla podróżnych, dosyć obszerny i przestronny. Między domem proboszcza, a miurem miejskim jest ogród nowo urządiziony. Szpital i proboszcz posiadają razem dwa łany ziemi52. Wiek XVI to okres wspaniałego rozwoju i rozkwi tu Chęcin. Niewielka kaplica szpitalna wzniesiona w odległych czasach, była już za skromna dla tego -ośrodka miejskiego. To też w połowie XVI w. -przystąpiono do budowy dużej świątyni szpitalnej. Wydaje się, że wykorzystano stary gmach poprzedniej świątyni i na jej zrębach rozbudowano nową. Nowowzniesiony ko ściół był już duży. Zerwano też z dawną tradycją i według ów czesnych zwyczajów nowo wzniesionej świątyni szpitalnej nadano nowy tytuł: św. Ducha. Został on konsekrowany i oddany do użytku. Akt erekcji pochodzi z 1569 r. i do połowy XIX w. prze chowywany był w aktach parafii Chęciny53. Po rozbudowie katpli- cy szpitalnej na kościół, zaszły w Chęcinach istotne zmiany. Stani sław Szafraniec, starosta chęciński, oddał kościół parafialny św. Bartłomieja innowiercom54. Wówczas po ugodzie proboszcza z burmistrzem i radą miejską, probositwo szpitalne zostaje złączo ne z kościołem św. Marii Magdaleny, który to kościół został wzniesiony z fundacji władz miejskich i znajdował się w odle głości 200 m. od szpitalnego, ale w obrębie murów miejskich. Po dokonaniu tej zmiany kościół św. 47 W. G r z e l i ń s k i , Monografia Chęcin, s 47 W. G r z e l i ń s k i , Monografia Chęcin, s. 53. 43 J. Ł a s k i, Liber Beneficiorum, s. 587. 43 J. Ł a s k i, Liber Beneficiorum, s. 587. 6. KOŚCIÓŁ ŚW. MARII MAGDALENY świątynia ta powstała z fundacji burmistrza i rajców miejskich. Świadczy o tym prawo prezenty przysługujące burmistrzowi i ra dzie miejskiej 64. Kościół ten posiadając niewielkie rozmiary, zo stał wzniesiony w oforęlbiie murów miejskich przy ulicy Niemiec kiej w pobliżu bramy miejskiej. Ponieważ Liber Beneficiorum Łaskiego nie wspomina o tym kościele, należy przypuszczać, że powstał on w połowie XVI w., co zdaje się potwierdzać kształt architektoniczny budowli 65. Decydującą rolę w dziejach kościoła św. Dudha odegrało spro wadzenie do Chęcin klarysek i założenie domu zakonnego w po bliżu kościoła św. Marii Magdaleny. Osiadły one w domu ofiaro wanym im po bezpotomnie zmarłym wójcie chęcińskim Kasprze Fodydze 59. Przez 30 lat przy klasztorze tym istniała tylko kaplica. Klaryski chcąc uzyskać dia siebie kościół św. Marii Magdaleny zaczęły prowadzić pertraktacje z proboszczem szpitalnym i radą miejską. Tymczasem w 1670 r. spalił się dach na kościele św. Du cha i kościół ten wymagał pilnego remontu. Za staraniem klary sek remontu tego dokonał ich protektor starosta chęciński Bidziń- ski. Odnowiony został również przy tym kościele dom dla pro boszcza szpitalnego60. Prowadzone przez szereg lat pertraktacje zostały uwieńczone powodzeniem. Klaryski 'Otrzymały na własność kościół św. Marii Magdaleny. Proboszcz szpitalny zamieszkał przy kościele św. Ducha, który od 1673 r. stał się powtórnie kościołem szpitalnym. Prawo patronatu i prezenty, które posiadał burmistrz i rada miejska dla kościoła św. Marii Magdaleny przeszło teraz na kościół św. Ducha. Odtąd też, jak świadczą chęcińskie księgi metrykalne, przy kościele tym rezydowali na stałe proboszczowie szpitalni. W 1787 r. szpital i kościół św. Ducha przeszedł pod wy łączną opiekę proboszcza parafii św. Bartłomieja i Jadwigi61, zaś proboszcz szpitalny został wikariuszem chęcińskim62. Okazją do fundacji tego kościoła były przyczyny prestiżowe, jak i utylitarne. Ponieważ w Chęcinach były już dwa kościoły ufundowane przez władców Polski, władze miejskie pragnęły za manifestować swą zamożność i znaczenie, wznosząc własnym ko sztem niewielki kościółek. Druga sprawa to względy praktyczne. Wielu kupców i podróżnych przybywało do Chęcin lub też zatrzy mywało się tu na krótki odpoczynek w czasie długich podróży. Przy kościele tym był zapewne dom gościnny, który spełniał rolę dziesiejszego hotelu. Obiekt ten tak został obmyślany, aby mógł stanowić konkurencję wolbec podobnego, który znajdował się po drugiej części murów miejskich na terenie probostwa szpitalnego. W czasie reformacji obydwa wielkie kościoły chęcińskie parafial ny i klasztorny zostały zajęte przez protestantów. Miasto oddaje wówczas kościół św. 5. KOŚCIÓŁ SW. DUCHA Pierwsza wzmianka o probostwie i szpitalu Ubogich w Chęci nach pochodzi z drugiej połowy XV w. 'Dnia 2 marca 1481 r. ks. Jiam Śmiech, chęcinianin, spełniając wolę swej matki Anny, uczy- tnił zapis na kościół szpitalny. Zapas polegał na darowaniu pola, łąk i sadzawek z obowiązkiem odprawiania Mszy za dobrodzie jów i fundatorów47. Kościół szpitalny, dom ubogich i probostwo szpitalne musiały być ufundowane znacznie wcześniej od wyżej wspomnianej daty. Wydaje się, że fundacja sięga przełomu XIV i XV w. Przypuszczenie to zdają się potwierdzać dwa fakty. yp ją ę p y 1) Pierwotny tytuł świątyni szpitalnej, to nie św. Ducha, ale Wniebowzięcia Najświętszej Maryi Panny48. ę ę 2) Świątynia ta na początku XVI w. nazwania jest w Liber Be- neficiorum Łaskiego nie kościołem, lecz kaplicą. Musiała być nie wielka, bo ufundowana została w odległych czasach, kiedy Chęci ny nie były zbyt wielkim miastem. Kazimierz Wielki pod koniec swego panowania, a może jeszcze wcześniej jego ojciec Władysław Łokietek, otoczył miasto Chęciny irairem. W średniowieczu szpitale i kościoły szpitalne budowane były z zasady poza murami miiasta, najczęściej w pobliżu bieżącej wody przy bramach miejskich. „Szpital” średniowieczny określa ny, jako hospicium, hospitale, Xenodochiium, czasem scthola obej mował nie tylko pomieszczenia dla starców i biedaków, ale także schronisko dla podróżnych, czasem pomieszczenie dla chorych. Ka żdy więc szpital — hospiicium składał się z sali lub sal dla pod opiecznych, pomieszczenia dla personelu — bractwa, kaplicy z cmentarzem 49. Usytuowanie szpitala chęcińskiego zgodne było z klasycznym wzorem sięgającym XIII w. w całej Zachodniej Europie. A więc kaplica wzniesiona była poza murami miasta przy bramie, od której zaczynał się trakt do Małogoszcza, Przedborza i na Śląsk, z którym Chęciny mogły swobodnie handlować. Miejsce to było przedłużeniem ulicy Niemieckiej i przy nim powstało później przedmieście. Posiadanie przez szpital własnej łaźni50, zdaje się potwierdzać, że budowany był on z myślą, a-by stanowił punkt za trzymania i noclegu dla podróżnych. Wszystkie te okoliczności zdają ;się przemawiać za bardzo wczesnym pochodzeniem tej fun dacji, a z nią świątyni szpitalnej. Najwcześniejszym terminem mo gły być -czasy ostatnich lat panowania Kazimierza Wielkiego lub K S . M A R IA N P A U L E W IC Z [15] K O ŚC IO Ł Y I K A P L IC E CHĘCIN [14] 224 225 Pieniądze te mają być dołączone do sumy złożonej przez poboż nych ludzi z miasta d .sąsiednich wsi na odnowienie kościoła św. Duiaha i św. Franciszka. 15 — Nasza Przeszłość t. 57 5. KOŚCIÓŁ SW. DUCHA Pieniądze te zostały żtożone w magi stracie chęcińskim na ręoe sławetnego Kaspra Fodygi, wójta chę cińskiego 56. On też zapewne, jako fachowiec murator i rzeźbiarz, kierował odbudową Obydwu świątyń. .Odbudowany na początku XVII w. kościół św. Ducha stał się na przeciąg 60 lat kościołem filialnym, gdyż funkcję kościoła szpitalnego spełniał nadal ko ściół św. Marii Magdaleny. Świadczą o tym Akta Archidiakona tu Kurzelowskiego. Dnia 8 marca 1624 r. sławetni burmistrz i raj cowie miasta Chęcin, posiadając prawo patronatu w kościele św. Marii Magdaleny, prezentują na wakujące probostwo szpitalne św. Marii Magdaleny ks. Tomasza Gabriela 57. W 1645 r. Maciej Maj z Polichna przeznacza część swojego pola do Xenodochii chęciń skiej pod wezwaniem św. Marii Magdaleny 58. Ostatecznie budynek kościoła św. Ducha zastał przejęty przez władze carskie i adaptowany na gmach świecki. W 1820 r. wła dze państwowe umieściły w nim biuro Sądu Policji Poprawczej. Później zaś został odprzedany osobom prywatnym63. Dzisiaj bu dynek ten stanowi własność rodziny Wilczyńskich, która w nim mieszka. Obok mego na terenie dawnego cmentarza znajduje się pralnia miejska. 62 Ostatni proboszcz szpitalny ks. Jan Adamiecki występuje kilka krotnie w metrykalnych .księgach parafialnych (L. B. 1774 -r.), jako proboszcz szpitalny, a później (L. B. 17612 r.) już tylko, jako wikariusz Chęciński. 6* W. G r z e l i ń s k i , Monografia Chęcin, s. 5. 65 Katalog zabytków sztuki w Polsce T. 3 z. 4 s. 1(7—418. 66 Fakt ten stwierdzają dokumenty Archiwum Kurzeflowskiego, cy towane wcześniej oraz zapis Macieja Maja wspomniany wyżej. 56 ADK, Akta Archidiakonatu Kurzelowskiego,, T. 3 s. 31. 57 Tamże s. 250. 58 W. G r z e l i ń s k i , Monografia Chęcin, s. 55. 59 M. P a u l e w i c z , E. K o s i k , Kasper Fodyga, s. 8. 60 A. K a r w a c k i , Materiały, s. 69. 61 W. G r z e l i ń s k i , Monografia Chęcin, s. 54— 55. 62 Ostatni proboszcz szpitalny ks. Jan Adamiecki występuje kilka krotnie w metrykalnych .księgach parafialnych (L. B. 1774 -r.), jako proboszcz szpitalny, a później (L. B. 17612 r.) już tylko, jako wikariusz Chęciński. 56 ADK, Akta Archidiakonatu Kurzelowskiego,, T. 3 s. 31. 57 Tamże s. 250. 58 W. G r z e l i ń s k i , Monografia Chęcin, s. 55. 59 M. P a u l e w i c z , E. K o s i k , Kasper Fodyga, s. 8. 60 A K a r w a c k i Materiały s 69 64 ADK, Akta Ardhaid. Kurzel., T. 3 s. 311), 260. 61 W. G r z e l i ń s k i , Monografia Chęcin, s. 54— 55. 64 ADK, Akta Ardhaid. Kurzel., T. 3 s. 311), 260. 65 Katalog zabytków sztuki w Polsce T. 3 z. 4 s. 1(7—418. 66 Fakt ten stwierdzają dokumenty Archiwum Kurzeflowskiego, cy towane wcześniej oraz zapis Macieja Maja wspomniany wyżej. ) 65 Katalog zabytków sztuki w Polsce T. 3 z. 4 s. 1(7—418. k d d k A h fl k 56 ADK, Akta Archidiakonatu Kurzelowskiego,, T. 3 s. 31. 57 Tamże s. 250. 6. KOŚCIÓŁ ŚW. MARII MAGDALENY Marii Magdaleny proboszczowi szpitalnemu, który przy nim osiada na przeszło sto lat i kościół ten staje się wówczas kościołem szpitalnym. Szpital przyjmuje teraz ■'nazwę szpitala św. Marii Magdaleny66. Na początku XVII w. w niewielkiej odległości od tego kościoła, również przy tej samej ulicy Niemieckiej, przy której był kościół św. Marii Magdaleny, wójt miasta Chęcin, murator, rzeźbiarz i gwarek chęciński Kaisper Fodyga, ibuduje Obszerny piętrowy dom. Umierając zaś bezpotomnie poleca, aby jego dom przekazano na cele pobożne. Jego bratanek, syn Sebastiana, ks. Henryk Fodyga, 87 M. P a u l e w i c z , E. Ko s i k , , Kasper Fodyga, s. 7—8. 68 Katalog zabytków sztuki w Polsce, T. 3 z.4 s. If7- 69 A. K a r w a c k i , Materiały, s. 71. 70 Tamże s. 59. 56 ADK, Akta Archidiakonatu Kurzelowskiego,, T. 3 s. 31. 57 Tamże s. 250. W G l i ń k i M fi Ch i K S . M A R IA N P A U L E W IC Z [16] 226 K O Ś C IO Ł Y I K A P L IC E CHĘCIN [17] 227 proboszcz w Mińsku koło Kielc i prebendarz kaplicy Trzech Kró li w Chęcinach spełnia .wolę zmarłego stryja, przekazując ten dom klaryskom sprowadzonym z Gniezna do Chęcin67. bernardynki z Wilna i one również objęły kościół po klaryskach. Do obecnej chwili świątynia ta jest kościołem klasztornym sióstr bernardynek, które utrzymują go i konserwują przy wydatnej pomocy władz państwowych, gdyż kościół stanowi obiekt zabytko wy, świadczący o bogatej i ciekawej przeszłości miasta Chęcin. y p y ę Klaryski mieszkając teraz w swoim klasztorze w pobliżu kościo ła św. Marii. Magdaleny, za wszelką cenę pragnęły pozyskać go dla siebie. Dopiero po trzydziestu latacih starań i zabiegów osiągnę ły sukces i w 1673 r. zawarły ugodę z radą miejską i proibaszazem szpitalnym, który zwolnił ten kościół i przeniósł się do kościoła św. Ducha. Starosta chęciński Bidizińslki, którego córka Teresa była ksienią klarysek chęcińskich, przystąpił do rozbudowy tego kościo ła. Podczas przebudowy prezbiterium kościół został dwukrotnie powiększony. W starej części kościoła wzniesiono olbszemy chór dla zakonnic, oraz urządzono krużganki wewnątrz kościoła od u- licy. Łączyły one chór z klasztorem. Wnętrze kościoła urządzono dosyć 'gustownie w stylu barokowym68. 71 J. Ł a s k i , Liber Beneficiorum, s. 587. 72 Informację zaczerpnięto w pracowni Atlasu Historycznego Koś cioła KUL. 73 J. Ł a s k i , Liber Beneficiorum, s. 587. 74 Tamże. 75 W. G r z e l i ń s k i , Monografia Chęcin, s. 41. 71 J. Ł a s k i , Liber Beneficiorum, s. 587. 72 Informację zaczerpnięto w pracowni Atlasu Historycznego Koś cioła KUL. 73 J. Ł a s k i , Liber Beneficiorum, s. 587. lu KAPLICA ZAM KOW A Drugą chronologicznie świątynią z terenu parafii Chęciny jest kaplica wzniesiona na zamku chęcińskim. Ponieważ zamek został wybudowany z polecenia księcia Władysława Łokietka zatem i ka plica też jest jego fundacji. Zamek miał charakter wybitnie mili tarny, wypadało, aby i kaplica zamkowa miała właściwy i odpo wiedni tytuł. Nadano jej więc wezwanie: Dziesięciu tysięcy św. żołnierzy męczenników71. Wezwanie to nie było zbyt częste i no siło go w Polsce zaledwie 20 świątyń, przeważnie kaplic zamko wych 72. y g y y Kiedy prace budowlane i adaptacyjne zostały już ukończone, kościół wraz z pięcioma ołtarzami został konsekrowany przez Mi kołaja Oborskiego, siufragana krakowskiego w 1685 r. Zezwolenie na konsekrację kościoła i ołtarzy, udzielił obcemu biskupowi arcy biskup gnieźnieński Stef an Wydżga. Wydarzenie to zostało uwie cznione na talbOicy marmurowej znajdującej się obok wielkiego ołtarza: y Z kaplicą związana była altaria i prebenda zamkowa uposażo na dosyć dobrze przez monarchów Polski. Im też przysługiwało prawo prezenty do tej kaplicy. Do uposażenia kaplicy i kapelana wchodziły dziesięciny ze wsi Skorków w parafii Małogoszcz oraz inna forma dochodów. Kapelan miał zastrzeżone mieszkanie przy kaplicy na zamku. Starosta był zobowiązany dostarczać mu wina do Mszy św. Od najdawniejszych czasów kapelan był zobowiąza ny odprawiać w kaplicy w ciągu tygodnia dwie Msze. Jedną o Dziesięciu tysiącach św. żołnierzy męczenników i drugą za zmar łych 7S. ołtarza: „Roku Pańskiego 1685 w czwartą niedzielę po Zielonych Świąt kach, która wypadła w tym roku 8 lipca Mikołaj Oborski biskup laodycejski, sufragan krakowski z pozwolenia Stefana Wydżgi, ar cybiskupa gnieźnieńskiego, prymasa Królestwa, konsekrował ko ściół ten panien zakonnych reguły św. Franciszka pod wezwaniem św. Marii Magdaleny; relikwie św. Jacka, św. Marcialisa, św. Wi talisa złożył w ołtarzach, których było pięć: wieilki ołtarz na cześć św. Marii Magdaleny; drugi Najśw. Maryi Panny i św. Anny, trzeci św. Franciszka i św. Józefa, czwarty św. Antoniego i św. Salomei, piąty św. Klary i św. Kunegundy. Rocznica konse kracji kościoła została naznaczona na pierwszą niedzielę września. „Biskup konsekrował wówczas również 4 dzwony do kościoła69. Starosta Stefan Bidiziński powiększył pierwotny dom Fodygi i klasztor połączył z kościołem. Dokupił ogród owocowy i całe za budowania -otoczył murami70. Urządzony wówczas cały ten o- biekt — kościół i klasztor służył klaryskom, aż do początku XX w. Po wymarciu zakonu, do którego władze carskie zabroniły przyjmować n a w ie j uszek, przez 30 lat klasztor srtał pusty, a ko ściół posiadał tylko kapelana. W 1930 r. 75 W. G r z e l i ń s k i , Monografia Chęcin, s. 41. 73 J. Ł a s k i , Liber Beneficiorum, s. 587. 74 Tamże. lu KAPLICA ZAM KOW A do klasztoru przybyły „Roku Pańskiego 1685 w czwartą niedzielę po Zielonych Świąt kach, która wypadła w tym roku 8 lipca Mikołaj Oborski biskup laodycejski, sufragan krakowski z pozwolenia Stefana Wydżgi, ar cybiskupa gnieźnieńskiego, prymasa Królestwa, konsekrował ko ściół ten panien zakonnych reguły św. Franciszka pod wezwaniem św. Marii Magdaleny; relikwie św. Jacka, św. Marcialisa, św. Wi talisa złożył w ołtarzach, których było pięć: wieilki ołtarz na cześć św. Marii Magdaleny; drugi Najśw. Maryi Panny i św. Anny, trzeci św. Franciszka i św. Józefa, czwarty św. Antoniego i św. Salomei, piąty św. Klary i św. Kunegundy. Rocznica konse kracji kościoła została naznaczona na pierwszą niedzielę września. „Biskup konsekrował wówczas również 4 dzwony do kościoła69. y W 1511 r. prebendariiuszem i kapelanem zamku chęcińskiego był Antoni z Unienia74. Król Zygmunt III przywracając w 1597 ko ściół św. św. Bartłomieja i Jadwigi katolikom, zatwierdził wszy stkie przywileje kaplicy zamkowej. Prebenda związana z tą ka plicą została wówczas przyłączona do probostwa św. św. Bartło mieja i Jadwigi. Odtąd też proboszcz miał obowiązek osobiście sprawować służbę Bożą przy ołtarzu na zamku. Ostatnim kapela nem — prebendarzem był Bartłomiej z Żarek75. Losy kaplicy zamkowej złączone były ściśle z losami zamku chęcińskiego. Kie dy ten stracił pozycję niezdobytej twierdzy i zaczął popadać w y W 1511 r. prebendariiuszem i kapelanem zamku chęcińskiego był Antoni z Unienia74. Król Zygmunt III przywracając w 1597 ko ściół św. św. Bartłomieja i Jadwigi katolikom, zatwierdził wszy stkie przywileje kaplicy zamkowej. Prebenda związana z tą ka plicą została wówczas przyłączona do probostwa św. św. Bartło mieja i Jadwigi. Odtąd też proboszcz miał obowiązek osobiście sprawować służbę Bożą przy ołtarzu na zamku. Ostatnim kapela nem — prebendarzem był Bartłomiej z Żarek75. Losy kaplicy zamkowej złączone były ściśle z losami zamku chęcińskiego. Kie dy ten stracił pozycję niezdobytej twierdzy i zaczął popadać w 71 J. Ł a s k i , Liber Beneficiorum, s. 587. 72 Informację zaczerpnięto w pracowni Atlasu Historycznego Koś cioła KUL. 73 J. Ł a s k i , Liber Beneficiorum, s. 587. 74 Tamże. K S . M A R IA N P A U L E W IC Z K O Ś C IO Ł Y I K A P L IC E CHĘCIN 228 [18] [19] 229 ruinę, razem z nim w ruinę popadła również i kaplica zanikowa, z której dzisiaj pozostały tylko ndkłe fragmenty pozwalające je dynie określić miejsce gdzie ona istniała. lu KAPLICA ZAM KOW A tektoniczne przyjęła w połowie XVII w. i w takim stanie prze trwała do naszych czasów. Współczesny historyk sztuki tak ją przedstawia: Położona na osi krużganku skrzydła północnego. Na planie kwadratu, o korpusie nieco wyższym od budynku klasztor nego, podsklepiona kryptami grobowymi, z niską apsydą od wscho du otwartą do wnętrza arkadą. Wnętrze kaplicy nakryte kopu łą wyodrębnioną gzymsem, na żagielkach z latarnią. W ścianach tanczcwych okrągłe okna. Belkowanie ścian, oraz promieniste, pła skie żefbra kopuły pokryte późnorenesansawą dekoracją stiukową ok. 1641 r. Nad tęczą stiukowy hienogram IHS i data 1641. Na dwu żagielkach późnorenesamsowe stiukowe kartusze z herbami: Gryf Jana Bnamidkiego i Nieczuja jego żony Anny Beaty z Wa- powakich. Kopuła kaplicy i półkopułka absydy lekko rozpłaszczo ne; latarnia kamienna z rzeźbionymi pilastrami hermowymi i głó wkami aniołków81. Po zamienieniiu całego zespołu, kaplica stała się barem. 2. KAPLICA SW. LEONARDA Obok kościoła franciszkańskiego znajdował się piętrowy klasz tor zabudowany w czworobok. Do wschodniego skrzydła wiryda- rza przylegała kaplica św. Leonarda76. Zdania historyków sztuki co do początków tej kaplicy są podzielone. Aleksander Król przy puszczał, że powstała, ona w trakcie rozbudowy klasztoru, doko nanej staraniem miecznika koronnego i starosty chęcińskiego Sta nisława Braniekiego w latach 1605—1607. Zdaniem A. Króla ka plicę św. Leonarda budował Kasper Fodyga11. J. Łoziński w pracy Grobowe kaplice kopułowe w Polsce, prze prowadzając dokładną analizę architektoniczną sądzi, że autorstwo Fodygi jest bardzo wątpliwe, jednakże kaplicę tę można zaliczyć do budowli pokrewnych jego twórczości78. Zarówno Król, jak i Łosiński przyjmują dwie fazy budowy kaplicy. Pierwsza według Króla miała miejsce na początku XVII w., zaś według Łozińskie go zbliżona jesit do 1641 r. Faza druga, to faza przebudowy i przy pada ona na 1641 r. Tę datę przyjmują historycy, gdyż jest ona umieszczona w kartuszu na wewnętrznym belkowaniu. 82 M. P a u l e w i c z , E. Kos i k; , Kasper Fodyga, s. 5— 7. 83 W. G r z e l i ń s k i , Monografia Chęcin, s. 50. 81 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 17. 79 A. K a r w a c k i , Materiały, s. 48. 80 Taimże. 78 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 15. 77 A. K r ó l , Kasper Fodyga budowniczy chęciński z początków XVII w. (w:) „Biuletyn Historii Sztuki”. Wairszawa 195)1 s. 107. 78 J. Ł o z i ń s k i , Kaplice kopułowe w Polsce 1520—1620, Warsza wa 1973 s. 48. 79 A. K a r w a c k i , Materiały, s. 48. 80 Taimże. 78 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 15. 77 A. K r ó l , Kasper Fodyga budowniczy chęciński z początków XVII w. (w:) „Biuletyn Historii Sztuki”. Wairszawa 195)1 s. 107. 78 J. Ł o z i ń s k i , Kaplice kopułowe w Polsce 1520—1620, Warsza wa 1973 s. 48. 77 A. K r ó l , Kasper Fodyga budowniczy chęciński z początków XVII w. (w:) „Biuletyn Historii Sztuki”. Wairszawa 195)1 s. 107. 79 A. K a r w a c k i , Materiały, s. 48. 80 T i ż V w. (w:) „ u ety sto S tu . Wa s awa 95) s. 07. 78 J. Ł o z i ń s k i , Kaplice kopułowe w Polsce 1520—1620, Warsza wa 1973 s. 48. 89 Katalog zabytków sztuki w Polsce, T. 3 z. 4 s. 14. 90 ADK, Sygn, PC 3/2, K. 31®. 3. KAPLICA TRZECH KRÓLI W predelli znajduje siię płaskorzeźba Hołdu Trzech Króli. Na ścianie znajduje się bogato ozdobione epitafium Kaspra i Zuzanny Fodygów89. Stan taki przetrwał do ostatnich dni. ny i ryt owa ny. W predelli znajduje siię płaskorzeźba Hołdu Trzech Króli. Na ścianie znajduje się bogato ozdobione epitafium Kaspra i Zuzanny Fodygów89. Stan taki przetrwał do ostatnich dni. przedstawicielem okresu późnego, swojskiego już renesansu. Wraz ze swym zespołem działał w schyłkowej fazie sztuki Odrodzenia, w której charakterystyczne elementy renesansowe przetwarzano na sposób rodzimy. Wzniesienie przez mieszczanina własnej kapli cy grobowej tłumaczyć można specjalną rolą Fodygi w Chęcinach znacznym jego bogactwem i kulturą 84. J. Łoziński tak oceniiia fakt wzniesienia tej kaplicy w Chęcinach: „Fodyga spolszczony korne- sek z Mesoooo działający w Chęcinach, dorobić się musiał znacz nej pozycji społecznej. Świadczy o tym 'nie tylko szumny tekst na pisu epitafijnego, głoszący między innymi, że nie był Fodyga osta tnim, lecz pierwszym w siwej sztuce, o czym mówią liczne wznie sione przez niego budowle. Dowodzi tego sam fakt, zupełnie odo sobniony, wystawienia sobie własnej kaplicy grolbowej” 85. W pod ziemiach tej kaplicy został pochowany fundator oraz jego żona Zuzanna z Grodzanowskioh. Po ukończeniu kaplicy jak świadczą Akta Archidiakonatu Kurzelowskiego1, poprosił Fodyga o poświę cenie kaplicy i oddał ją na „chwałę Bożą”. Przy kaplicy tej u- fundował altarię86, którą później arcybiskup gnieźnieński Henryk Firlej z Dąbrowicy erygował na prebendę 87. i Zuzanny Fodygów89. Stan taki przetrwał do ostatnich dni. 4. KAPLICA MATKI BOSKIEJ ANIELSKIEJ W POLICHNIE W połowie XIX w. na terenie parafii Chęciny w odległości 6 km. od kościoła parafialnego, w Polichnie, zostaje wybudowana kapli ca. Początki jej są bardzo skromne. Michał Łasa buduje w pobli żu swego domu drewnianą kaplicę. Fundator pragnie, aby kaplica została poświęcona w święto św. Michała 29 września 1851 r. i aby otrzymała za patrona św. Michała. Konsystorz Biskupi w Kiel cach wyraża na to zgodę90. Kiedy po 30 latach kaplica ta chyli się ku upadkowi, wśród mieszkańców wsi powstaje myśl wybudo wania nowej kaplicy, znacznie większej i trwałej, która by wy starczyła dla całej wsi. Zamiar ten zrealizowano w 1882 r. wzno sząc nową trwałą kaplicę. W 1965 r. kaplica otrzymała własnego kapłana, który spełnia tu funkcje duszpasterskie dla 900 wier nych, zaś kaplica została kościołem rektoralnym. Patronem tej świątyni jest Matka Boska Anielska/. j ą y yg ę Pierwszym prebendarzem tej kaplicy był członek rodziny fun datora, syn brata Sebastiana — ks. Henryk Fodyga 88. , g f ę , 88 Bibl. Jagieł. Rkps 5478 — Aota Judiciaria Advocatialia Chencinen- sis, K. 69— TO. 84 A. K r ó 1, Kasper Fodyga budowniczy, s. 91—ilOl. 85 J. Ł o z i ń s k i , Kaplice kopułowe, s. l®4. , p p , 86 ADK Akita Archid. Kurzei., T. 3 s. 42. , 87 W. G r z e l i ń s k i , Monografia Chęcin, s. 50. 3. KAPLICA TRZECH KRÓLI Chęciny w drugiej połowie XVI w. przeżywają okres wspaniałe go rozwoju. Przybywają tu i osiedlają się liczni cudzoziemcy. Wśród nich znajdują się dwaj bracia Kasper i Sebastian Fodygo- wie. Przybyli oni z Włoch z miastecźka Mesocoo. Obydwaj z za wodu byli muratorami i rzeźbiarzami. Znaleźli tu dla siebie pra cę, założyli tu rodziny, żeniąc się z Polkami i weszli w skład mieszczaństwa chęcińskiego. Kasper Fodyga dzięki swym umie jętnościom zawodowym, pracowitości, i gospodarności, dorobił się znacznego majątku i wszedł w skład paitrycjatu miejskiego. Przez szereg lait pełnił on funkcję wójta miasrta Chęcin. Pod koniec życia, kiedy Jan Płaza zorganizował na nowo upadłe górnictwo kruszsczowe w starostwie chęcińskim, Kasper Fodyga zostaje gwar kiem. Lokuje on wówczas swoje kapitały, zabrane w ciągu praco witego życia, w rozwój chęcińskiego gómidtwa. Wspaniały ten gest został wynagrodzony. Obfitość rudy, zwłaszcza w pierwszych la tach po rozpoczęciu prac górniczydh sprawiła, że wójt i gwarek chęciński Kasper Fodyga stał się bardzo zamożnym człowiekiem 82. Posiadając wystarczająoe zasoby pieniężne, przystąpił on wów czas do budowy własnej kaplicy. Kaplica ta została wzniesiona w kościele parafialnym św. św. Bartłomieja i Jadwigi i ukończona została w 1614 r. 83. Na cześć swojego patrona Kaspra — Fodyga wybrał wezwanie Trzech Króli. A. Król tak charakteryzuje syl wetkę fundatora i budowniczego tej kaplicy: Kasper Fodyga był W rzeczywistości kaplica ta powstała znacznie wcześniej niż twierdzą obydwaj historycy sztuki. W 1598 r., w nok po odebra niu kościoła i klasztoru protestantom, wizytował go O. Donat. Zastaje on wówczas kaplicę św. Leonarda. Pisze o niej: „In ambi- tu capella s. Leonardi, ad ąuiam est maxima devotio hominum. Sacrarifum muratuim cum testidine” 79. Protestanci posiadali klasz tor od 1581 r. Kaplica musiała powstać znacznie wcześniej przed zagarnięciem klasztoru, skoro miejscowa ludność tak była do niej przywiązana. Powstała zapewne przynajmniej w połowie XVI w., a jej budowa miała podwójny cel. Miała ona służyć mieszkańcom klasztoru, jako kaplica domowa, zaś jej podziemia stanowiły miej sce wiecznego spoczynku licznej szlachty z okolic Chęcin. O ko ściele franciszkańskim, a zapewne też i o kaplicy św. Leonarda, pisze w 1612 r. wizytator O. Gośki: „Multi nobiles in hac eodesia sepulti nulla epitaphia haibent” 80. Kaplica rozbudowywana i upiększana ostateczne kształty archi- K O Ś C IO Ł Y I K A P L IC E CHĘCIN [21] KS. M A R IA N P A U L E W IC Z 231 230 [20] ny i ryt owa ny. 84 A. K r ó 1, Kasper Fodyga budowniczy, s. 91—ilOl. 85 J. Ł o z i ń s k i , Kaplice kopułowe, s. l®4. 86 ADK Akita Archid. Kurzei., T. 3 s. 42. 87 W. G r z e l i ń s k i , Monografia Chęcin, s. 50. 88 Bibl. Jagieł. Rkps 5478 — Aota Judiciaria Advocatialia Chencinen- sis, K. 69— TO. 5. KAPLICA CMENTARNA y y yg Kaplica ta .odegrała poważną rolę w życiu parafii. Dzięki jej istnieniu rozpowszechniło się nadawanie w parafii imion Kasper, Melchior i Baltazar. Założona przy niej przez fundatora preiben- da należała do najbogatszych prebend chęcińskich. Pod względem architektonicznym kaplica ta stanowi niepoślednie dzieło sztuki. Położona na wyższym od nawy południowej poziomie, podsklepdo- na jest kryptą grobową. Wzniesiona na planie kwadratu kryta ko pułą na żagielkatih z latarnią. Ściany tarczowe z okrągłymi okna mi od wschodu i południa. Kopula jest żebrowana promieniście. Na marmurowym fryzie belkowania obiegającego wokół kaplicę — napis o Zbudowaniu kaplicy w 1614 r. W żagielkach kopuły w stiukowych obramieniach późnorenesansowe kartusze z gmerkami Fodygi i herbami Prawdzie (Zaremba?) jego żony. Ponad wej ściem na ścianie tarczowej malowidło: Hołd Trzech Króli z XVII w. Otwór wejściowy ostrotukowy, ujęty od strony nawy profilo waną archiwoltą na konsolach. Zewnętrzne ściany gładkie, kopu ła lekko rozpłaszczona z latarnią o arkadowych oknach między pilasterkami. Ołtarz z kieleckiego marmuru i białego wapienia, pó- źnorenesansofwy związany z architekturą kaplicy; bogato rzeźbi o- W 1807 r. zostaje założony w Chęcinach nowy cmentarz grze balny. Do roku 1965 nie posiadał on kaplicy. Nową kaplicę wzniósł na cmentarzu w latach 1965— 1970 ks. Franciszek Kudełko, pro boszcz i dziekan chęciński. Pełni ona funkcję kaplicy cmentarnej.
https://openalex.org/W4254967707	https://dro.deakin.edu.au/articles/journal_contribution/Corrigendum_Topical_Ophthalmic_Formulation_of_Trichostatin_A_and_SurR9-C84A_for_Quick_Recovery_Post-alkali_Burn_of_Corneal_Haze/20777614/1/files/37026778.pdf	English	null	Corrigendum: Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze	Frontiers in pharmacology	2,018	cc-by	1,363	Corrigendum : Topical Ophthalmic Formulation of Trichostatin A and SurR9 C84A for Quick Recovery Post-alkali Burn of Corneal Haze AUTHOR(S) Kislay Roy, Prasad Neerati, Chun Hei Antonio Cheung, Rupinder K Kanwar, Rajat Sandhir, Jagat Kanwar PUBLICATION DATE 18-12-2018 HANDLE 10536/DRO/DU:30131940 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Corrigendum : Topical Ophthalmic Formulation of Trichostatin A and SurR9- C84A for Quick Recovery Post-alkali Burn of Corneal Haze 10536/DRO/DU:30131940 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B CORRECTION published: 18 December 2018 doi: 10.3389/fphar.2018.01434 CORRECTION Kislay Roy 1, Prasad Neerati 2, Chun Hei Antonio Cheung 3, Rupinder K. Kanwar 1, Rajat Sandhir 4 and Jagat R. Kanwar 1* Kislay Roy 1, Prasad Neerati 2, Chun Hei Antonio Cheung 3, Rupinder K. Kanwar 1, Rajat Sandhir 4 and Jagat R. Kanwar 1* 1 Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research, Centre for Molecular and Medical Research, School of Medicine, Faculty of Health, Deakin University, Geelong, VIC, Australia, 2 Drug Metabolism & Clinical Pharmacokinetics Division, Department of Pharmacology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India, 3 Department of Pharmacology and Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 4 Department of Biochemistry, Panjab University, Chandigarh, India Keywords: survivin, haze, scarring, wound healing, cytokines, trichostatin-A Edited and reviewed by: Salvatore Salomone, Università degli Studi di Catania, Italy Correspondence: Jagat R. Kanwar jagat.kanwar@deakin.edu.au Edited and reviewed by: Salvatore Salomone, Università degli Studi di Catania, Italy A Corrigendum on Correspondence: Jagat R. Kanwar jagat.kanwar@deakin.edu.au Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze by Roy, K., Neerati, P., Cheung, C. H. A., Kanwar, R. K., Sandhir, R., and Kanwar, J. R. (2017). Front. Pharmacol. 8:223. doi: 10.3389/fphar.2017.00223 Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 29 October 2018 Accepted: 19 November 2018 Published: 18 December 2018 Citation: Roy K, Neerati P, Cheung CHA, Kanwar RK, Sandhir R and Kanwar JR (2018) Corrigendum: Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze. Front. Pharmacol. 9:1434. doi: 10.3389/fphar.2018.01434 Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 29 October 2018 Accepted: 19 November 2018 Published: 18 December 2018 Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Immediately after publication of the original article, the authors found that the incorrect versions of Figure 1B and Table 2 were included due to human error. The correct version of Figure 1 and Table 2 are included in this Corrigendum. Immediately after publication of the original article, the authors found that the incorrect versions of Figure 1B and Table 2 were included due to human error. The correct version of Figure 1 and Table 2 are included in this Corrigendum. Prasad Neerati was not included as an author in the published article. HPLC data in Table 2 was repeated and now Prasad Neerati is included in the list of authors in the article. The authors apologize for these errors and state that this does not change the scientiﬁc conclusions of the article in any way. The original article has been updated. Received: 29 October 2018 Accepted: 19 November 2018 Published: 18 December 2018 Frontiers in Pharmacology \| www.frontiersin.org December 2018 \| Volume 9 \| Article 1434 Citation: Citation: Roy K, Neerati P, Cheung CHA, Kanwar RK, Sandhir R and Kanwar JR (2018) Corrigendum: Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze. Front. Pharmacol. 9:1434. doi: 10.3389/fphar.2018.01434 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 © 2018 Roy, Neerati, Cheung, Kanwar, Sandhir and Kanwar. 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. December 2018 \| Volume 9 \| Article 1434 Frontiers in Pharmacology \| www.frontiersin.org Roy et al. Topical Ophthalmic Formulation for Alkali Burn FIGURE 1 \| Change in corneal tissue integrity post-alkali burn: (A) Gene expression analysis to determine the effective dosage of TSA and SurR9-C84A using ex vivo rat eyes. (B) Rat eyes were exposed to 1 M NaOH for 1 min and then treated with TSA or SurR9-C84A or a combination of both [TSA+SurR9-C84A(SR9)] for 40 min. The excised eyes were used to determine the opacity and inﬂammatory scores and H&E staining was performed to determine the effect of alkali burn and treatments on corneal tissue integrity. (C) Immunoﬂuorescence staining for expression of clathrin in conjunctival layer and claudin in corneal layer was performed to determine the effect of alkali burn and treatments on corneal tissue integrity. (D) Western blotting analysis conﬁrmed that both decrease in expressions of clathrin and claudin in rat corneal tissue lysates. Each blot was repeated thrice and a representative blot has been presented. (E) Band density analysis of western blot was performed using image J software. The experiments were conducted in ﬁve animals per treatment group. The H&E staining was performed on ﬁve slides obtained from each animal and a representative image has been presented. Five images of each treatment were taken for immunoﬂuorescence studies and a representative image has been presented. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Frontiers in Pharmacology \| www.frontiersin.org December 2018 \| Volume 9 \| Article 1434 Citation: \| FIGURE 1 \| Change in corneal tissue integrity post-alkali burn: (A) Gene expression analysis to determine the effective dosage of TSA and SurR9-C84A using ex vivo rat eyes. (B) Rat eyes were exposed to 1 M NaOH for 1 min and then treated with TSA or SurR9-C84A or a combination of both [TSA+SurR9-C84A(SR9)] for 40 min. The excised eyes were used to determine the opacity and inﬂammatory scores and H&E staining was performed to determine the effect of alkali burn and treatments on corneal tissue integrity. (C) Immunoﬂuorescence staining for expression of clathrin in conjunctival layer and claudin in corneal layer was performed to determine the effect of alkali burn and treatments on corneal tissue integrity. (D) Western blotting analysis conﬁrmed that both decrease in expressions of clathrin and claudin in rat corneal tissue lysates. Each blot was repeated thrice and a representative blot has been presented. (E) Band density analysis of western blot was performed using image J software. The experiments were conducted in ﬁve animals per treatment group. The H&E staining was performed on ﬁve slides obtained from each animal and a representative image has been presented. Five images of each treatment were taken for immunoﬂuorescence studies and a representative image has been presented. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. December 2018 \| Volume 9 \| Article 1434 Frontiers in Pharmacology \| www.frontiersin.org 2 Topical Ophthalmic Formulation for Alkali Burn Roy et al. TABLE 2 \| Concentration of TSA in tissues and plasma after topical application in eye. Time (min) Plasma (ng/mL, mean ± SD) Aqueous humor (ng/mL, mean ± SD) Cornea (µg/g, mean ± SD) Conjunctiva (µg/g, mean ± SD) Tear ﬂuid (µg/g, mean ± SD) 2 103.61 ± 28.37 119.43 ± 37.92 216.83 ± 78.05 267.88 ± 75.51 333.42 ± 69.72 10 263.87 ± 80.96 112.94 ± 15.10 291.02 ± 109.25 261.29 ± 66.47 208.93 ± 30.82 30 109.16 ± 33.00 62.80.24 ± 7.29 762.86 ± 385.68 178.23 ± 35.22 154.98 ± 73.25 Peaks were referenced with commercial standard from Assay Matrix Pty Ltd. TABLE 2 \| Concentration of TSA in tissues and plasma after topical application in eye*. December 2018 \| Volume 9 \| Article 1434 Frontiers in Pharmacology \| www.frontiersin.org 3
https://openalex.org/W1985960960	https://europepmc.org/articles/pmc4133652?pdf=render	English	null	Redefining Strategies to Introduce Tolerance-Inducing Cellular Therapy in Human beings to Combat Autoimmunity and Transplantation Reactions	Frontiers in immunology	2,014	cc-by	5,578	nja ten Brinke1, Irma Joosten2, S. Marieke van Ham1, Cees van Kooten3 and Berent 1 Department of Immunopathology, Division Research and Landsteiner Laboratory, Sanquin Blood Supply, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands atory for Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, Netherlands tment of Nephrology, Leiden University Medical Center, Leiden, Netherlands 4 Laboratory for Translational Immunology, Center for Molecular and Cellular Immunology, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht Netherlands 4 Laboratory for Translational Immunology, Center for Molecular and Cellular Immunology, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht, Netherlands WRONG INCENTIVES bl h h Publications in high impact journals enhance the career of young academic scientist and their esteem and improve their chances for subsequent funding. Currently, these journals have a preference for highly innovative studies, which are by nature often funda- mental. Studies in human subjects are less straightforward and relatively seldom make it to the top journals in the ﬁeld. Thus, the current academic reward structure has created a wrong incen- tive for researchers, namely, to keep their research focused on what yields the highest chance of a next top publication and not on what directly or indirectly might be beneﬁcial for translation into human application. This can be considered as a detrimental development for the design of novel clinical therapies. HURDLES IN CLINICAL TRANSLATION PERSPECTIVE ARTICLE Redeﬁning strategies to introduce tolerance-inducing cellular therapy in human beings to combat autoimmunity and transplantation reactions Correspondence: It is becoming increasingly clear, however, that the intrinsic nature of tolerance-inducing cel- lular products leads to unique requirements for safe and efﬁcient clinical translation. Here, we highlight some of the most notable hurdles in translation of tolerance-inducing cellular therapies and deﬁne some prerequisites that may substantially move the ﬁeld forward. Reviewed by: Reviewed by: Yi-chi Kong, Wayne State University School of Medicine, USA Nick David Jones, University of Birmingham, UK Luis Graca, University of Lisbon, Portugal Reviewed by: Yi-chi Kong, Wayne State University School of Medicine, USA Nick David Jones, University of Birmingham, UK Reviewed by: Yi-chi Kong, Wayne State University School of Medicine, USA Nick David Jones, University of Birmingham, UK Luis Graca, University of Lisbon, Portugal Correspondence: p Anja ten Brinke, Department of Immunopathology, Division Research, Sanquin Blood Supply, P.O. Box 9190, 1006 AD Amsterdam, Netherlands e-mail: a.tenbrinke@sanquin.nl Keywords: tolerance, regulatoryT cells, dendritic cells, regulatory agencies, autoimmunity, transplantation Tolerance-inducing cellular therapies hold great promise for the treatment of patients with chronic inﬂammation, as seen in autoimmune diseases, and for the prevention of graft rejection and graft-versus-host-disease (GvHD) following transplantation (1, 2). Such treatments possess the key for a true restoration of the immune balance and thus may prevent or minimize the life-long use of immunosuppressive drugs and therewith associated adverse side-effects. Their potential is supported by a vast amount of data from in vitro human assays and experimental models (3–7). The ﬁrst cautious steps to clinical translation have been undertaken by phase I and II studies with both regulatory T-cells (Tregs) and tolerogenic dendritic cells (tolDC) in GvHD, organ transplanta- tion, type I diabetes, and RA (2, 8–11). It is becoming increasingly clear, however, that the intrinsic nature of tolerance-inducing cel- lular products leads to unique requirements for safe and efﬁcient clinical translation. Here, we highlight some of the most notable hurdles in translation of tolerance-inducing cellular therapies and deﬁne some prerequisites that may substantially move the ﬁeld forward. a number of essential and intricate steps, which require optimal ﬁne-tuning. These steps serve to demonstrate proof-of-concept for the therapeutic potential of the product and to gain optimal information on the safety of the product. Failures or hurdles at any of these steps will substantially slow down the translation of the tolerance-inducing cell therapies (Figure 1 and Table 1). Tolerance-inducing cellular therapies hold great promise for the treatment of patients with chronic inﬂammation, as seen in autoimmune diseases, and for the prevention of graft rejection and graft-versus-host-disease (GvHD) following transplantation (1, 2). Such treatments possess the key for a true restoration of the immune balance and thus may prevent or minimize the life-long use of immunosuppressive drugs and therewith associated adverse side-effects. Their potential is supported by a vast amount of data from in vitro human assays and experimental models (3–7). The ﬁrst cautious steps to clinical translation have been undertaken by phase I and II studies with both regulatory T-cells (Tregs) and tolerogenic dendritic cells (tolDC) in GvHD, organ transplanta- tion, type I diabetes, and RA (2, 8–11). Edited by: Clinical translation of tolerance-inducing cell therapies requires a novel approach focused on innovative networks, patient involvement, and, foremost, a fundamental paradigm shift in thinking from both Academia, and Industry and Regulatory Agencies.Tolerance-inducing cell products differ essentially from conventional drugs. They are personalized and target interactive immunological networks to shift the balance toward tolerance. The human cell products are often absent or fundamentally different in animals. This creates important limitations of pre-clinical animal testing for safety and efﬁcacy of these products and calls for novel translational approaches, which require the combined efforts of the different par- ties involved. Dedicated international and multidisciplinary consortia that focus on clinical translation are of utmost importance. They can help in informing and educating regulatory policy makers on the unique requirements for these cell products, ranging from pre- clinical studies in animals to in vitro human studies. In addition, they can promote reliable immunomonitoring tools.The development of tolerance-inducing cell products requires not only bench-to-bedside but also reverse translation, from bedside back to the bench. Redeﬁning strategies to introduce tolerance-inducing cellular therapy in human beings to combat autoimmunity and transplantation reactions Anja ten Brinke1, Irma Joosten2, S. Marieke van Ham1, Cees van Kooten3 and Berent Jan Prakken4 CONFUSED REGULATORS Hurdles How to move forward Wrong incentive: human studies are less attractive because of academic reward structure Educate journals, granting organizations, and academic evaluators by creating awareness about the limitations of animal models and value of studies involving human materials Promote long-term, supervised grant structure for collaborative research focused on human translational studies Confused regulators: cellular therapies are treated similarly as conventional drug Create awareness that cellular therapies need new evaluation tools Men are not mice: problems cured in animals do not necessarily translate to therapy for human beings Create awareness about differences between human beings and mice Educate about the limitations and added value of mice models Use the human model: invest in monitoring of clinical studies Scattered research by many different research groups Promote extensive international collaborative networking Insufﬁcient patient partnering Involve patients/patients’ organizations more closely in the development of cellular therapies FIGURE 1 \| Steps and hurdles in clinical translation of tolerance-inducing cellular therapies. Educate journals, granting organizations, and academic evaluators by creating awareness about the limitations of animal models and value of studies involving human materials Promote long-term, supervised grant structure for collaborative research focused on human translational studies the novelty of these therapies. All cell-based therapeutic products (irrespective whether they aim to induce immunological tolerance or immune activation) are essentially different from conventional chemical drugs. Still, for efﬁcacy and safety of cell products, regulatory agencies mostly rely on the models used for testing conventional drugs. This leads to the request for similar product requirements in extensive animal studies on dosage, conventional toxicology, and pharmacodynamics. Many of the characteristics of chemical compounds (solubility, diffusion capacity, turnover into toxic side products) that are tested in such studies, however, do not apply to cells. Therefore, these studies are often of little use for predicting efﬁcacy or safety of a cellular product upon infusion. These studies create additional costs, slow down clin- ical translation, and hamper effective progression of cell-based therapies. CONFUSED REGULATORS To improve human health using cellular therapies, basic scien- tiﬁc discoveries must be translated into pre-clinical studies [e.g., in vitro or in vivo (animal) model systems] and subsequently be applied in human subjects in clinical studies. This process entails Currently,regulatory bodies treat cellular therapy in a similar fash- ion as conventional drugs. Unfamiliarity with tolerance-inducing cell therapies is at the basis of this view and is readily explained by August 2014 \| Volume 5 \| Article 392 \| 1 www.frontiersin.org Redeﬁning strategies for tolerance-inducing cellular therapy ten Brinke et al. FIGURE 1 \| Steps and hurdles in clinical translation of tolerance-inducing cellular therapies. Table 1 \| How to move forward. Hurdles How to move forward Wrong incentive: human studies are less attractive because of academic reward structure Educate journals, granting organizations, and academic evaluators by creating awareness about the limitations of animal models and value of studies involving human materials Promote long-term, supervised grant structure for collaborative research focused on human translational studies Confused regulators: cellular therapies are treated similarly as conventional drug Create awareness that cellular therapies need new evaluation tools Men are not mice: problems cured in animals do not necessarily translate to therapy for human beings Create awareness about differences between human beings and mice Educate about the limitations and added value of mice models Use the human model: invest in monitoring of clinical studies Scattered research by many different research groups Promote extensive international collaborative networking Insufﬁcient patient partnering Involve patients/patients’ organizations more closely in the development of cellular therapies the novelty of these therapies. All cell-based therapeutic products (irrespective whether they aim to induce immunological tolerance or immune activation) are essentially different from conventional MEN ARE NOT MICE Up to today, much emphasis has been put on the use of experi- mental animal models as a necessary step toward the clinic not FIGURE 1 \| Steps and hurdles in clinical translation of tolerance-inducing cellular therapies. FIGURE 1 \| Steps and hurdles in clinical translation of tolerance-inducing cellular therapies. FIGURE 1 \| Steps and hurdles in clinical translation of tolerance-inducing cellular therapies. Table 1 \| How to move forward. MEN ARE NOT MICE Also, tolerance-induci cells theoretically could even become pro-inﬂammatory in t pro-inﬂammatory environment of autoimmune inﬂammatio This asks for additional safety precautions and additional forms experimental testing. Some human cell types, however, are no existent in animals or show great differences in molecular phen type and function. For instance, human- and mouse-induced Tr have different regulators of their transcription and show diffe ent stability (19, 20). This may lead to wrong leads and clues f development of novel therapies in human beings. Interestingly enough, the absence of appropriate small an mal models has not hampered the HIV research ﬁeld in ma ing tremendous advances. On the contrary, it may even ha stimulated a more rapid translation from bench-to-bedside. HOW TO MOVE FORWARD We foresee several ways to speed up the translation of toleranc inducing cell therapies into the clinic, as discussed below. CHANGE INCENTIVES The changed environment requires a transformation in t appraisal of science. Because of the limitations of animal studi for the ﬁnal translation toward human beings, academic eval ators and journals need to re-evaluate the value of the compl and often more messy studies in human beings. Likewise, as si gle research groups cannot perform such extensive human studi granting organizations should give priority support to large inte national and multidisciplinary consortia to prevent overlap and accelerate the process. EDUCATE REGULATORS Therapeutic cells have different mechanisms of action and a d ferent safety proﬁle compared to conventional chemical drugs “biologicals”andthusneedtoﬁtdifferentregulatoryrequiremen Currently, awareness is slowly arising that such therapies requi new requirements and evaluative tools (21). It is imperative th academia, industry, and regulators work together to adapt the re ulatory policy to meet these requirements. Initiatives such as fro Burroughs Welcome Fund (BWF) (22), which funds academ research on innovative approaches for assessing the safety an efﬁcacy of new therapies, and as a result, promotes regulatory po icy decisions based on the state-of-the-art science, are a promisi start. USE A MODEL AS A MODEL Itisclearthatmenarenotmice.If wewanttocureahumandisea we should keep this as a primary focus when considering the u of experimental models. Though seemingly obvious, it requir a paradigm shift in thinking, not only from researchers but al from journals and regulators. To date, major journals and regul tors almost always request validation in animal models as a gold standard. We believe that for the development of cellular thera to the success of biologicals, e.g., TNFα blocking therapies. MEN ARE NOT MICE Ini- tial interesting observations that TNFα blockage diminished IL-1 production in in vitro human rheumatoid synovial cultures led to extensive studies in mouse models for rheumatoid arthritis, which have resulted into very successful treatment of rheumatoid arthri- tis with TNFα blocking agents in human beings [as reviewed in Ref. (13)]. Also studies in mice supported evidence that inhibitory molecules like CTLA-4 are involved in immune control and facili- tated the use of anti-CTLA-4 antibodies in clinical practice to treat cancer (14). which are not directly life threatening. Also, tolerance-inducing cells theoretically could even become pro-inﬂammatory in the pro-inﬂammatory environment of autoimmune inﬂammation. This asks for additional safety precautions and additional forms of experimental testing. Some human cell types, however, are non- existent in animals or show great differences in molecular pheno- type and function. For instance, human- and mouse-induced Treg have different regulators of their transcription and show differ- ent stability (19, 20). This may lead to wrong leads and clues for development of novel therapies in human beings. tated the use of anti CTLA 4 antibodies in clinical practice to treat cancer (14). Clear differences exist between biologicals and cell-based prod- ucts. First, biologicals are generated in batches, while cell-based products are personalized and often consist of modiﬁed cells of individual patients. This obviously complicates animal testing of the latter. Biologicals are also different to cell-based products in terms of mechanism of action. Whereas biologicals initially target one single molecule, cellular products target multiple effec- tor molecules and cells that act in highly interactive networks to shift the immunological balance toward immune activation (anti-cancer therapy) or toward immune tolerance. This fact has important consequences when realizing that the human immune system differs more from rodent models than often is assumed in genetic make-up and immunological maturity. A recent study clearly underscored that genome-wide inﬂammatory responses in some major diseases/disease models signiﬁcantly differed between human beings and mice (15). As a consequence, experimental models – though useful in many other ways – mimic poorly human disease. Also for biologicals, the different genetic make- up of the target of the biological in animal models compared to human beings has created a false sense of safety in the past. The development of anti-CD40 ligand antibodies to prevent trans- plant rejection and treat SLE was halted when the ﬁrst studies in human beings showed thrombo-embolic complications (16, 17). MEN ARE NOT MICE Subsequent studies indicate that this is caused by the activation of surface FcγRIIa on human platelets, an FcR variant not present on mice platelets (18). Next to genetic differences, also dissimilar maturation of the immune systems in human beings and caged animals may play a role. Mice are mostly studied at young age and are not continuously exposed to pathogens. Consequently, they are more immunological naïve compared to adult patient populations. This further limits the suitability of mouse models as the crucial preparatory step for bringing immunotherapy to the clinic; an experienced and environmentally educated immune system will most certainly react differently to immune interven- tions. Since cell-based products act on an interactive network of immunological targets, described differences in genetic make-up and immunological maturity make especially evaluation of cell- based products in animal models difﬁcult, and may even create a false sense of safety of the product. Furthermore, inadvertently the preferred choice of administration routes often differs in mice versus men, again questioning if ﬁndings can be translated from mouse studies to application in human beings. Th li i f l i d i ll l h f Interestingly enough, the absence of appropriate small ani- mal models has not hampered the HIV research ﬁeld in mak- ing tremendous advances. On the contrary, it may even have stimulated a more rapid translation from bench-to-bedside. We foresee several ways to speed up the translation of tolerance- inducing cell therapies into the clinic, as discussed below. CHANGE INCENTIVES The changed environment requires a transformation in the appraisal of science. Because of the limitations of animal studies for the ﬁnal translation toward human beings, academic evalu- ators and journals need to re-evaluate the value of the complex and often more messy studies in human beings. Likewise, as sin- gle research groups cannot perform such extensive human studies, granting organizations should give priority support to large inter- national and multidisciplinary consortia to prevent overlap and to accelerate the process. EDUCATE REGULATORS h ll h Therapeutic cells have different mechanisms of action and a dif- ferent safety proﬁle compared to conventional chemical drugs or “biologicals”andthusneedtoﬁtdifferentregulatoryrequirements. Currently, awareness is slowly arising that such therapies require new requirements and evaluative tools (21). It is imperative that academia, industry, and regulators work together to adapt the reg- ulatory policy to meet these requirements. Initiatives such as from Burroughs Welcome Fund (BWF) (22), which funds academic research on innovative approaches for assessing the safety and efﬁcacy of new therapies, and as a result, promotes regulatory pol- icy decisions based on the state-of-the-art science, are a promising start. www.frontiersin.org MEN ARE NOT MICE the novelty of these therapies. All cell-based therapeutic products (irrespective whether they aim to induce immunological tolerance or immune activation) are essentially different from conventional chemical drugs. Still, for efﬁcacy and safety of cell products, regulatory agencies mostly rely on the models used for testing conventional drugs. This leads to the request for similar product requirements in extensive animal studies on dosage, conventional toxicology, and pharmacodynamics. Many of the characteristics of chemical compounds (solubility, diffusion capacity, turnover into toxic side products) that are tested in such studies, however, do not apply to cells. Therefore, these studies are often of little use for predicting efﬁcacy or safety of a cellular product upon infusion. These studies create additional costs, slow down clin- ical translation, and hamper effective progression of cell-based therapies. Up to today, much emphasis has been put on the use of experi- mental animal models as a necessary step toward the clinic, not in the least by regulatory bodies. The use of animal models to learn about mechanisms of action, deﬁne therapeutic targets, and design potential therapeutics is beyond doubt. Meta-analysis has shown that only about a third of highly cited animal research is translated at the level of human randomized trials (12). This is in part due to the fact that studies on animals do not always predict the effects of immunomodulatory therapies in human beings cor- rectly. Speciﬁcally, for tolerance-inducing therapies, its use needs redeﬁning. It is important to distinguish the value of animal models based on the type of immunomodulatory therapy used, i.e., biologi- cals versus cell-based products. Studies in mice have contributed Frontiers in Immunology \| Immunological Tolerance August 2014 \| Volume 5 \| Article 392 \| 2 Redeﬁning strategies for tolerance-inducing cellular therapy ten Brinke et al. to the success of biologicals, e.g., TNFα blocking therapies. Ini- tial interesting observations that TNFα blockage diminished IL-1 production in in vitro human rheumatoid synovial cultures led to extensive studies in mouse models for rheumatoid arthritis, which have resulted into very successful treatment of rheumatoid arthri- tis with TNFα blocking agents in human beings [as reviewed in Ref. (13)]. Also studies in mice supported evidence that inhibitory molecules like CTLA-4 are involved in immune control and facili- tated the use of anti-CTLA-4 antibodies in clinical practice to treat cancer (14). Clear differences exist between biologicals and cell-based prod- ucts. MEN ARE NOT MICE First, biologicals are generated in batches, while cell-based products are personalized and often consist of modiﬁed cells of individual patients. This obviously complicates animal testing of the latter. Biologicals are also different to cell-based products in terms of mechanism of action. Whereas biologicals initially target one single molecule, cellular products target multiple effec- tor molecules and cells that act in highly interactive networks to shift the immunological balance toward immune activation (anti-cancer therapy) or toward immune tolerance. This fact has important consequences when realizing that the human immune system differs more from rodent models than often is assumed in genetic make-up and immunological maturity. A recent study clearly underscored that genome-wide inﬂammatory responses in some major diseases/disease models signiﬁcantly differed between human beings and mice (15). As a consequence, experimental models – though useful in many other ways – mimic poorly human disease. Also for biologicals, the different genetic make- up of the target of the biological in animal models compared to human beings has created a false sense of safety in the past. The development of anti-CD40 ligand antibodies to prevent trans- plant rejection and treat SLE was halted when the ﬁrst studies in human beings showed thrombo-embolic complications (16, 17). Subsequent studies indicate that this is caused by the activation of surface FcγRIIa on human platelets, an FcR variant not present on mice platelets (18). Next to genetic differences, also dissimilar maturation of the immune systems in human beings and caged animals may play a role. Mice are mostly studied at young age and are not continuously exposed to pathogens. Consequently, they are more immunological naïve compared to adult patient populations. This further limits the suitability of mouse models as the crucial preparatory step for bringing immunotherapy to the clinic; an experienced and environmentally educated immune system will most certainly react differently to immune interven- tions. Since cell-based products act on an interactive network of immunological targets, described differences in genetic make-up and immunological maturity make especially evaluation of cell- based products in animal models difﬁcult, and may even create a false sense of safety of the product. Furthermore, inadvertently the preferred choice of administration routes often differs in mice versus men, again questioning if ﬁndings can be translated from mouse studies to application in human beings. which are not directly life threatening. USE A MODEL AS A MODEL Itisclearthatmenarenotmice.If wewanttocureahumandisease, we should keep this as a primary focus when considering the use of experimental models. Though seemingly obvious, it requires a paradigm shift in thinking, not only from researchers but also from journals and regulators. To date, major journals and regula- tors almost always request validation in animal models as a golden standard. We believe that for the development of cellular therapy this is not the right approach. Even “humanized” mouse models can only mimic the human system and never completely reﬂect a human disease with its enormous heterogeneity and complexity (23). Animal models should be used for what they are developed for, namely, as model for mechanistic pathways and not as model The application of tolerance-inducing cellular therapy for autoimmunity is different compared to cellular therapy for other indications. While anti-cancer cellular therapy treats terminally ill patients, without alternative treatment options, tolerance- inducing cellular products target patients with chronic diseases, August 2014 \| Volume 5 \| Article 392 \| 3 Redeﬁning strategies for tolerance-inducing cellular therapy ten Brinke et al. AUTHOR CONTRIBUTIONS Anja ten Brinke, Irma Joosten, S. Marieke van Ham, Cees van Kooten, and Berent Jan Prakken wrote the paper. AUTHOR CONTRIBUTIONS for a speciﬁc disease. As such, they can be instrumental for speciﬁc issues that cannot be addressed in human beings directly, like the in vivo interaction of cellular products with other immune cells and local tissues and effects thereof. Animal models may be specif- ically valuable to evaluate issues as homing, longevity, and stability of the tolerance-inducing cellular products. Still,deﬁnite proof for these issues needs to be obtained in clinical trials in human beings. Therefore, while being a valuable source for mechanistic data and safety data on product stability, animal models should not be used as a single decisive requirement for tolerance-inducing cellular therapy development. ACKNOWLEDGMENTS The views expressed are inspired by an international symposium organized by the authors entitled:“Lessons learned from immune regulation; toward tolerance-inducing cellular therapies” held at the Royal Netherlands Academy of Arts and Sciences in Ams- terdam on 16 and 17 October 2013. The authors received the grants for their work from Sanquin Blood Supply (internal not- for-proﬁt grant) (Anja ten Brinke, S. Marieke van Ham), not-for- proﬁt Landsteiner Foundation for Blood Transfusion Research (S. USE A MODEL AS A MODEL Marieke van Ham), Dutch Kidney Foundation (Cees van Kooten, Irma Joosten),the Netherlands Organization of Scientiﬁc Research (NWO)(CeesvanKooten),theEuropeanUnion(CeesvanKooten, Berent Jan Prakken), Dutch Top Institute Pharma (Irma Joosten) and Leo Pharma (Denmark) (Irma Joosten), the Dutch Arthritis Foundation (Berent Jan Prakken), the Child Health program of the UMC Utrecht (Berent Jan Prakken), STW technology devel- opment (Berent Jan Prakken), the EU FP7 Framework program (Berent Jan Prakken), and European grant for European coopera- tion in science and technology (Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies) (Anja ten Brinke, S. Marieke van Ham). SCIENTIFIC COLLABORATION 4. Bonham CA, Peng L, Liang X, Chen Z, Wang L, Ma L, et al. Marked prolonga- tion of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig. J Immunol (2002) 169:3382–91. doi:10.4049/jimmunol.169.6.3382 The development of cellular therapies can only be realized through extensive international collaborative networking. Experience and data should be shared to prevent repetition, waste of funding, and to reach consensus on difﬁcult questions. This requires a different mind-set from the general competitive nature of basic biomed- ical research. Promising initiatives in this respect are the ONE study, a uniﬁed approach evaluating cellular immunotherapy in solid organ transplantation,the Immune Tolerance Network (ITN, sponsored by NIAID), the recently initiated EU COST Action A FACTT (Action to Focus and Accelerate tolerance-inducing cel- lular therapies), and the development of broad platforms for biological studies such as UCAN-U (Understanding Childhood Arthritis Network) (26). 5. MoreauA,Varey E,Beriou G,Hill M,Bouchet-Delbos L,Segovia M,et al. Tolero- genic dendritic cells and negative vaccination in transplantation: from rodents to clinical trials. Front Immunol (2012) 3:218. doi:10.3389/ﬁmmu.2012.00218 6. Singer BD, King LS, D’Alessio FR. Regulatory T cells as immunotherapy. Front Immunol (2014) 5:46. doi:10.3389/ﬁmmu.2014.00046 7. Gordon JR, Ma Y, Churchman L, Gordon SA, Dawicki W. Regulatory dendritic cells for immunotherapy in immunologic diseases. Front Immunol (2014) 5:7. doi:10.3389/ﬁmmu.2014.00007 8. Hutchinson JA, Riquelme P, Sawitzki B, Tomiuk S, Miqueu P, Zuhayra M, et al. Cutting edge: immunological consequences and trafﬁcking of human regula- tory macrophages administered to renal transplant recipients. J Immunol (2011) 187:2072–8. doi:10.4049/jimmunol.1100762 9. Brunstein CG, Miller JS, Cao Q, McKenna DH, Hippen KL, Curtsinger J, et al. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety proﬁle and detection kinetics. Blood (2011) 117:1061–70. doi:10.1182/blood-2010-07-293795 REFERENCES 1. Stenger EO, Turnquist HR, Mapara MY, Thomson AW. Dendritic cells and reg- ulation of graft-versus-host disease and graft-versus-leukemia activity. Blood (2012) 119:5088–103. doi:10.1182/blood-2011-11-364091 2. Di IM, Falzetti F, Carotti A, Terenzi A, Castellino F, Bonifacio E, et al. Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood (2011) 117:3921–8. doi:10.1182/blood-2010-10-311894 3. Taylor PA, Lees CJ, Blazar BR. The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood (2002) 99:3493–9. doi:10.1182/blood.V99.10.3493 USE THE HUMAN MODEL Efforts should be made to obtain data from human studies. For example, migration potential, dosing, and life-span of tolerance- inducing cell therapies can be deduced from experiences with cellular therapy in cancer. Sometimes, a case-report in human beings can be as valuable and informative as a study in a mouse model (24). Certainly, the ﬁrst trial on tolerance-inducing cell therapies will yield valuable information. Phase “0” studies using very low dosage of the product may be speciﬁcally considered to establish the stability of the tolerance-promoting capacity of the cellular product. Finally, to establish clinical efﬁcacy of novel tolerance-inducing cell therapies, it is imperative to deﬁne reliable surrogate outcome parameters that reﬂect effective immunother- apy. This needs to arise from reverse translation,i.e.,from bedside- to-bench, and thus again through studies in human beings (25). For that reason, we strongly advocate that development of cell- speciﬁc immunomonitoring technology in human beings is fur- ther advanced and promoted. Thus, clinical and animal model- based observations ideally should complement each other and lead to insights, which would not be possible by either one alone. 1. Stenger EO, Turnquist HR, Mapara MY, Thomson AW. Dendritic cells and reg- ulation of graft-versus-host disease and graft-versus-leukemia activity. Blood (2012) 119:5088–103. doi:10.1182/blood-2011-11-364091 Redeﬁning strategies for tolerance-inducing cellular therapy ten Brinke et al. 24. Martin S, Wolf-Eichbaum D, Duinkerken G, Scherbaum WA, Kolb H, Noordzij JG,et al. Development of type 1 diabetes despite severe hereditary B-lymphocyte deﬁciency. N Engl J Med (2001) 345:1036–40. doi:10.1056/NEJMoa010465 13. Feldmann M, Maini SR. Role of cytokines in rheumatoid arthritis: an edu- cation in pathophysiology and therapeutics. Immunol Rev (2008) 223:7–19. doi:10.1111/j.1600-065X.2008.00626.x 14. Peggs KS, Quezada SA, Korman AJ, Allison JP. Principles and use of anti- CTLA4 antibody in human cancer immunotherapy. Curr Opin Immunol (2006) 18:206–13. doi:10.1016/j.coi.2006.01.011 25. Ledford H. Translational research: the full cycle. Nature (2008) 453:843–5. doi:10.1038/453843a 26. Available from: http://www.onestudy.org/; http://www.immunetolerance.org/; http://www.cost.eu/domains_actions/bmbs/Actions/BM1305; http://www. ucan-u.org 15. Seok J,Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, et al. Genomic responses in mouse models poorly mimic human inﬂammatory diseases. Proc Natl Acad Sci U S A (2013) 110:3507–12. doi:10.1073/pnas.1222878110 Natl Acad Sci U S A (2013) 110:3507–12. doi:10.1073/pnas.12228 27. Braun S. Gene-based therapies of neuromuscular disorders: an update and the pivotal role of patient organizations in their discovery and implementation. J Gene Med (2013) 15:397–413. doi:10.1002/jgm.2747 16. Biogen Says It has Stopped Ongoing Trials of Anti-CD40 Ligand Monoclonal Antibody. Biogen Inc. Press Release (1999). 17. IDEC Pharmaceuticals Announces A Clinical Hold on Ongoing Clinical Trials of Its IDEC-131 Antibody. IDEC Pharmaceuticals Press Release (2002). Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 18. Robles-Carrillo L, Meyer T, Hatﬁeld M, Desai H, Davila M, Langer F, et al. Anti-CD40L immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol (2010) 185:1577–83. doi:10.4049/jimmunol.0903888 Received: 22 May 2014; accepted: 31 July 2014; published online: 15 August 2014. Received: 22 May 2014; accepted: 31 July 2014; published online: 15 August 2014. Received: 22 May 2014; accepted: 31 July 2014; published online: 15 August 2014. Citation: ten Brinke A, Joosten I, van Ham SM, van Kooten C and Prakken BJ (2014) Redeﬁning strategies to introduce tolerance-inducing cellular therapy in human beings to combat autoimmunity and transplantation reactions. Front. Immunol. 5:392. doi: 10.3389/ﬁmmu.2014.00392 19. INVOLVE THE STAKEHOLDERS Patients (and patient organizations) are the true stakeholders of cellular therapy development. They should be more closely involved in risk and beneﬁt analysis of cellular therapies. There are various examples of patient organizations as true catalysts for novel development,for example,in the ﬁeld of muscular dystrophy (27). We propose that basic scientists, clinicians, regulators, indus- try, and patients collaboratively develop guidelines for strategic development of deﬁned cellular therapies. 10. Marek-Trzonkowska N, Mysliwec M, Siebert J, Trzonkowski P. Clinical applica- tion of regulatory T cells in type 1 diabetes. Pediatr Diabetes (2013) 14:322–32. doi:10.1111/pedi.12029 11. Geissler EK. The ONE Study compares cell therapy products in organ transplan- tation: introduction to a review series on suppressive monocyte-derived cells. Transplant Res (2012) 1:11. doi:10.1186/2047-1440-1-10 Transplant Res (2012) 1:11. doi:10.1186/2047-1440-1-10 12. Hackam DG, Redelmeier DA. Translation of research evidence from animals to humans. JAMA (2006) 296:1731–2. doi:10.1001/jama.296.14.1731 Frontiers in Immunology \| Immunological Tolerance August 2014 \| Volume 5 \| Article 392 \| 4 Redeﬁning strategies for tolerance-inducing cellular therapy August 2014 \| Volume 5 \| Article 392 \| 5 Redeﬁning strategies for tolerance-inducing cellular therapy Pfoertner S, Jeron A, Probst-Kepper M, Guzman CA, Hansen W, Westendorf AM, et al. Signatures of human regulatory T cells: an encounter with old friends and new players. Genome Biol (2006) 7:R54. doi:10.1186/gb-2006-7- 7-r54 Redeﬁning strategies to introduce tolerance-inducing cellular therapy in human beings to combat autoimmunity and transplantation reactions. Front. Immunol. 5:392. doi: 10.3389/ﬁmmu.2014.00392 ThisarticlewassubmittedtoImmunologicalTolerance,a sectionof thejournalFrontiers in Immunology. 20. Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol (2006) 24:209–26. doi:10.1146/annurev.immunol.24.021605.090547 ThisarticlewassubmittedtoImmunologicalTolerance,a sectionof thejournalFrontiers in Immunology. 21. Sung NS, Burris JE. Regulatory science innovation: a rate-limiting step in trans- lation. Sci Transl Med (2012) 4:150fs35. doi:10.1126/scitranslmed.3004719 Copyright © 2014 ten Brinke, Joosten, van Ham, van Kooten and Prakken. 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 publica- tion 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 © 2014 ten Brinke, Joosten, van Ham, van Kooten and Prakken. 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 publica- tion 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. 22. Available from: http://www.bwfund.org/grant-programs/regulatory-science 23. Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol (2012) 12:786–98. doi:10.1038/nri3311 August 2014 \| Volume 5 \| Article 392 \| 5 www.frontiersin.org
https://openalex.org/W2074844687	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0038067&type=printable	English	null	Low Pathogenic Avian Influenza Isolates from Wild Birds Replicate and Transmit via Contact in Ferrets without Prior Adaptation	PloS one	2,012	cc-by	12,064	Abstract Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with a 2,3 linkage [Neu5Ac(a2,3)Gal]. Despite the lack of a2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for a 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential. kens JA, Humberd-Smith J, Gordy JT, Bradley KC, et al. (2012) Low Pathogenic Avian Influenza Isolates from Wild Birds Replicate and errets without Prior Adaptation. PLoS ONE 7(6): e38067. doi:10.1371/journal.pone.0038067 Editor: Elankumaran Subbiah, Virginia Polytechnic Institute and State University, United States of America Received September 22, 2011; Accepted April 30, 2012; Published June 1, 2012 Copyright: 2012 Driskell 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 Centers for Disease Control (CDC) grant 5U19Cl00040102 and National Institutes of Health contract HHSN266200700006C. Abstract The CDC provided CDC approval of animal protocols for this study and reviewed this manuscript prior to submission for publication. Otherwise, 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: smt@uga.edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: smt@uga.edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: smt@uga.edu . These authors contributed equally to this work. Low Pathogenic Avian Influenza Isolates from Wild Birds Replicate and Transmit via Contact in Ferrets without Prior Adaptation Elizabeth A. Driskell1., Jennifer A. Pickens2., Jennifer Humberd-Smith2, James T. Gordy2, Konrad C. Bradley3, David A. Steinhauer3, Roy D. Berghaus4, David E. Stallknecht4, Elizabeth W. Howerth1, Stephen Mark Tompkins2* 1 Department of Pathology, University of Georgia, Athens, Georgia, United States of America, 2 Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America, 3 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America, 4 Department of Population Health, University of Georgia, Athens, Georgia, United States of America PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 Both H1N9 and H6N1 wild bird influenza viruses replicated in the upper respiratory tract of ferrets Both H1N9 and H6N1 demonstrated replication in the upper respiratory tract of ferrets, evident by nasal washes and immunohistochemistry (IHC). Although both H1N9 and H6N1 replicated in the nasal cavity, H1N9 replication was more robust, reaching consistently higher titers than H6N1 for days 3 and 5 pi with shedding occurring consistently longer (Figure 1A, B and Table 1). Lesions of influenza infection were present in the nasal turbinates of ferrets infected with H1N9 and H6N1 (Figure 2A), however, only a single ferret in the H6N1 group (day 3 pi) had lesions while nasal turbinates of all four ferrets infected with H1N9 had lesions (both days 3 and 7 pi). This correlates with the viral titers obtained from nasal washes of these ferrets, as viral shedding quickly declined over a short period of time in this group with some ferrets shedding virus only up to day 3 pi (Figure 1). Lesions in the nasal turbinates included infiltration of the submucosa with mild to moderate numbers of lymphocytes with fewer plasma cells and occasional submucosal edema on both days 3 and 7 pi, with an increased amount of inflammation on day 7 pi for H1N9 (Figure 2A). The presence of influenza viral antigen was confirmed by abundant strong intranuclear and frequent intracytoplasmic staining on IHC for the nucleoprotein (NP) of influenza A in the nasal turbinates epithelium (Figure 2B). Positive staining occurred on day 3 pi in the nasal turbinates of ferrets that had lesions and was not present by day 7 pi for both viruses. Here we examine the potential for infection and transmission of an H1N9 (A/Ruddy Turnstone/DE/1171/02abbreviated H1N9) and an H6N1 (A/Ruddy Turnstone/DE/892/02abbreviated H6N1) AIV. Using sequence, in vitro binding, and glycan microarray analysis we determined the receptor specificity of these viruses. Using the ferret model, the most representative animal model of human influenza virus infection, we performed in vivo assessment of the potential for infection, replication, and transmission of these viruses in mammals. Despite a dominant a2,3 (avian) binding specificity we demonstrate that both of these viruses replicate in both the upper and lower respiratory tract of ferrets, inducing pulmonary lesions, but resulting in little morbidity. Moreover, we demonstrate that one of these viruses (H1N9) is able to transmit via direct contact, despite its dominant avian a2,3 SA binding preference. Both H1N9 and H6N1 wild bird influenza viruses replicated in the upper respiratory tract of ferrets These findings support the recent findings of others [37] and taken together demonstrate that AIV circulating in wild bird populations can transmit to mammals, albeit not always causing clinical disease, and that transmissibility of AIV to mammals is not restricted to specific subtypes (i.e. H5N1 or H7). Further investigation of these circulating AIVs is warranted to better understand their zoonotic potential. LPAIVs Replicate and Transmit in Ferrets However, mean lymphocytes (mean+/ 2standard error) decreased in both H6N1 (3.50+/20.06 pre inoculation to 3.26+/20.06 day 1 pi) and H1N9 (3.76+/ 20.06 pre inoculation to 3.61+/20.09 day 1 pi) inoculated groups compared to the allantoic inoculated group (3.46+/ 20.05 pre inoculation to 3.45+/20.06 day 1 pi) for day 1 pi, and then increased for the H1N9 (4.06+/20.16 day 7 pi) inoculated group compared to the allantoic inoculated group (3.67+/20.11 day 7 pi) and remained elevated through day 18 pi (Table S1). they have also been used for numerous studies of AIV that have resulted in human disease [20–22]. In this study, two wild bird AIVs (H1N9 and H6N1 subtypes) that exhibited robust pulmo- nary replication in mice were further studied in a ferret model to better assess pathogenesis and transmission capacity in mammals. p g p y Viral contributors to host range restriction and virulence of AIVs in mammals have been demonstrated to be a multifactorial. The interaction between the major viral glycoprotein, the hemagglutinin (HA) and the host cell sialic acid receptors is considered critical for establishing an influenza infection, and species specific binding restrictions have been identified. Influenza viruses of avian origin preferentially bind terminal sialic acids with a a2,3 linkage located in cells in the gastrointestinal tract of birds and on the ciliated cells and type II pneumocyte in the human respiratory tract [23–28]. Conversely, human influenza viruses exhibit preferential binding to terminal sialic acids with a a2,6 SA linkage located most prominently on non-ciliated cells of the human upper respiratory tract (nasopharynx and trachea) [23,27,29–32]. It is thought that the receptor specificity of influenza viruses is a large component of host restriction; where in some AIV cases (H5, H7, and H9 subtypes), the viruses are able to infect and cause disease in humans yet exhibit poor human to human transmission [14,33–35]. The amino acid residues contributing to a2,3 versus a2,6 SA binding specificity have been described for some viruses and mutation analysis has shown some of these residues to be directly involved with altering viral receptor specificity. In human H3 strains, amino acids Leu226 and Ser228 (H3 numbering) results in a2,6 SA binding, where avian strains that preferentially bind a2,3 SA receptors exhibit a Gln226 and Gly228 amino acid sequence [26,27,32,36]. Amino acid residues 138, 190, 194, and 225 (H3 numbering) have also been shown to be differentially conserved in avian and human influenza viruses [32]. Both H1N9 and H6N1 wild bird influenza viruses replicated in the lower respiratory tract of ferrets There was evidence of replication of both H1N9 and H6N1 in the lung of inoculated ferrets, with similar histopathologic progression of lesions for both viruses. Influenza was detected in the lung for both viruses for all ferrets day 3 pi using virus isolation in ECEs, but was not isolated for day 7 pi from the lung for either group of ferrets (Table 2). For both viruses, histopathologic change in the lung on day 3 pi was characterized by a small amount of mucus admixed with neutrophils and macrophages within the lumens of bronchi and large bronchioles with peribronchial inflammation, primarily lymphocytes, that surrounded and PLoS ONE \| www.plosone.org LPAIVs Replicate and Transmit in Ferrets LPAIVs Replicate and Transmit in Ferrets ferrets took an additional two to six days to return to pre-infection weight. In contrast, six out of seven ferrets infected with H1N9 had mild weight loss most prominent at day 1 post inoculation (pi) (Table 1) in which it took up to four days to return to pre-infection weight. The average temperature of H1N9 and H6N1 inoculated ferrets was most elevated on day 1 pi (Table 1). Direct contact H1N9 ferrets had elevated temperatures that did correlate with shedding of virus in nasal washes, while direct contact H6N1 ferrets, which did not become infected, had rare, inconsistent temperature elevations. Sneezing was not observed in any group of ferrets and all ferrets remained bright and alert through the duration of the study. There were no statistically significant differences in the total or differential leukocyte parameters for group by day interactions. However, mean lymphocytes (mean+/ 2standard error) decreased in both H6N1 (3.50+/20.06 pre inoculation to 3.26+/20.06 day 1 pi) and H1N9 (3.76+/ 20.06 pre inoculation to 3.61+/20.09 day 1 pi) inoculated groups compared to the allantoic inoculated group (3.46+/ 20.05 pre inoculation to 3.45+/20.06 day 1 pi) for day 1 pi, and then increased for the H1N9 (4.06+/20.16 day 7 pi) inoculated group compared to the allantoic inoculated group (3.67+/20.11 day 7 pi) and remained elevated through day 18 pi (Table S1). ferrets took an additional two to six days to return to pre-infection weight. In contrast, six out of seven ferrets infected with H1N9 had mild weight loss most prominent at day 1 post inoculation (pi) (Table 1) in which it took up to four days to return to pre-infection weight. The average temperature of H1N9 and H6N1 inoculated ferrets was most elevated on day 1 pi (Table 1). Direct contact H1N9 ferrets had elevated temperatures that did correlate with shedding of virus in nasal washes, while direct contact H6N1 ferrets, which did not become infected, had rare, inconsistent temperature elevations. Sneezing was not observed in any group of ferrets and all ferrets remained bright and alert through the duration of the study. There were no statistically significant differences in the total or differential leukocyte parameters for group by day interactions. Introduction highly transmissible in the human population. Recent studies have demonstrated the high compatibility of avian and human in- fluenza reassortants in vitro and in vivo and generation of viable reassortants in vivo in ferrets, further raising the concern of the natural generation of a pandemic strain [15–18]. Examining the capacity of a spectrum of wild bird AIVs to infect mammals is necessary to complete our understanding of AIV host range restrictions and to better define potential risks of mammalian infection and viral reassortment. The host and virulence range for avian influenza viruses (AIV) continues to surprise, with numerous cases of direct transmission from birds to mammals that result in a range of disease including pneumonia, conjunctivitis, and occasionally systemic disease [1– 3]. Although transmission of AIV to humans resulting in disease has been limited to poultry adapted viruses, there is evidence of both direct transmission of AIV to other mammalian species [3–5] and experimental evidence that numerous AIV hemagglutinin (HA) subtypes can infect mammals [5–10]. However, there remains a great void of knowledge regarding the capacity of AIV to infect mammals, especially related to AIVs from the wild bird reservoir. Human AIV infections have been limited to the H5, H7, and H9 subtypes [2,11–14] and these viruses are of concern because they have a pandemic potential if they become We have previously screened wild bird AIVs in a mouse model and demonstrated their varying capacity to replicate in the lung of mice, with some isolates exhibiting robust pulmonary replication regardless of HA subtype and causing mild clinical disease [19]. Ferrets are a better model for influenza infection and transmission in humans as they are naturally susceptible to the virus and have a similar distribution of sialic acid glycans in the respiratory tract; PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 1 June 2012 \| Volume 7 \| Issue 6 \| e38067 June 2012 \| Volume 7 \| Issue 6 \| e38067 LPAIVs Replicate and Transmit in Ferrets LPAIVs Replicate and Transmit in Ferrets LPAIVs Replicate and Transmit in Ferrets Figure 1. Nasal shedding and direct contact transmission of wild bird influenza viruses in ferrets. Seven ferrets were intranasally inoculated with 56105 PFUs of either H6N1 (A) or H1N9 (B) and nasal washes were collected and titered on MDCK cells (days post-inoculation portion of graph). Three naı¨ve ferrets were paired with three of the inoculated ferrets 24 hours post inoculation for each virus group (days post-direct contact portion); nasal washes were collected titered on MDCK cells. Both H6N1 and H1N9 demonstrated replication in the upper respiratory tract of the ferrets, however, viral shedding was consistently greater in magnitude and duration for H1N9. H1N9 demonstrated direct contact transmission, but H6N1 did not transmit to direct contact ferrets. doi:10 1371/journal pone 0038067 g001 Smaller bronchioles were frequently filled with neutrophils, macrophages, and cellular debris and lined by a mixture of ectatic and very plump epithelial cells, indicative of early repair after previous epithelial damage (Figure 2D, E). Smaller bronchioles still had intraluminal inflammatory exudate and epithelial regenera- tion on day 7 pi. Peribronchiolar alveoli of affected bronchioles were occasionally filled by macrophages, but notably, there were minimal alveolar changes. Some pulmonary vessels had small perivascular cuffs composed primarily of lymphocytes. On both day 3 and day 7 pi for both viruses, the caudal lung lobes were more affected in severity and extent than the cranial lung lobes. Importantly, only segmental areas of the lung were affected in ferrets inoculated with either virus, with some lung lobes in individual ferrets having no histopathologic lesions at all. There was evidence of viral replication in the trachea, with rare neutrophils present in the epithelium for both H1N9 and H6N1 and rare positive intranuclear epithelial immunoreactivity for the NP of influenza for one H1N9 inoculated ferret. Lung in the three inoculated ferrets from repeat transmission studies was examined microscopically at day 21 pi; these tissues had no significant lesions, indicating that complete resolution of pulmonary damage and inflammation had occurred by this time. As there was evidence of pulmonary damage with regeneration, we suspected that viral replication in the lung had already peaked by day 3 pi. A titer could not be obtained for the lung via TCID50 assay for day 3 pi for either virus (limit of detection = 1.3 or 1.0 log10 TCID50/g) despite positive virus isolation. Extra-respiratory detection of wild bird influenza viruses in ferrets Few changes in other organs were observed on histopathology. There was mild to moderate gross enlargement of tracheobron- chial lymph nodes with microscopic lesions of moderate follicular hyperplasia as a result of the antigenic stimulation in all ferrets (both H1N9 and H6N1, days 3 and 7 pi). Perivascular cuffs of lymphocytes were present in the olfactory nerves and olfactory bulbs of the brain for one of the two ferrets infected with H6N1 on day 7 pi and one of the two ferrets infected with H1N9 on day 7 pi, although no influenza antigen was detected via immunohis- tochemistry on day 3 pi in the olfactory bulb for any of the ferrets. However, these lesions in combination with positive virus isolation on olfactory bulb in one of the ferrets infected with H6N1 on day 3 pi, are supportive of probable direct extension of virus from the infected nasal epithelium, as has been shown in numerous experimental intranasal influenza inoculations (Table 2) [6,21,38]. Virus isolation was also performed on several other organs with sporadic positive results for rectal swabs, liver, and spleen, despite the absence of histopathologic lesions in these organs (Table 2). Figure 1. Nasal shedding and direct contact transmission of wild bird influenza viruses in ferrets. Seven ferrets were intranasally inoculated with 56105 PFUs of either H6N1 (A) or H1N9 (B) and nasal washes were collected and titered on MDCK cells (days post-inoculation portion of graph). Three naı¨ve ferrets were paired with three of the inoculated ferrets 24 hours post inoculation for each virus group (days post-direct contact portion); nasal washes were collected titered on MDCK cells. Both H6N1 and H1N9 demonstrated replication in the upper respiratory tract of the ferrets, however, viral shedding was consistently greater in magnitude and duration for H1N9. H1N9 demonstrated direct contact transmission, but H6N1 did not transmit to direct contact ferrets. doi:10 1371/journal pone 0038067 g001 doi:10.1371/journal.pone.0038067.g001 occasionally infiltrated peribronchial glands (Figure 2G). There was positive intranuclear immunoreactivity against the NP of influenza A in small numbers of bronchiolar cells in the lungs of ferrets infected with H1N9 and H6N1 on day 3 pi (Figure 2F). No influenza antigen was present via IHC in the lung of ferrets infected with either of the two viruses on day 7 pi, supporting that virus was cleared from the lung by that time point. LPAIVs Replicate and Transmit in Ferrets Therefore, two additional ferrets were inoculated with H1N9 and lung examined at day 2 pi. In the day 2 pi ferret lung, a virus titer was obtained using a 1:3 dilution scheme (Table 1). Histopathologic changes in the lung on day 2 pi also confirmed suspicion of early viral replication, damage, and clearance, as the affected bronchiolar epithelium had necrosis and sloughing without indication of the regeneration that was observed on day 3 pi (Figure 2C). Positive intranuclear immunoreactivity against the NP of influenza A was also observed in small numbers of bronchiolar cells in the lungs for day 2 pi. Wild bird influenza viruses replicated in ferrets but exhibited low virulence Infection in H1N9 and H6N1 inoculated ferrets and in H1N9 contact ferrets was demonstrated with presence of virus in nasal washes and seroconversion despite minimal clinical signs (Table 1). Three ferrets infected with H6N1 had transient, mild weight loss that was most prominent day 1 pi (Table 1), but two of these June 2012 \| Volume 7 \| Issue 6 \| e38067 PLoS ONE \| www.plosone.org 2 LPAIVs Replicate and Transmit in Ferrets Table 1. Morbidity, seroconversion, and respiratory viral replication of ferrets inoculated with wild bird avian influenza viruses H1N9 and H6N1. Inoculated animals Clinical parameters Virus shedding Seroconversion Virus Weight loss (max %, average %) Range temperature increasea Sneezing Virus detection in nasal wash (peak log10 TCID50/mL)b Average TCID50/g virus in lung (day p.i.)c Number with seroconversion (HI titers) Number with seroconversion (MN titers) H6N1 3/7 (6.3, 5.8) 0.1–1.6 0/3 7/7 (5.2) Not detected (3,7) 3/3 (1:160, 1:160, 1:160) 3/3 (1:2560, 1:1280, 1:640) H1N9 6/7 (12.8, 4.5) 0.1–1.7 0/3 7/7 (5.8) 5.1 (2); Not detected (3,7) 3/3 (1:40, 1:40, 1:40) 3/3 (1:320, 1:640, 1:320) Allantoic fluid 0/3 0.1–2.4d 0/3 0/3 NDe 0/3f ND Direct contact animals H6N1 1/3 (2.7, ,0) 0.4–1.4 0/3 0/3 ND 0/3 0/3 H1N9 2/3 (6.0, ,0) 0.1–1.5 0/3 3/3 (6.0) ND 3/3 (1:20, 1:40, 1:80) 3/3 (1:320, 1:640, 1:640) aTemperature is in degrees Celsius. bLimit of detection for nasal wash 1.5 log10 TCID50/mL. cLimit of detection for lung day 7 pi both viruses and day 3 pi for H6N1 is 1.3 TCID50/g; for days 2 and 3 pi for H1N9 is 1.0 TCID50/g. dOne control ferret potentially had an unrelated infection, but remained influenza seronegative. eNot done. f June 2012 \| Volume 7 \| Issue 6 \| e38067 3 PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 Extra-respiratory detection of wild bird influenza viruses in ferrets Rare peribronchiolar glands contained cellular debris and necrosis of the glandular epithelium, with presence of influenza antigen in the epithelial cells demonstrated via IHC on day 3 pi (Figure 2H). PLoS June 2012 \| Volume 7 \| Issue 6 \| e38067 4 PLoS ONE \| www.plosone.org LPAIVs Replicate and Transmit in Ferrets Figure 2. Histopathologic lesions and influenza antigen localization in ferrets inoculated with wild bird influenza viruses. A) Nasal turbinates of ferrets inoculated with H6N1 or H1N9 demonstrated moderate submucosal inflammation (asterisk) (H1N9, d3pi). B) There is widespread strong intranuclear and some intracytoplasmic positive immunoreactivity for the nucleoprotein of influenza A on immunohistochemistry in ferrets inoculated with H6N1 or H1N9 on day 3 pi (H1N9, d3pi). C) Epithelial damage in the lung was early for ferrets inoculated with H1N9, with necrosis in the bronchioles (arrow) and inflammation (asterisks) within and around bronchioles on day 2 pi. D and E) There was evidence of early repair with regeneration of bronchiolar epithelium (arrow) and persistence of inflammation (asterisks) on day 3 pi for ferrets inoculated with H6N1 and H1N9 (H1N9, d3pi). The arrow highlights the stretched and plump bronchiolar epithelial cells, indicating regeneration. F) Presence of influenza antigen was confirmed in ferrets inoculated with H6N1 and H1N9 by strong positive intranuclear staining of bronchiolar epithelial cells with immunohistochemistry on day 3 pi (H6N1, d3pi). G) Inflammation around larger airways in the lung was also present in ferrets inoculated with H6N1 and H1N9, with prominent periglandular bronchial inflammation (asterisk) (H6N1, d7pi). H) There was strong positive intranuclear staining for the nucleoprotein of influenza in the peribronchial glandular epithelial cells on immunohistochemistry on day 3 pi in ferrets inoculated with H6N1 and H1N9 (H1N9, d3pi). Figure 2. Histopathologic lesions and influenza antigen localization in ferrets inoculated with wild bird influenza viruses. A) Nasal turbinates of ferrets inoculated with H6N1 or H1N9 demonstrated moderate submucosal inflammation (asterisk) (H1N9, d3pi). B) There is widespread strong intranuclear and some intracytoplasmic positive immunoreactivity for the nucleoprotein of influenza A on immunohistochemistry in ferrets inoculated with H6N1 or H1N9 on day 3 pi (H1N9, d3pi). C) Epithelial damage in the lung was early for ferrets inoculated with H1N9, with necrosis in the bronchioles (arrow) and inflammation (asterisks) within and around bronchioles on day 2 pi. Wild bird H1N9 influenza virus transmitted via contact between ferrets California/04/2009) generated no detectable titer. Red blood cells (RBCs) from most species express both a2,3 and a2,6 linked sialic acids, but equine erythrocytes are unique in that they exhibit predominantly a2,3 linked SA receptors. Both H6N1 and H1N9 agglutinated guinea pig and turkey RBCs to similar levels as compared to a human influenza. Turkey erythrocytes contain a mixture of a2,3 and a2,6 linked sialic acid linked receptors and guinea pig RBCs express largely a2,6 linked sialic acids with lower levels of a2,3 linked sialic acid receptors [41]. While the mixed a2,3 and a2,6 linkages on these RBCs precludes determination of definitive a2,6 linked sialic acid binding, the lack of clear changes in binding to erythrocytes expressing predominantly a2,6 linked sialic acids suggests limited binding to these glycans. Interestingly, H1N9 exhibited contact transmission between ferrets consistently in all three ferret pairs, but H6N1 did not directly transmit (Figure 1). All three direct contact ferrets that became infected with H1N9 had similar peak viral titers (average peak of 5.2 log10 TCID50 for transmission ferrets and average peak of 5.4 log10 TCID50 for inoculated ferrets) with similar length of shedding time (5 days for both groups) in the nasal wash compared to inoculated ferrets, although the time point of transmission varied greatly between pairs. Transmission variability may be somewhat explained by the varied time point of peak virus in the inoculated ferrets, which matched the pattern of transmission to the paired direct contact ferret (e.g. the later the peak virus in the inoculated ferret, the later the paired contact ferret had indication of transmission). Again, in ferrets that were infected via direct contact transmission, ferret health including clinical signs and morbidity parameters (temperature/weight loss) were minimally affected (Table 1). Direct contact transmission of H1N9 was repeated with a second study, and subsequently confirmed by two out of three direct contact ferrets becoming infected (data not shown). To further define the receptor specificity of the viruses, glycan microarrays were utilized to determine the precise sialyl-oligosac- charide binding profile for H1N9 and H6N1 as compared to a previously defined seasonal H1N1 human influenza virus, A/ Pennsylvania/08/2008 [40]. Purified and fluorescently labeled viruses were submitted to Core H of the Consortium for Functional Glycomics and binding was assessed against 511 glycans (Table S2). Extra-respiratory detection of wild bird influenza viruses in ferrets D and E) There was evidence of early repair with regeneration of bronchiolar epithelium (arrow) and persistence of inflammation (asterisks) on day 3 pi for ferrets inoculated with H6N1 and H1N9 (H1N9, d3pi). The arrow highlights the stretched and plump bronchiolar epithelial cells, indicating regeneration. F) Presence of influenza antigen was confirmed in ferrets inoculated with H6N1 and H1N9 by strong positive intranuclear staining of bronchiolar epithelial cells with immunohistochemistry on day 3 pi (H6N1, d3pi). G) Inflammation around larger airways in the lung was also present in ferrets inoculated with H6N1 and H1N9, with prominent periglandular bronchial inflammation (asterisk) (H6N1, d7pi). H) There was strong positive intranuclear staining for the nucleoprotein of influenza in the peribronchial glandular epithelial cells on immunohistochemistry on day 3 pi in ferrets inoculated with H6N1 and H1N9 (H1N9, d3pi). doi:10.1371/journal.pone.0038067.g002 PLoS ON PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 5 LPAIVs Replicate and Transmit in Ferrets Table 2. Presence of influenza virus in ferret organs by virus isolation in ECEs for ferrets infected with wild bird avian influenza viruses H6N1 and H1N9. Virus Days post inoculation Lung Rectal swab Intestine Olfactory bulb Liver Spleen H6N1 3 2/2 2/7 0/2 1/2 1/2 0/2 5 NDa 1/5 ND ND ND ND 7 0/2 0/5 ND ND ND ND H1N9 3 2/2 1/7 0/2 0/2 2/2 1/2 5 ND 1/5 ND ND ND ND 7 0/2 1/5 ND ND ND ND For each organ listed, the number of animals with a positive isolation of virus out of the number of animals tested is shown. aNot done for this sample. doi:10.1371/journal.pone.0038067.t002 Table 2. Presence of influenza virus in ferret organs by virus isolation in ECEs for ferrets infected with wild bird avian influenza viruses H6N1 and H1N9. Table 2. Presence of influenza virus in ferret organs by virus isolation in ECEs for ferrets infected with wild bird avian influenza viruses H6N1 and H1N9. For each organ listed, the number of animals with a positive isolation of virus out of the number of animals tested is shown. aNot done for this sample For each organ listed, the number of animals with a positive isolation of virus out of the number of animals tested is shown. aNot done for this sample. doi:10.1371/journal.pone.0038067.t002 Wild bird influenza viruses exhibited typical AIV receptor specificity Effective transmission by the H1N9 virus raised questions regarding potential mechanisms for transmission. Segment four, encoding the HA gene was sequenced for each virus to examine receptor specificity as compared to HA sequences defined in the literature. Both H6N1 and H1N9 viruses contained glutamic acid (E) and glycine (G) at positions 190 and 225 of the HA, in contrast to human influenza strains, A/North Carolina/1/1918 and A/ Pennsylvania/08/2008, that contains aspartic acid (D) at both 190 and 225 position (Table 3) that is associated with to a2,6 linked sialic acid receptor specificity [39,40]. The glutamine (Q) at position 226 and glycine (G) at position 228 of the HA are characteristic of avian strains but have been also been identified in human strains and have been shown to influence a2,3 linked sialic acid receptor specificity. The H1N9 and H6N1 viruses contained the HA amino acid residues most commonly present in avian influenza strains that exhibit a2,3 linked sialic acid specificity. Wild bird H1N9 influenza virus transmitted via contact between ferrets The binding motif for all three viruses shows that the H1N9 and H6N1 predominantly bind oligosaccharides that contain N-acetylneuraminic acid a2,3 moieties with little binding observed for the a2,6 and a2,8 linked sialic acid containing glycans (Fig. 3A,B). There was limited N-glyconeur- aminic acid (NeuGc) recognition for the H1N9 and H6N1 with only the a2,3 NeuGc glycans demonstrated binding. The NeuGc moieties are the predominant sialic acid species on equine erythrocytes, where the H1N9 and H6N1 a2,3 NeuGc SA binding preference corroborates the horse erythrocyte agglutina- tion data (Table 4) [42]. In contrast, the human control strain (A/ Pennsylvania/08/2008) demonstrated the predicted a2,6 linked sialic acid receptor binding properties (Fig. 3C) with minor a2,3 linked sialic acid binding, which is common among human influenza viruses. This uncharacteristic a2,3 linked sialic acid receptor specificity is often the result of fucosylated and sulfated modifications to the oligosaccahrides enhancing suboptimal sialic acid receptor recognition, which, as previously shown, appears to be the case for the human control strain (A/Pennsylvania/08/ 2008) [40]. Wild bird influenza viruses exhibited typical AIV receptor specificity PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 Discussion Ruddy Turnstone) can also directly infect and replicate in mammals, even with the potential for direct contact mammal to mammal transmission (Table 1, Figure 1). In the study by Nam et al the A/AB/Kor/CN5/09 (H6N5) virus also replicated to high titers in the upper respiratory tract of ferrets and caused middle disease. Similar to our findings with the H1N9 virus, the H6N5 virus showed limited transmission to contact naı¨ve ferrets [37]. Interestingly, the PB1 segment of the H6N5 virus was similar to and possibly derived from the H5N1 highly pathogenic avian influenza viruses circulating in Asia. In contrast, comparison of the sequences from the AIVs described here demonstrates that the segments are all of LPAIV origin, unrelated to HPAI viruses (data not shown). The H6N5 virus was isolated from feces, and so the species of origin is unknown. It would be Our viruses were passaged in embryonated chicken eggs, which may lend to some adaptive mutations; however, we kept serial passages low to minimize alteration of the original viruses. Interestingly, these viruses replicated to relatively high titers in the upper respiratory tract of the ferret and induced lesions in both the upper and lower respiratory tracts, but minimal disease was observed clinically with complete resolution of pulmonary lesions (Table 1, Figures 1–2). Importantly, lesions and antigen localiza- tion in the lung indicate there was no alveolar replication of the virus. Alveolar localization of influenza with alveolar lesions has been associated with increased virulence [47]. Additionally, the results of virus titration and histopathology comparing days 2 and 3 pi is supportive that infection was rapid, minimal, and had rapid clearance in the lower respiratory tract. These observations may have played a role in the minimal induction of disease for these two viruses in the ferret. A study that examined a variety of AIVs of the H6 subtype not only demonstrated replication with variable morbidity in ferrets, but also showed no correlation between the ability to infect the ferrets and the source of the virus (e.g. wild bird vs. poultry) [7]. This study provides additional indication that the source of virus may not be as important of a factor in transmission to mammals, regardless of subtype, although many other factors would play a role in natural transmission including host interactions and amount of virus shed. Avian influenza virus infections and H7 infectious in particular have presented as conjunctivitis [2]. Discussion To functionally assess the sialic acid receptor specificity of H1N9 and H6N1, we examined the erythrocyte binding of the avian influenza strains. Both viruses were able to agglutinate equine erythrocytes to a 512 HAU/ml titer (Table 4), while the human influenza strains (A/New Caledonia/20/1999 and A/ It has been demonstrated that some low pathogenic H6, H7, and H9 AIV subtypes have the ability to replicate in ferrets; however studies that have demonstrated this examined primarily poultry adapted isolates and have limited scope in examining the June 2012 \| Volume 7 \| Issue 6 \| e38067 June 2012 \| Volume 7 \| Issue 6 \| e38067 PLoS ONE \| www.plosone.org 6 LPAIVs Replicate and Transmit in Ferrets ble 3. Comparison of critical amino acids involved in receptor specificity of influenza hemagglutinin. Hemagglutinin Amino Acidsa Virus Isolate ID HA Subtype Host 190 225 226 228 Accession No. A/Ruddy Turnstone/DE/1171/2002 H1N9 H1 Avian E G Q G CY116631 A/Ruddy Turnstone/DE/892/2002 H6N1 H6 Avian E G Q G CY116633 A/Duck/Alberta/35/1976 H1 Avian E G Q G AF091309 A/South Carolina/1/1918 H1 Human D D Q G AF117241 A/New Caledonia/20/1999 H1 Human N D Q G AB304818 A/Pennsylvania/08/2008 H1 Human D D Q G FJ549047 A/California/04/2009 H1 Human D D Q G FJ966082 aHemagglutinin residues using H3 numbering. doi:10.1371/journal.pone.0038067.t003 interesting to identify the potential reservoirs of this virus through live bird surveillance. full pathogenesis of these infections [6,7,10,43]. Van Hoven et al [43] demonstrated that a two H1N1 AIVs isolated a ducks and a mallard could infect ferrets and replicate to high titers in the upper respiratory tract of ferrets. We have previously shown that current circulating North American wild bird LPAIVs do have a capacity to infect and replicate in mammals using a mouse model of infection, although they caused little or no disease [19]. More recently, Nam et al [37] identified an H6N5 isolated from a fecal sample of an aquatic bird in Korea, A/AB/Kor/CN5/09 (H6N5), that infected mice, replicated to high titer and caused mortality without adaptation. Interestingly, this virus also disseminated to extrapulmonary tissues. From the previous work we selected two viruses of distinct subtype for further study in the ferret model, which most accurately emulated human infection and transmission of influenza virus [44–46]. We have expanded upon previous work by demonstrating that subtypes of ‘‘lesser concern’’ isolated from migratory shore birds (i.e. June 2012 \| Volume 7 \| Issue 6 \| e38067 Discussion While this route of administration is understudied in ferrets, Belser et al [48] recently explored infection of ferrets with human influenza viruses and avian influenza viruses isolated from human infections. Both Table 4. Agglutination of erythrocytes from different animal species by human and avian influenza vir Table 4. Agglutination of erythrocytes from different animal species by human and avian influenza viruses. Hemagglutination Titersa Virus Isolate ID HA Subtype Host Turkey Equine Guinea Pig A/Ruddy Turnstone/DE/1171/2002 H1N9 H1 Avian 1024 512 512 A/Ruddy Turnstone/DE/892/2002 H6N1 H6 Avian 1024 512 256 A/New Caledonia/20/1999 H1 Human 1024 0 256 A/California/04/2009 H1 Human 64 0 16 A/Pennsylvania/08/2008 H1 Human 128 0 32 aHemagglutination titers are provide as the reciprocal of the highest virus dilution generating agglutination. doi:10.1371/journal.pone.0038067.t004 June 2012 \| Volume 7 \| Issue 6 \| e38067 7 7 LPAIVs Replicate and Transmit in Ferrets h d i i i i fl i i l di b H1 l l i f i i hi Figure 3. Glycan binding analysis of wild bird avian or human influenza viruses. Influenza viruses wer cells, purified on a 25% sucrose cushion by ultracentrifugation, and labeled with Alexa488 before being app organized based on Neu5GC, a2,3 SA, a2,6 SA and a2,8 SA glycan structures and represented by different color analysis was performed by Core H of the Consortium for Functional Glycomics. A) A/Ruddy Turnstone/DE/1171/0 892/02 (H6N1), C) A/Pennsylvania/08/2008 (H1N1). doi:10.1371/journal.pone.0038067.g003 Figure 3. Glycan binding analysis of wild bird avian or human influenza viruses. Influenza viruses were propagated in Madin-Darby kidney cells, purified on a 25% sucrose cushion by ultracentrifugation, and labeled with Alexa488 before being applied to the microarray. The data was organized based on Neu5GC, a2,3 SA, a2,6 SA and a2,8 SA glycan structures and represented by different color schemes. Glycan microarray binding analysis was performed by Core H of the Consortium for Functional Glycomics. A) A/Ruddy Turnstone/DE/1171/02 (H1N9), B) A/Ruddy Turnstone/DE/ 892/02 (H6N1), C) A/Pennsylvania/08/2008 (H1N1). doi:10 1371/journal pone 0038067 g003 explore ocular infection in this study; however in light of the demonstrated infection and transmission with H7 influenza viruses, this should be an area of future study. human and avian-origin influenza viruses, including subtypes H1, H3, H5, and H7, and HPAI and LPAI types could infect via the ocular route and spread to the pulmonary tract. Interestingly, the H7 influenzas transmitted to naı¨ve contact ferrets [48]. Discussion We did not June 2012 \| Volume 7 \| Issue 6 \| e38067 PLoS ONE \| www.plosone.org 8 LPAIVs Replicate and Transmit in Ferrets Pulmonary replication of these two AIVs was rapid in onset and rapid to resolve regardless of magnitude of virus and pulmonary lesions. This rapid transient infection may be related to inoculation methods, which place a large dose of virus in an anesthetized ferret where it becomes inhaled deep into the respiratory tract. This confounds risk assessment, as natural exposure would be through a fomite or aerosol droplet from another infected individual. Early pulmonary viral titers and histopathology have not been examined in transmission ferrets in other transmission experiments or in this study. This would be an interesting component to evaluate to aid in determining the pulmonary replication capacity of these AIVs in a more realistic transmission setting. [19]). Other experiments have demonstrated replication of AIVs in the upper and lower respiratory tract of ferrets with some viruses having higher replication in the nasal turbinates and others with higher replication in the lung [6,7,21]. Clearly, cellular tropism in an influenza infected host is complex, and while the cellular SA ligand for HA binding is certainly an important component, additional mechanisms are likely at work. In our study, the H1N9 subtype AIV exhibited efficient direct contact transmission between ferrets. Aerosol transmission was not explored in this study due to lack of a validated aerosol transmission model in our facilities, however, it would be an interesting next step. Many experiments have established the importance of receptor binding in influenza transmission, dem- onstrating limited or no contact or aerosol transmission in viruses that have a binding preference for a2,3 linked SA over a2,6 linked SA [10,43,56,57]. Both the H1N9 and H6N1 viruses have avian specific a 2,3 linked SA receptor binding as shown in the erythrocyte binding assays. There are no previously defined HA amino acid residues that would suggest an altered receptor specificity and this assumption is supported by the glycan microarray analysis, where H1N9 dominantly bound glycans having a2,3 SA linkages. However, there may be other un- identified amino acids in the HA or other viral gene segments that mediated the efficient direct transmission of the H1N9 in ferrets, where other H1N1 viruses failed to efficiently transmit via contact [43]. Direct contact H6N1 ferrets did not become infected and did not seroconvert (Figure 1). Discussion It is thought that the avian influenza preference for binding a2,3 linked SA receptors compared to the human influenza preference for binding a2,6 linked SA receptors provides somewhat of a barrier to transmission in ferrets and humans due to the paucity of a2,3 linked SA in the upper respiratory tract. However, there is a presence of a2,3 linked SA in the lower respiratory tract of both these species, and it has been proposed that if enough influenza can be deposited in the lower respiratory tract, pulmonary infection will predominate in these species [47]. We did observe infection and replication in the lung of these ferrets for both H6N1 and H1N9 that supports viral attachment in the lung (bronchioles, but not alveoli), however, we also observed robust upper respiratory tract infections that were more pro- ductive with higher viral titers present than compared to the lung despite the alpha a2,3 SA binding preference (Table 1, Figures 1– 2).We and others typically observe that ferrets infected with human influenza viruses have viral replication restricted to the upper respiratory tract [52–54]. The absence of pulmonary infection is confirmed by negative virus isolation, absence of lesions on histopathology, and absence of viral antigen on IHC. Very mild pulmonary infections have been demonstrated in other laboratories with human influenza viruses in experimental ferret infections, however severe pneumonia is only associated with highly virulent human influenza viruses or highly pathogenic avian influenza viruses [38,47,55]. In this study, neither H6N1 nor H1N9 virus appeared to infect alveolar epithelial cells (Figure 2) yet, both viruses did infect mouse and feline alveolar epithelial cells in other in vivo experimental trials in our laboratory (submitted and Our study, in combination with additional studies of AIV infections in ferrets, indicates that there is a capacity for wild bird AIVs, subtype notwithstanding, to directly infect mammals with minimal clinical signs. The results support the potential for direct interspecies transmission or formation of a viable AIV reassortant. Although we have demonstrated the low virulence and rapid clearance of these AIVs, possibilities for reassortment in suscep- tible wild and domestic mammalian species make these species of particular interest and worth further investigation. Furthermore, the variable magnitude of seroconversion despite productive influenza infection could make surveillance and monitoring for mammalian infection with AIVs difficult. Discussion Perhaps this is due to lower levels of viral shedding for a shorter period of time in H6N1 infected ferrets as compared to H1N9 infected ferrets. Few studies have examined hematologic parameters in in- fluenza infected ferrets, but investigation into this is worthy as it may be a good measure of clinical disease [49]. Examining clinical pathologic findings of such a small sample size can be difficult, given the marked variation between individuals. Nonetheless, there was a trend in both groups H6N1 and H1N9 compared to the allantoic inoculated group of a decrease in lymphocytes on day 1 pi that resolved by day 3 pi for individual animals. Lymphope- nia is well established to occur in the very early stages of viral infections. Indeed, it has been demonstrated that experimental ferret infections with HPAI H5N1 viruses result in a profound lymphopenia days 3 and 5 pi [50]. Also, ferrets infected with a variety of H1N1 influenza viruses had a decrease in lymphocytes days 3 and 7 pi [49]. For H1N9 inoculated animals, lymphocytes were increased between days 7 pi through 18 pi for many individuals. Lymphocytosis has not been described in ferrets experimentally infected with influenza, although this finding seems logical, given that chronic antigenic stimulation can induce increases in lymphocytes resulting in peripheral lymphocytosis. The viral polymerase has also been suggested to potentially have a role in efficient avian to mammalian transmission, replication, and localization of viral infection based upon differences in temperature for optimal replication, tissue/species tropism for replication, rate of replication, and effect on efficiency of viral nuclear transport [43,58–64]. Sequence analysis of H1N9 PB2 found avian specific Glu627 and Asp701 residues (GenBank Accession ACY79819; data not shown), suggesting that there may be other genetic features contributing to the robust upper respiratory replication and transmission of this H1N9 virus in ferrets. Infectivity of AIVs in mammals and humans is thought to be reliant on the viral hemagglutinin binding sialic acid (SA) residues on host cells, and differences in binding between mammalian versus avian influenza viruses are suggested to be partially responsible for host specificity and localization of infection [24,51]. Discussion Together, these studies support the need for expanded analysis of influenza viruses from their reservoir species as understanding of the mechanisms of infection and transmission is incomplete and subsequent risk assessment imperfect. PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 Viruses Avian influenza viruses used were cloacal swab isolates from wild birds in the United States acquired from Southeastern Cooperative Wildlife Disease Study (Athens, GA). Viruses were isolated from cloacal swabs in 9 day old embryonated chicken eggs (ECE) at 37uC for 72 hours and then minimally passaged (3 or fewer passages) in ECEs. Select virus isolates were screened in a previous study in BALB/c mice [19]. Two viruses that exhibited efficient pulmonary replication and induced pulmonary lesions in BALB/c mice were selected for in vivo studies in ferrets: A/Ruddy Turnstone/DE/892/02 (H6N1 subtype; NCBI Taxonomy ID: 680602; abbreviated H6N1) and A/Ruddy Turnstone/DE/1171/ 02 (H1N9 subtype; NCBI Taxonomy ID: 680596; abbreviated H1N9). The original low passage isolates, once selected by screening methods, were grown once more in 9 to 10 day old ECE to generate a stock of the virus. Stock viruses were aliquoted and stored at 280uC until use. Stock virus titers were determined by plaque assay on MDCK cells. Infection and direct transmission study y Seven ferrets were inoculated per virus (four for tissue examination and three for transmission study) and three additional naive ferrets were used to assay direct contact transmission. In the contact trials, one direct contact ferret was housed with one inoculated ferret as paired cage mates. An additional three ferrets were mock infected with allantoic fluid in PBS for negative controls for nasal washes, serology, and complete blood counts (CBC). Inoculated ferrets were lightly anesthetized with isoflurane and intranasally inoculated with 56105 PFU in 500 mL of sterile PBS (250 mL of per nostril) with either H6N1 or H1N9. Direct contact ferrets were placed with inoculated ferrets twenty-four hours post inoculation. An additional study was performed with H1N9 as previously described to confirm direct contact trans- mission, using three inoculated and three direct contact ferrets. Temperatures monitored for four days to establish baseline then temperature, weights, and complete blood counts were monitored in inoculated ferrets on days 1, 3, 5, 7, 10, 13, 18, and 21 pi and in direct contact ferrets on days 2, 4, 6, 9, 12, 17, and 20 post contact (pc). Nasal washes were sampled from ferrets on days 1, 3, 5, 7, 10 pi or days 2, 4, 6, 9, 12, and 17 pc to monitor for viral infection. For nasal washes, ferrets were anesthetized with 4 mg ketamine via intramuscular injection and 1 mL of sterile PBS with penicillin (4000 U/ml) (Calbiochem, Gibbstown, NJ), streptomy- cin (800 mg/ml) (Sigma, St. Louis, MO), polymyxin B (400 U/ml) (MP Biochmemicals, LLC, Solon, OH), and gentamicin (100 mg/ ml) (Gibco, Carlsbad, CA) was introduced into the nostrils to induce sneezing and collected in specimen cups. For repeat study ferrets, temperature, weights, and nasal washes were performed for Glycan microarray binding y y g Viruses were cultured at an MOI of 0.01 on MDCKs for 72 hours in 16 Minimal Essential Medium supplemented with 1 mg/ml TPCK [L-(tosylamido-2-pheyl) ethyl chloromethyl ke- tone]-treated trypsin (Worthington Biochemical Corporation, Lakewood, NJ). The viral supernatant (10 ml) was collected and centrifuged at 5,000 RPM for 5 minutes to remove cell debris before viral purification. Virus in the culture supernatant was purified on a 25% sucrose cushion and resuspended in 16 PBS with 1 mM EDTA. Briefly, each virus was purified through a 25% sucrose gradient by high speed centrifugation at 28,000 rpm at 4uC for 3 hours. The purified viruses were resuspended in1XPBS with 1 mM EDTA on ice for 4 hours and stored at 280uC. Viral titers were determined by standard plaque assay on MDCK cells. Approximately 107 PFU of each purified strain were labeled with 25 mg of Alexa488 dye in 1 M NaHCO3 (pH 9) for 1 hour. To remove residual dye, each sample was dialyzed in a 7000 MWCO Slide-A-Lyzer MINI dialysis cassette (Thermo Scientific) against PBS with 1 mM EDTA overnight. The labeled viruses were analyzed via glycan microarray by the Core H of the Consortium of Functional Glycomics (www.functionalglycomics.org), where 70 ml of labeled virus was added to glycan microarray slide and incubated at 4uC for 1 hour. Each microarray was scanned by Perkin-Elmer ProScanAray that detected SA binding peaks designated as relative fluorescent units (RFUs). Sequencing Total viral RNA was extracted from AIV infected allantoic fluid using the RNeasy kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer’s protocol. One-step RT-PCR was performed on viral RNA using a universal primer set (Uni12/Inf-1 59- GGGGGGAGCAAAAGCAGG-39 and Uni13/Inf-1 59- CGGGTTATTAGTAGAAACAAGG-39) as previously described [65]. All 8 segments were generated, the HA segment was excised and gel purified using the QIAquick Gel Extraction kit (QIAGEN, Inc., Valencia, CA). The HA was sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) with subtype specific primers (primer sequences available upon re- quest). Some sequences for the H1N9 and H6N1 viruses were already available on GenBank. The accession numbers are GU050646.1, GU050647.1, GU050624.1, GU050623.1, GU050622.1, GU050620.1, GU050619.1, GU050618.1, and GU050621.1 (A/ruddy turnstone/Delaware/892/2002 segment 1 and segment 2, and A/ruddy turnstone/Delaware/1171/2002 segments, 1, 2, 3, 5, 6, 7, and 8 respectively). HA sequences are available at GenBank. Segments sequenced for this study and submitted to GenBank include A/ruddy turnstone/Delaware/ 892/2002 segments 3, 4, 6, and 8 and A/ruddy turnstone/ Delaware/1171/2002 segment 4 (GenBank Accession numbers CY116632, CY116633, CY116634, CY116633, and CY116631, respectively). Ethics statement These studies were conducted in strict accordance with guidelines approved by the Institutional Animal Care and Use Committee and supported through the Office of Animal Care and use of the University of Georgia, following guidelines established June 2012 \| Volume 7 \| Issue 6 \| e38067 June 2012 \| Volume 7 \| Issue 6 \| e38067 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 9 LPAIVs Replicate and Transmit in Ferrets by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care; Accreditation Date: 3/2/2011), licensed by the USDA (USDA #57-R-005), and maintaining an Assurance of Compliance with the U.S. De- partment of Health and Human Services (PHS Assurance #A3437-01). The protocols utilized for these studies were approved by the Institutional Animal Care and Use Committees of the University of Georgia and the Centers for Disease Control and Prevention. by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care; Accreditation Date: 3/2/2011), licensed by the USDA (USDA #57-R-005), and maintaining an Assurance of Compliance with the U.S. De- partment of Health and Human Services (PHS Assurance #A3437-01). The protocols utilized for these studies were approved by the Institutional Animal Care and Use Committees of the University of Georgia and the Centers for Disease Control and Prevention. Ferrets Castrated male Fitch ferrets (Triple F Farms, Sayre, PA), 3 months old and seronegative to circulating human H1N1 and H3N2 influenza viruses, were used for the study. Ferrets were housed in a BSL2 facility in HEPA filtered isolator caging (Allentown, Allentown, NJ). A subcutaneous temperature tran- sponder (BMDS, Seaford, DE) was implanted in each ferret for identification and temperature measurement. Determination of viral titers Nasal washes were immediately tested via real time RT-PCR to aid in determining days for sample collection. Briefly, viral RNA was extracted from nasal wash by using RNeasy mini kit (QIAGEN, Inc., Valencia, CA) and the Qiagen one-step RT- PCR kit was used for RRT-PCR with a Stratagene MX300P/ 3005P thermocyler and Mx Pro QPCR software (La Jolla, CA). Reaction mixture and PCR cycling protocol is available upon request. An influenza virus matrix gene specific primer and probe set were used as follows: primer M+25, sequence AGA TGA GTC TTC TAA CCG AGG TCG; primer M-124, sequence TGC AAA AAC ATC TTC AAG TCT CTG; and probe M+64, sequence FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA [66] (Biosearch Technologies, Novato, CA). Four ferrets per virus were humanely euthanized (two on day 3 pi and two on day 7 pi per virus) and lung, nasal turbinate, liver, spleen, and olfactory bulb were sampled under sterile conditions and frozen at 280uC for virus isolation. Based on histopathology and viral titers from the lung, we suspected early pulmonary infection with rapid clearance, therefore an additional two ferrets were intranasally inoculated as previously described with H1N9 and humanely euthanized at day 2 pi with fresh lung collected to examine the earlier time point. Tissues were later homogenized in 1 mL PBS with antibiotics, clarified by centrifugation, and 100 mL of clarified homogenate was inoculated into 9 to 10 day old ECEs for virus isolation (4 eggs per sample, 72 hour incubation). Nasal washes and clarified lung homogenate were titrated in MDCK cells with serial 1:10 or 1:3 dilutions with a 1.5 log10TCID50/mL (nasal wash) and 1. 3 to 1.0 log10 TCID50/gram (lung). Acknowledgments The authors wish to thank the Animal Resources personnel at the College of Veterinary Medicine, University of Georgia, for excellent animal husbandry. We would also like to thank histotechnicians in the Veterinary Pathology Department at University of Georgia for their assistance, especially Abbie Butler for her outstanding immunohistochemistry support. Additional thanks go to Taiana Costa, Deb Carter and Jon Gabbard for superior assistance with animal work and Cheryl Jones for excellent technical support at the College of Veterinary Medicine, University of Georgia. The authors would also like to acknowledge The Consortium for Functional Glycomics for support of the glycan array analysis. Serology and hematology At day 21 pi (day 20 pc), blood was collected and seroconver- sion was determined via hemagluttination inhibition (HI) and microneutralization (MN) assays. All blood samples were analyzed the same day as sample collection. Complete blood counts were performed using VetScan analyzer (Abaxis, Union City, CA). Leukocyte counts were log transformed and statistically analyzed using repeated measures ANOVA (Stata version 11.0) to examine differences between viral groups and days pi. Degrees of freedom for F-tests of repeated measures factors were adjusted using the Greenhouse-Geisser estimate of epsilon to correct for any departures from the sphericity assumption. All testing assumed a two-sided alternative hypothesis and P-values,0.05 were considered significant. 1. Beigel JH, Farrar J, Han AM, Hayden FG, Hyer R, et al. (2005) Avian influenza A (H5N1) infection in humans. The New England Journal Of Medicine 353: 1374–1385. 2. Belser JA, Bridges CB, Katz JM, Tumpey TM (2009) Past, present, and possible future human infection with influenza virus A subtype H7. Emerging Infectious Diseases 15: 859–865. Histopathology and immunohistochemistry Histopathology and immunohistochemistry Lung (cranial and caudal lobes), trachea, tracheobronchial lymph node, esophagus, heart, spleen, liver, stomach, small intestine, large intestine, pancreas, mesenteric lymph node, kidneys, adrenal gland, bladder, brain, and nasal turbinates were collected on days 3 and 7 pi from inoculated ferrets (two ferrets per virus per day, the same ferrets as described for virus isolation in fresh tissues). The additional two ferrets that were intranasally inoculated with H1N9 and humanely euthanized at day 2 pi had the same set of tissues collected for histopathology and immuno- histochemistry. All tissues were preserved in 10% neutral buffered formalin. Tissues were routinely processed, embedded and stained with hematoxylin and eosin. Immunohistochemical staining was performed on lung, trachea, and nasal turbinates for all ferrets. Immunohistochemistry was performed using a commercially available goat polyclonal antibody to the nucleoprotein of influenza A virus at a 1:10,000 dilution (Biodesign International, Sako, Maine). Tissues were deparaffinized and blocked with a commercial protein blocking agent (Dako Cytomation, Carpin- teria, CA) and a linked strepavidin-biotin immunoperoxidase system was used for immunolabeling. The reaction was visualized with 3, 39-diaminobenzidine substrate (Dako Cytomation, Car- pinteria, CA). Author Contributions Conceived and designed the experiments: EAD JAP DES EWH SMT. Performed the experiments: EAD JAP JHS JTG KCB EWH. Analyzed the data: EAD JAP KCB DAS RDB EWH SMT. Contributed reagents/ materials/analysis tools: KCB DAS RDB DES EWH SMT. Wrote the paper: EAD JAP EWH SMT. 7. Gillim-Ross L, Santos C, Chen Z, Aspelund A, Yang CF, et al. (2008) Avian influenza H6 viruses productively infect and cause illness in mice and ferrets. Journal Of Virology 82: 10854–10863. 6. Belser JA, Lu X, Maines TR, Smith C, Li Y, et al. (2007) Pathogenesis of avian influenza (H7) virus infection in mice and ferrets: enhanced virulence of Eurasian H7N7 viruses isolated from humans. Journal Of Virology 81: 11139–11147. 3. Klopfleisch R, Wolf PU, Uhl W, Gerst S, Harder T, et al. (2007) Distribution of lesions and antigen of highly pathogenic avian influenza virus A/Swan/ Germany/R65/06 (H5N1) in domestic cats after presumptive infection by wild birds. Veterinary Pathology 44: 261–268. J gy 8. Hinshaw VS, Webster RG, Easterday BC, Bean WJ Jr. (1981) Replication of avian influenza A viruses in mammals. Infection and Immunity 34: 354–361. 4. Keawcharoen J, Oraveerakul K, Kuiken T, Fouchier RA, Amonsin A, et al. (2004) Avian influenza H5N1 in tigers and leopards. Emerging Infectious Diseases 10: 2189–2191. 9. Joseph T, McAuliffe J, Lu B, Jin H, Kemble G, et al. (2007) Evaluation of replication and pathogenicity of avian influenza a H7 subtype viruses in a mouse model. Journal Of Virology 81: 10558–10566. Erythrocyte binding assays Fresh turkey, guinea pig, and equine erythrocytes were thoroughly washed with 16 PBS and resuspended to 1% v/v in 16 PBS/.5%BSA. A standard hemagglutination assay was performed for each AIV isolate against all types of erythrocytes, and appearance of agglutination was scored after a 60-min incubation period. PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e38067 10 LPAIVs Replicate and Transmit in Ferrets inoculated ferrets and transmission ferrets on days 1, 3, 5, 7, 9, 11, 13 and 15 pi and on days 2, 4, 6, 8, 10, 12, and 14 pc. Repeat study ferrets were humanely euthanized at day 21 pi (day 20 pc) and samples of lung from all ferrets were fixed in neutral buffered formalin for histopathology. 5. Song D, Kang B, Lee C, Jung K, Ha G, et al. (2008) Transmission of avian influenza virus (H3N2) to dogs. Emerging Infectious Diseases 14: 741–746. Supporting Information Table S1 Total and differential leukocyte counts for ferrets inoculated with wild bird avian influenza viruses H1N9 and H6N1. (DOCX) Table S1 Total and differential leukocyte counts for ferrets inoculated with wild bird avian influenza viruses H1N9 and H6N1. (DOCX) Table S2 Raw Glycan Microarray Data. (DOCX) LPAIVs Replicate and Transmit in Ferrets Rowe T, Leon AJ, Crevar CJ, Carter DM, Xu L, et al. (2010) Modeling host responses in ferrets during A/California/07/2009 influenza infection. Virology 401: 257–265. 28. Ito T, Suzuki Y, Mitnaul L, Vines A, Kida H, et al. (1997) Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species. Virology 227: 493–499. 53. Sweet C, Bird RA, Coates DM, Overton HA, Smith H (1985) Recent H1N1 viruses (A/USSR/90/77, A/Fiji/15899/83, A/Firenze/13/83) replicate poorly in ferret bronchial epithelium. Brief report. Archives Of Virology 85: 305–311. 29. 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Maines TR, Lu XH, Erb SM, Edwards L, Guarner J, et al. References 4. Keawcharoen J, Oraveerakul K, Kuiken T, Fouchier RA, Amonsin A, et al. (2004) Avian influenza H5N1 in tigers and leopards. Emerging Infectious Diseases 10: 2189–2191. PLoS ONE \| www.plosone.org 11 June 2012 \| Volume 7 \| Issue 6 \| e38067 June 2012 \| Volume 7 \| Issue 6 \| e38067 PLoS ONE \| www.plosone.org 64. Gabriel G, Herwig A, Klenk HD (2008) Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathogens 4: e11. 65. Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, et al. (2009) Single- reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses. Journal Of Virology 83: 10309–10313. 63. Subbarao EK, London W, Murphy BR (1993) A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. Journal Of Virology 67: 1761–1764. 61. 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https://openalex.org/W2414674499	https://www.hydrol-earth-syst-sci.net/21/1017/2017/hess-21-1017-2017.pdf	English	null	Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina, USA, using multi-satellite data fusion	Hydrology and earth system sciences	2,017	cc-by	17,147	Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina, USA, using multi-satellite data fusion Yun Yang1, Martha C. Anderson1, Feng Gao1, Christopher R. Hain2, Kathryn A. Semmens3, William P. Kustas1, Asko Noormets4, Randolph H. Wynne5, Valerie A. Thomas5, and Ge Sun6 1USDA ARS, Hydrology and Remote Sensing Laboratory, Beltsville, MD, USA 2Marshall Space Flight Center, Earth Science Branch, Huntsville, AL, USA 3Nurture Nature Center, Easton, PA, USA 1USDA ARS, Hydrology and Remote Sensing Laboratory, Beltsville, MD, USA 2Marshall Space Flight Center, Earth Science Branch, Huntsville, AL, USA 3Nurture Nature Center, Easton, PA, USA 1USDA ARS, Hydrology and Remote Sensing Laboratory, Beltsville, MD, USA 2Marshall Space Flight Center, Earth Science Branch, Huntsville, AL, USA 3Nurture Nature Center, Easton, PA, USA 4Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, USA 5Department of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA 6Eastern Forest Environmental Threat Assessment Center, Southern Research Station, USDA Forest Service, Raleigh, NC, USA 6Eastern Forest Environmental Threat Assessment Center, Southern Research Station, USDA Forest Service, Raleigh NC USA Correspondence to: Yun Yang (yun.yang@ars.usda.gov) Received: 27 April 2016 – Discussion started: 1 June 2016 Accepted: 19 January 2017 – Published: 17 February 2017 Received: 27 April 2016 – Discussion started: 1 June 2016 Accepted: 19 January 2017 – Published: 17 February 2017 Accepted: 19 January 2017 – Published: 17 February 2017 sat ET retrievals due to cloud cover and/or the scan-line corrector (SLC) failure on Landsat 7. The retrieved daily ET time series agree well with observations at two Amer- iFlux eddy covariance ﬂux tower sites in a managed pine plantation within the modeling domain: US-NC2 located in a mid-rotation (20-year-old) loblolly pine stand and US- NC3 located in a recently clear-cut and replanted ﬁeld site. Root mean square errors (RMSEs) for NC2 and NC3 were 0.99 and 1.02 mm day−1, respectively, with mean absolute errors of approximately 29 % at the daily time step, 12 % at the monthly time step and 0.7 % over the full study pe- riod at the two ﬂux tower sites. Analyses of water use pat- terns over the plantation indicate increasing seasonal ET with stand age for young to mid-rotation stands up to 20 years, but little dependence on age for older stands. Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina, USA, using multi-satellite data fusion An accounting of consumptive water use by major land cover classes rep- resentative of the modeling domain is presented, as well as relative partitioning of ET between evaporation (E) and tran- spiration (T ) components obtained with the TSEB. The study provides new insights about the effects of management and land use change on water yield over forested landscapes. Abstract. As a primary ﬂux in the global water cycle, evap- otranspiration (ET) connects hydrologic and biological pro- cesses and is directly affected by water and land manage- ment, land use change and climate variability. Satellite re- mote sensing provides an effective means for diagnosing ET patterns over heterogeneous landscapes; however, limita- tions on the spatial and temporal resolution of satellite data, combined with the effects of cloud contamination, constrain the amount of detail that a single satellite can provide. In this study, we describe an application of a multi-sensor ET data fusion system over a mixed forested/agricultural land- scape in North Carolina, USA, during the growing season of 2013. The fusion system ingests ET estimates from the Two-Source Energy Balance Model (TSEB) applied to ther- mal infrared remote sensing retrievals of land surface temper- ature from multiple satellite platforms: hourly geostationary satellite data at 4 km resolution, daily 1 km imagery from the Moderate Resolution Imaging Spectroradiometer (MODIS) and biweekly Landsat thermal data sharpened to 30 m. These multiple ET data streams are combined using the Spatial and Temporal Adaptive Reﬂectance Fusion Model (STARFM) to estimate daily ET at 30 m resolution to investigate sea- sonal water use behavior at the level of individual forest stands and land cover patches. A new method, also exploit- ing the STARFM algorithm, is used to ﬁll gaps in the Land- 1 Introduction Finally, the range in spatial resolution and coverage of existing TIR data sources enables mapping of ET from the plot or ﬁeld scale (< 100 m resolution) up to continental or global coverage at 1–5 km resolution. p g g Current forest ET estimation methods span a range of spa- tial scales: from individual plants, to tower footprints, to wa- tershed scales (Fang et al., 2015). These methods include in situ measurement, simulation using hydrologic and land sur- face models which are normally driven by weather data and estimation from satellite remote sensing data. Techniques for measuring ET include weighing lysimeters (Wullschleger et al., 1998), sap ﬂow (Klein et al., 2014; Smith and Allen, 1996) and plant chambers (Cienciala and Lindroth, 1995), soil water budgets (Cuenca et al., 1997), eddy covariance (EC; Baldocchi et al., 2001) and catchment water balance (Pan et al., 2012). While EC is a widely used observation method and provides an important data source to many re- search ﬁelds (Baldocchi et al., 2001), it measures turbulent ﬂuxes over a relatively small footprint area (102–104 m2), which is determined by the microclimate conditions around the ﬂux tower and the instrument height. Catchment water balance is also a frequently used method, calculating ET from long-term precipitation and streamﬂow observations with the assumption that the soil water storage change is neg- ligible (Domec et al., 2012; Wilson et al., 2001). All these observation methods have their inherent advantages and lim- itations, especially when considering both temporal and spa- tial resolution issues. The Atmosphere–Land Exchange Inverse Model (ALEXI; Anderson et al., 1997, 2007a, b) and associated ﬂux dis- aggregation algorithm (DisALEXI; Anderson et al., 2004; Norman et al., 2003) are examples of a multiscale energy balance modeling approach that can utilize LST data from multiple satellite platforms with TIR sensing capabilities. The regional ALEXI model uses time-differential measure- ments of morning LST rise from geostationary satellites to estimate daily ﬂux patterns at 3–10 km resolution and conti- nental scales. Using higher-resolution LST information from polar-orbiting systems, DisALEXI enables downscaling of ALEXI ﬂuxes to ﬁner scales, better resolving land use and moisture patterns over the landscape and better approximat- ing the spatial scale of ground-based ﬂux observations. Land- sat data (30–120 m) can be used to retrieve ET at the ﬁeld scale, which is particularly useful for water management ap- plications. 1 Introduction sume the plant growth rate is static. This assumption can result in errors in simulating ET dynamics, especially over shorter time periods (seasonally, monthly, weekly or daily) (Méndez-Barroso et al., 2014; Tian et al., 2010). Often phys- ical process-based models involve hundreds of input vari- ables/parameters, many of which are not easily measured or known in a spatially distributed manner at watershed and re- gional scales. Although models can be calibrated using local or watershed-scale observations, there is the often-mentioned problem of equiﬁnality, where different sets of parameters during calibration give the same simulation results due to the inherent complexity of the system (Von Bertalanffy, 1968; Beven and Freer, 2001). Evapotranspiration (ET) is a major component of the water balance and connects hydrologic and biological processes (Hanson et al., 2004; Wilson et al., 2001). ET varies with climate, vegetation type and phenological stage and is di- rectly affected by land management strategies and climate change (Pereira et al., 2002). ET is also a key variable in most ecohydrological models and ecosystem service assessments (Abramopoulos et al., 1988; Kannan et al., 2007; Olioso et al., 1999; Tague and Band, 2004; Sun et al., 2011a, b). In spite of the importance of ET, routine estimation of ET at high spatial (plot level) and temporal (daily) resolution has not yet been achieved with acceptable accuracy over land- scape and regional scales (Wang and Dickinson, 2012). ) Mapping ET using satellite remote sensing data has been widely applied since the 1980s due to growing interest in the spatial dynamics of water use at the watershed and regional scales (Kalma et al., 2008). Of particular interest in the wa- ter resource community are surface energy balance methods based on remotely sensed land-surface temperature (LST) retrieved from thermal infrared (TIR) imagery, which pro- vides proxy information regarding the surface moisture sta- tus (Hain et al., 2011; Anderson et al., 2012a). LST captures signals of crop stress and variable soil evaporation that are often missed by crop coefﬁcient remote sensing techniques, which are based on empirical regressions with reﬂectance- based vegetation indices. Furthermore, diagnostic estimates of ET from the surface energy balance provide an indepen- dent estimate of landscape water use that is a valuable bench- mark for comparison with estimates based on water balance or hydrologic modeling (Hain et al., 2015; Yilmaz et al., 2014). Published by Copernicus Publications on behalf of the European Geosciences Union. 1018 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1019 Figure 1. Schematic diagram of the ALEXI and DisALEXI modeling schemes. The left panel shows TSEB as employed to partition the income radiometric temperature (TRAD(θ), θ is view angle) into canopy (subscript “c”) and soil (subscript “s”) components based on vegetation coverage (f (θ)). Sensible heat (H) is regulated by the aerodynamic resistance (Ra), bulk leaf boundary layer resistance (Rx) and soil surface boundary layer resistance (Rs). ALEXI combines the TSEB and ABL models to estimate air temperature (TA) at the blending height. The right panel represents the disaggregation of ALEXI output to ﬁner scales based on LST and f (θ) information from Landsat and MODIS. Figure 1. Schematic diagram of the ALEXI and DisALEXI modeling schemes. The left panel shows TSEB as employed to partition the income radiometric temperature (TRAD(θ), θ is view angle) into canopy (subscript “c”) and soil (subscript “s”) components based on vegetation coverage (f (θ)). Sensible heat (H) is regulated by the aerodynamic resistance (Ra), bulk leaf boundary layer resistance (Rx) and soil surface boundary layer resistance (Rs). ALEXI combines the TSEB and ABL models to estimate air temperature (TA) at the blending height. The right panel represents the disaggregation of ALEXI output to ﬁner scales based on LST and f (θ) information from Landsat and MODIS. val (approximately daily) but is too coarse (1 km in the TIR bands) for ﬁeld-scale ET estimation. Cammalleri et al. (2013) proposed a data fusion method to combine ET estimates de- rived from geostationary, MODIS and Landsat TIR data, at- tempting to exploit the spatiotemporal advantages of each class of satellite to map daily ET at a sub-ﬁeld scale. This ET fusion approach has been successfully applied over rain- fed and irrigated corn, soybean and cotton ﬁelds (Cammal- leri et al., 2013, 2014), as well as irrigated vineyards (Sem- mens et al., 2015). The work described here constitutes the ﬁrst application to forest land cover types, representing a sub- stantially different roughness and physiological regime than that of shorter crops. This presents a modeling challenge in terms of accurately deﬁning turbulent exchange coefﬁcients, as well as describing radiation transport through the canopy. vancement, based on data fusion, for ﬁlling gaps in Landsat- based ET retrievals due to partial cloud cover as well as the scan-line corrector (SLC) failure in Landsat 7. Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina This tech- nique facilitates more complete use of the existing Landsat archive for investigating water use dynamics at the landscape scale. www.hydrol-earth-syst-sci.net/21/1017/2017/ 1 Introduction However, due to the lengthy revisit interval (8 to 16 days), further lengthened by cloud contamination, the number of useful Landsat scenes that can be acquired during a growing season is limited. The Moderate Resolution Imag- ing Spectroradiometer (MODIS) has a shorter revisit inter- Another group of forest ET estimation methods is em- pirically based, establishing a relationship between ET with other parameters: for example, precipitation, reference ET and vegetation indices (leaf area index – LAI, normalized difference vegetation index – NDVI and enhanced vegeta- tion index – EVI) (Johnson and Trout, 2012; Mutiibwa and Irmak, 2013; Nemani and Running, 1988; Sun et al., 2011a; Zhang et al., 2004). Many studies have applied process-based ecohydrological models to estimate ET (Chen and Dudhia, 2001; Tague and Band, 2004; Tian et al., 2010). These mod- els usually estimate ET from potential ET, which is then downregulated based on weather data and soil and vegeta- tion characteristics. However, with the focus on predicting runoff and the soil water proﬁle, studies using hydrologic models generally do not evaluate the performance of ET sim- ulation. To simplify the physical processes, many models as- Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 2.2 Data processing and fusion system Roughness length (Zm) impacts the aerodynamic resis- tance (Ra), which is the resistance to heat transport across the layer between the nominal momentum exchange sur- face within the canopy and the height of the air tempera- ture boundary condition (ZT (m)). The aerodynamic resis- tance (Ra) can be expressed as Eq. (3) (Brutsaert, 1982): In this study, ET retrievals generated with DisALEXI using TIR data from MODIS (near daily, at 1 km resolution) and Landsat (periodic, sharpened to 30 m resolution) have been fused to produce daily Landsat-scale ET time series. The major components of the processing stream are described in greater detail below, including a data-mining sharpener (DMS; Gao et al., 2012b) tool that is used to improve the spatial resolution of the LST inputs to DisALEXI, the Spatial and Temporal Adaptive Reﬂectance Fusion Model (STARFM; Gao et al., 2006) and a gap-ﬁlling procedure that is applied to ALEXI and MODIS and Landsat-DisALEXI re- trievals prior to disaggregation and fusion. The gap-ﬁlling and fusion processes are schematically represented in Fig. 2. Ra = h ln ZT −d Zm −ψh ih ln Zu−d Zm −ψm i k2u , (3) (3) where k is the von Karman constant (0.4); u (m s−1) is the wind speed measured at height Zu (m); d (m) is the dis- placement height; Zm (m) is the roughness length, which can be estimated from the nominal canopy height (hc(m)), Zm ≈hc/8 (Shaw and Pereira, 1982); and ψh and ψm are the stability corrections for heat and momentum transport, re- spectively. Additional boundary layer resistances linking the bulk canopy and the soil surface to the in-canopy momentum exchange node (RX and RS, respectively; see Fig. 1) are de- ﬁned as in the series TSEB model formulation described in Norman et al. (1995). Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina This time-differential approach used in ALEXI reduces model sensitivity to errors in LST retrieval due to atmo- spheric and surface emissivity effects, but it does constrain ET estimates to the relatively coarse spatial scales typical of geostationary satellites. To estimate ET at the ﬁner scales re- quired for many management applications, the ALEXI ﬂuxes can be spatially disaggregated using the DisALEXI approach (Anderson et al., 2004; Norman et al., 2003). DisALEXI uses, as an initial estimate, air temperature estimates diag- nosed by ALEXI at a nominal blending height at the interface between the TSEB and ABL submodels, along with high spa- tial resolution images of surface temperature data and veg- etation cover fraction from polar-orbiting or airborne sys- tems, to run the TSEB at sub-pixel scales over each ALEXI pixel area. The TSEB ﬂuxes are reaggregated and com- pared with the ALEXI pixel ﬂux, and the air temperature boundary condition is iteratively modiﬁed until the ﬂuxes are consistent at the ALEXI pixel scale. More details on the ALEXI/DisALEXI multiscale modeling system can be found in Anderson et al. (2004, 2011, 2012b). using the Beer–Lambert law. The surface energy balance for the canopy, soil and the combined system is represented in Eq. (2): using the Beer–Lambert law. The surface energy balance for the canopy, soil and the combined system is represented in Eq. (2): Rn,s = Hs + λEs + G (2a) Rn„c = Hc + λEc (2b) Rn = H + λE + G, (2c) (2a) (2b) (2c) (2c) where the subscripts “c” and “s” represent ﬂuxes from the canopy and soil components, and Rn is net radiation, λE is latent heat ﬂux, H is sensible heat and G is the soil heat ﬂux (all in units of W m−2). Component surface temperatures in Eq. (1) are used to constrain Rn, H and G; canopy transpi- ration (λEc) is initially estimated with a modiﬁed Priestley– Taylor approach under the unstressed conditions assumption, and then iteratively downregulated if Tc indicates canopy stress, ruled by the assumption that condensation under day- time clear-sky conditions is unlikely, while soil evapora- tion (λEs) is computed as a residual to the soil energy budget. Further information regarding the TSEB model formulation is provided by Kustas and Anderson (2009). 2.1 Thermal-based multiscale ET retrieval The regional ALEXI and the associated DisALEXI models are based on the the Two-Source Energy Balance (TSEB) land-surface representation of Norman et al. (1995), with further reﬁnements by Kustas and Norman (1999, 2000). The combined modeling system is described schematically in Fig. 1. Rather than treating the land surface as a homo- geneous surface, the TSEB partitions modeled surface ﬂuxes and observed directional radiometric surface temperature be- tween soil and vegetation components: In this paper, ALEXI and DisALEXI are applied over a commercially managed loblolly pine (Pinus taeda) planta- tion, representing a range in stand age, to estimate daily ﬁeld- scale ET using the data fusion methodology. Retrieved 30 m ET time series are evaluated at two ﬂux tower sites, sited in mature and recently clear-cut pine stands. The primary sci- ence objectives are to (1) study the accuracy of ALEXI and DisALEXI ET retrievals over forested sites, (2) evaluate the models’ ability to capture the dynamics of ﬂuxes over the contrasting canopy structures in both pine and the adjacent vegetation and (3) investigate the utility of daily ﬁeld-scale ET retrievals for water resource management in forested sys- tems. Additionally, we present a novel methodological ad- TRAD(Ø)4 = f (Ø)T 4 c + [1 −f (Ø)]T 4 s , (1) (1) where Ø is the thermal view angle, f (Ø) is the fractional vegetation cover apparent at the thermal view angle, TRAD is the directional radiometric temperature, Tc is the canopy tem- perature and Ts is the soil temperature (K). In remote sensing applications, f (Ø) can be estimated from retrievals of LAI www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1020 2.2.1 Data-mining sharpener (DMS) In both the Landsat and MODIS imaging systems, the TIR sensors have signiﬁcantly lower spatial resolution than the shortwave instruments on the same platform. For Landsat, TIR resolution varies from 60 m (Landsat 7) to 100 m (Land- sat 8) to 120 m (Landsat 5), while the shortwave images are processed to 30 m. For MODIS, TIR resolution is 1 km while the shortwave resolution is 250 m. Particularly for Landsat, there is a beneﬁt to mapping ET at 30 m rather than the na- tive TIR resolution, as boundaries in land cover and moisture variability are much better deﬁned. The regional-scale ALEXI model applies the TSEB in time-differential mode using measurements of morning LST rise obtained from geostationary platforms (Anderson et al., 1997, 2007a). Energy closure over this morning period is ob- tained by coupling the TSEB with a simple model of atmo- spheric boundary layer (ABL) development (Fig. 1). In this study, instantaneous morning ﬂuxes from ALEXI have been upscaled to daily total latent heat ﬂux by conserving the ra- tio of λE to solar radiation, following the recommendations of Cammalleri et al. (2014). Daily latent heat ﬂux (in energy units of MJ m−2 day−1) is converted to ET (in mass units of mm day−1) by dividing by the latent heat of vaporization (λ = 2.45 MJ kg−1). The DMS sharpening tool implemented within the ET fu- sion package enables this higher-resolution mapping. The DMS technique creates regression trees between TIR band brightness temperatures and shortwave spectral reﬂectances both globally across the full scene and within a localized moving window (Gao et al., 2012b). The original TIR data Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1021 Figure 2. Flowchart describing the Landsat gap-ﬁlling and data fu- sion method. The arrows represent the methods applied. The boxes represent the datasets with different spatial and temporal charac- teristics created during the process. The dashed boxes indicate ET products with partially ﬁlled scenes (due to clouds or SLC gaps); solid boxes identify gap-ﬁlled scenes and the thick box highlights the ﬁnal gap-ﬁlled 30 m daily product. timated at time t0. ALEXI and MODIS-DisALEXI Gaps in the daily ET maps from ALEXI and MODIS- DisALEXI were ﬁlled using the method described by Ander- son et al. (2012b). Daily reference ET is ﬁrst calculated us- ing the Food and Agriculture Organization (FAO) Penman– Monteith formulation for a grass reference site (Allen et al., 1998). The ratio of actual-to-reference ET (fRET) is com- puted and then smoothed and gap ﬁlled at each pixel using a Savitzky–Golay ﬁlter. Gap-ﬁlled daily ET is recovered by multiplying this fRET series by daily reference ET. 2.2.2 Spatial and Temporal Adaptive Reﬂectance Fusion Model (STARFM) The STARFM algorithm fuses spatial information from Landsat imagery with temporal information from the coarser but more frequently collected MODIS imagery to produce daily estimates at Landsat-like scale. STARFM was origi- nally designed for application to surface reﬂectance images, but has demonstrated utility in fusing higher-order satellite products as well, as long as there is sufﬁcient consistency between the Landsat and MODIS retrievals. 2.2.1 Data-mining sharpener (DMS) (xi, yj) is the pixel location, M(xi, yj, t0) is the MODIS pixel value at time t0, M(xi, yj, tk) is the MODIS pixel value at time tk and L(xi, yj, tk) is the Land- sat pixel value at time tk. Wijk is the weighting factor that determines how much each pixel in the moving window con- tributes to the estimation of the center pixel value. In the ET fusion system, STARFM uses the weighting function derived from Landsat ET and MODIS ET retrieved on the same date and MODIS ET on the prediction date to get Landsat-like ET estimations on all prediction dates between Landsat over- passes. 2.2.3 ET gap-ﬁlling methods Spatiotemporal gaps in TIR-based ET retrievals occur for a variety of reasons, including cloud cover, frequency of sen- sor overpass, limitations imposed to avoid distortions in LST data acquired at large off-nadir view angles and other sensor issues. Prior to disaggregation and fusion, the input ET ﬁelds must be gap ﬁlled, both spatially and temporally, to the extent possible to ensure relatively gap-free output time series. Figure 2. Flowchart describing the Landsat gap-ﬁlling and data fu- sion method. The arrows represent the methods applied. The boxes represent the datasets with different spatial and temporal charac- teristics created during the process. The dashed boxes indicate ET products with partially ﬁlled scenes (due to clouds or SLC gaps); solid boxes identify gap-ﬁlled scenes and the thick box highlights the ﬁnal gap-ﬁlled 30 m daily product. Due to the high temporal frequency of data acquisition from both geostationary and MODIS systems, the ALEXI and DisALEXI–MODIS retrievals can be reasonably gap ﬁlled and interpolated to daily time steps in all but the cloudi- est of circumstances. Time intervals between clear-sky Land- sat acquisitions are too lengthy in general, motivating the need for data fusion to ﬁll temporal gaps. Spatial gaps in Landsat ET retrievals have been ﬁlled using a method based on STARFM, as described below. are sharpened from their native spatial resolution to ﬁner res- olution with DMS, with the choice of using all of the avail- able shortwave bands or a subset of these bands or even a single vegetation index as input to the regression tree. The sharpened results from the global and local models are com- bined based on a weighting factor calculated from the resid- uals of the two sharpened results. Landsat-DisALEXI First, ET data from both MODIS and Landsat retrievals are extracted onto a common 30 m grid. A moving searching window method is then used in STARFM to estimate values at the center pixel of the moving window: To ensure optimal spatial coverage in the fused 30 m daily time series, the Landsat-based ET retrievals on Landsat over- pass dates must also be gap ﬁlled to the extent possible. Gaps in Landsat ET result from cloud cover, or in the case of Land- sat 7, missing pixels due to the SLC failure that has been oc- curring since May 2003, resulting in striped gaps in all but the center of each scene. In the case of Landsat, the time interval between usable overpasses may be too long to jus- tiﬁably use the fRET approach applied to the ALEXI and MODIS time series. Therefore, an alternate method has been L xp/2yp/2t0 = p X i=1 p X j=1 n X k=1 Wijk × M xi,yj,t0 +L xi,yj,tk −M xi,yj,tk , (4) (4) where p is the size of the moving window and (xp/2, yp/2) is the center pixel of the moving window that needs to be es- www.hydrol-earth-syst-sci.net/21/1017/2017/ www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina eloped to ﬁll cloud gaps/stripes to create ﬁlled scenes for estion into STARFM. ﬂectance and brightness temperature as inputs to produce cloud, cloud shadow, water and snow mask for Landsat im- developed to ﬁll cloud gaps/stripes to create ﬁlled scenes for ingestion into STARFM. ﬂectance and brightness temperature as inputs to produce cloud, cloud shadow, water and snow mask for Landsat im- ages (Zhu and Woodcock, 2012). Cloud physical properties are ﬁrst used to identify potential cloud pixels and clear-sky pixels and then normalized temperature, spectral variability and brightness probability functions are combined to esti- mate cloudy area. The cloud shadow area is derived from the darkening effect of the cloud shadows in the near-infrared band, view angle of the satellite sensor and the solar illu- mination angle. In this study, we ﬂagged pixels with Fmask class 2 (cloud_shadow) and 4 (cloud) in the cloud mask ﬁle as cloud impacted. ﬂectance and brightness temperature as inputs to produce cloud, cloud shadow, water and snow mask for Landsat im- ages (Zhu and Woodcock, 2012). Cloud physical properties are ﬁrst used to identify potential cloud pixels and clear-sky pixels and then normalized temperature, spectral variability and brightness probability functions are combined to esti- mate cloudy area. The cloud shadow area is derived from the darkening effect of the cloud shadows in the near-infrared band, view angle of the satellite sensor and the solar illu- mination angle. In this study, we ﬂagged pixels with Fmask class 2 (cloud_shadow) and 4 (cloud) in the cloud mask ﬁle as cloud impacted. The method involves running STARFM for the partly cloudy or striped prediction date using Landsat-retrieved ET from surrounding clear dates. The cloud/stripe-impacted ar- eas in the Landsat retrieval are then ﬁlled as a weighted function of the STARFM estimated Landsat-like ET and the Landsat-retrieved ET. This weighting is implemented to re- duce impacts of bias that may exist between the STARFM estimate and the actual retrieval in the area of the gap, which could otherwise result in a notably patchy ﬁll. The weighting function is computed within a moving window, predicting ET at the center pixel. 3.1 Study area The study area (Fig. 3) is located over the Parker Tract in the lower coastal plain of North Carolina. The Parker Tract consists of loblolly pine plantations of different ages and na- tive hardwood forests (Noormets et al., 2010). The planta- tions are commercially managed for timber production by the Weyerhaeuser Company. The study area is ﬂat, about 3 m a.s.l. and has been ditched (fourth-order ditches at 100 m spacing) to manage the water table and improve tree pro- ductivity (Domec et al., 2012). The soil is Belhaven series Histosol, with a 50–85 cm organic layer over coarse glacial outwash sand (Sun et al., 2010). The study area is classiﬁed as outer coastal plain mixed forest province (Bailey, 1995). The long-term (1945–2008) monthly temperature ranges be- tween 26.6 ◦C in July and 6.4 ◦C in January, with an annual mean temperature of 15.5 ◦C. The long-term annual precipi- tation is around 1320 ± 211 mm, relatively evenly distributed throughout the year. Filled Value = AL −AS + p X i p X j Wi,j × Si,j , (5) (5) where AL is the average of pixels in the moving window in Landsat-retrieved ET, AS is the average of pixels in the mov- ing window in STARFM fused ET on the same day as the Landsat-retrieved ET, i and j is the pixel location in the mov- ing window, p is the moving window size, W is the weight- ing score and S is the STARFM value. The searching distance is predeﬁned based on the hetero- geneity of the study area. A larger searching distance nor- mally requires a longer computing time and can result in more random noise. A searching distance that is too small might not be able to provide a sufﬁcient number of similar surrounding pixels to predict the value of the center pixel. As described above, pixels that are far away from the center pixel have lower weighting than pixels that are close to the center pixel. When the gap area is large and contiguous (more than 80 % of the moving window), there are not enough good pixels that can provide useful information for the gap ﬁll. In this case, the gaps are left unﬁlled. Evaluation of the DisALEXI ET estimates was performed at two AmeriFlux tower sites in this area: US-NC2 (35– 48◦N, 76–40◦W) and US-NC3 (35–48◦N, 76–39◦W). Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina The weighting value of each pixel in the moving window is calculated based on land cover type, spatial distance to the center predicting pixel and pixel value and is then normalized to a 0–1 value. Pixels that have the same land cover type as the prediction pixel, are nearby and have a similar value are assigned a higher weighting score. The resulting ﬁlled value is computed as www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1022 3.2 Micrometeorological and land management data At both NC2 and NC3, energy ﬂuxes were measured us- ing an open-path eddy covariance system, which includes a CSAT3 three-dimensional sonic anemometer (Campbell Sci- entiﬁc instrument – CSI, Logan, UT, USA1), a CR5000 data logger (CSI), an infrared gas analyzer (IRGA, Model LI- 7500, LI-COR, Lincoln, NE, USA) and a relative humidity and air temperature sensor (model HMP-45C; Vaisala Oyj, Helsinki, Finland) (Sun et al., 2010). Soil heat ﬂux was mea- sured at NC2 with three heat ﬂux plates (model HFT3, CSI, Logan, UT, USA) at the depth of 2 cm. The soil heat ﬂux plates were placed in three contrasting microsites – one in a row of trees, in relative shade, another between rows in a mostly open environment and one about halfway in be- tween. Measurements of G at the NC3 site are not available for 2013 due to an instrument failure. Net radiation was mea- sured with four-component net radiometers (Kipp & Zonen CNR-1, Delft, the Netherlands) at each of the two towers. Precipitation was measured by two tipping bucket types of rain gages (TE-525, CSI; Onset data-logging rain gage, On- set Computer Corporation, USA). LST data from the GOES imager instruments were used to run ALEXI over the continental US for 2013 at 4 km reso- lution (Anderson et al., 2007a). In addition, MODIS (4-day) LAI products (MCD15A3) were aggregated from 1 to 4 km and interpolated to daily scale using the smoothing algorithm developed by Gao et al. (2008). LAI is used to estimate f (Ø) for Eq. (1), to compute radiation transmission to the soil surface and to assign land cover class-dependent vegetation heights for roughness parameterization (see Anderson et al., 2007a). MODIS products used in the MODIS disaggregation include instantaneous swath LST (MOD11_L2; Wan et al., 2004), geolocation data (MOD03), NDVI (MOD13A2; Huete et al., 2002), LAI (MCD15A3; Myneni et al., 2002), albedo (MCD43GF; Schaaf et al., 2010; Sun et al., 2017) and land cover (MCD12Q1; Friedl et al., 2002). The LST swath data, at 1 km spatial resolution, were converted to geographic coordinates using an interactive data language (IDL)-based MODIS reprojection tool. The NDVI product (1 km spatial resolution) is produced at 16-day intervals, LAI (1 km) at 4 days and albedo (1 km) at 8 days. All data were quality checked using a data quality ﬁlter. The MODIS NDVI, LAI and albedo data were bilinearly interpolated to estimate daily values. 3.3 ALEXI/DisALEXI model inputs The ET estimation process involves fusion of data from three major geostationary and polar-orbiting satellite sys- tems: GOES, MODIS and Landsat. In addition, each ET re- trieval pulled meteorological inputs (air temperature, wind speed, vapor pressure, atmospheric pressure and insolation) from a common gridded dataset, generated at hourly time steps and relatively coarse spatial resolution (32 km) as part of the North American Regional Reanalysis (NARR). Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina story leaf area. The mostly herbaceous understory contained 85 ± 52 g C m−2 at NC3. story leaf area. The mostly herbaceous understory contained 85 ± 52 g C m−2 at NC3. story leaf area. The mostly herbaceous understory contained 85 ± 52 g C m−2 at NC3. were used statistically to assess the relationships between water use and stand age. A 60 m buffer inside the edge of each ﬁeld was applied to exclude the pixels mixed with roads or other ﬁelds. All the other pixels were used to assess the re- lationships between water use and stand age. Both NC ﬂux towers are equipped with similar instrumen- tation, and biophysical data are collected routinely. These measurements are described below. This study focuses on data collected during the 2013 growing season, starting after the launch of Landsat 8 on 11 February 2013 and continuing until 8 November 2013. 3.1 Study area US- NC2 is a mid-rotation plantation stand with 90 ha area, which was established after clear-cutting a previous rotation of loblolly pine, replanted with 2-year-old seedlings at 1.5 m by 4.5 m spacing in 1992. The stand has been fertilized twice – at establishment and in 2010, following a thinning in 2009. The tree density during the study period in 2013 was 171 trees per hectare with a standing biomass of 42.6 t C ha−1 in the overstory and 6.5 t C ha−1 in the understory. The un- derstory was composed of red maple, greenbrier and vol- unteer loblolly pine. US-NC3 was established in 2013 in a stand that was clear-cut in 2012, located approximately 1.5 km from US-NC2. US-NC3 was replanted with seedling loblolly pines after the clear cut. Trees in the US-NC2 site were 22 years old in 2013, were 19.0 m tall and had a mean LAI of 3.77 m2 m−2, whereas NC3 was freshly planted with 2-year-old seedlings, which were 0.2 m tall and had no over- Previous Landsat gap-ﬁlling techniques have focused on ﬁlling spectral reﬂectance ﬁelds. Chen et al. (2011) applied a similar weighting function in a moving window to ﬁll the Landsat 7 SLC-off images using an appropriate thematic mapper (TM) image or SLC-on enhanced thematic mapper plus (ETM+) image. Roy et al. (2008) used both MODIS BRDF/Albedo products and Landsat observations to predict Landsat reﬂectance with a semi-physical fusion approach. In contrast, the methods described here are a novel application of data fusion to ﬁlling SLC-off gaps in ET retrievals. The cloud mask used in this study is the Fmask (func- tion of mask) data from the Level 2 surface reﬂectance prod- uct distributed by EROS (Earth Resources Observation and Science) center. Fmask uses Landsat top-of-atmosphere re- Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ 1023 Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 Table 1. Landsat overpass dates used in the study. Table 1. Landsat overpass dates used in the study. Sensor Landsat 7 Landsat 8 Landsat 8 Landsat 8 Landsat 8 Landsat 8 Landsat 8 Landsat 8 DOY 96 104 136 152 200 248 312 328 % Cloudiness 0.1 0 16.4 5.1 23.8 0.3 0.1 0.1 % SLC gap 37 n/a n/a n/a n/a n/a n/a n/a n/a means not applicable. n/a means not applicable. STARFM ET. The white rectangular box located in the north- east area of both ﬁgures contains no data because more than 40 % of Landsat pixels within the 4 km ALEXI ET pixel were affected by a large water body. cluding seasonal maximum and minimum vegetation height (used in the surface roughness formulations), leaf size and leaf absorptivity in the visible, near-infrared (NIR) and TIR bands following Cammalleri et al. (2013). For DisALEXI us- ing Landsat, the 30 m national land cover dataset (NLCD) for 2006 (Fry et al., 2011; Wickham et al., 2013) was used. For the MODIS disaggregation, the NLCD was resampled to 1 km resolution using the dominant class in each pixel. In each of these cases, the spatial patterns within miss- ing regions in the direct retrievals appear reasonably recon- structed with the ET gap-ﬁlling method, with no obviously patchy artifacts in the gap-ﬁlled ET images. Even linear structures were restored: for example, roads and ﬁeld bound- aries. The gap-ﬁlling method works even better when the small or linear objects are very different from surrounding pixels. The choice of moving window size can affect the gap- ﬁlling results and may require scene-based adjustment, with the need to balance the risk of inappropriate candidate pixel selection if the window is too large, or a lack of candidate pixels if the window is too small. 3.2 Micrometeorological and land management data MODIS LST was sharpened using NDVI to reduce the off-nadir pixel smearing effect. Flux observations at 30 min time steps were quality checked, as judged by atmospheric stability and ﬂux station- arity (Noormets et al., 2008). The 30 min data were then gap ﬁlled using the monthly regression between observed and po- tential ET models created from good quality observed data. The energy imbalance problem was checked and the average closure ratio of the 30 min dataset at the NC2 site was 0.88 during daytime when net radiation was larger than 0. Since there were no soil heat ﬂux observations at the NC3 site, there was no closure information and the observed latent heat was used to compare with the simulated data. The 30 min en- ergy ﬂuxes during the daytime were summed up to get daily energy ﬂuxes for validation. Landsat 8 thermal infrared and shortwave surface re- ﬂectance data from 2013 used to run DisALEXI were ob- tained from USGS. Eight relatively cloud-free (> 75 %) Landsat scenes (path 14 and row 35) and clear conditions over the tower site were available during the 2013 growing season, including one Landsat 7 scene and seven from Land- sat 8 (Table 1). Landsat-scale LAI was retrieved from Land- sat shortwave surface reﬂectance data using MODIS LAI products as reference (Gao et al., 2012a). LST was sharp- ened to 30 m using the blue, green, red, near infrared, SWIR1 and SWIR2 bands (refer to Sect. 2.2.1 for more details about Landsat LST sharpening). Stand age maps and tree planting history for the study area were obtained from the Weyerhaeuser Company. The stand age ranges from 1 to 89 years, with most stands un- der 30 years of age. Since this information is proprietary, the stand age maps cannot be displayed; however, these data 1The use of trade, ﬁrm, or corporation names in this article is for the information and convenience of the reader. Such use does not constitute an ofﬁcial endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suit- able. Land cover type was used in both the Landsat and MODIS disaggregation to set pixel-based vegetation parameters in- www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 1024 Y. 3.2 Micrometeorological and land management data Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1024 Figure 3. A Landsat 8 true color image (5 September 2013) showing the North Carolina study area. The yellow crosses indicate the location of the NC2 and NC3 ﬂux towers. Figure 3. A Landsat 8 true color image (5 September 2013) showing the North Carolina study area. The yellow crosses indicate the location of the NC2 and NC3 ﬂux towers. Table 1. Landsat overpass dates used in the study. 4.1 Performance of the Landsat gap-ﬁlling algorithm The left image is Landsat-retrieved ET with clouds masked using the Fmask data layer and the right image has been processed through the Landsat gap-ﬁlling method. with or without closure enforcement. As mentioned earlier, closure could not be assessed at NC3 due to failure among the soil heat ﬂux instrumentation. At NC2, closure by resid- ual resulted in an increase in observed ET by approximately 12 % on average. The gap-ﬁlling method described in Sect. 2.2.3 relies on the inputs from both the original Landsat ET and the STARFM predictions, which in turn rely on a ﬁlled MODIS image on the target date as well as a MODIS-Landsat image pair on a surrounding date. If the Landsat image in the input pair also has gaps, additional pairs can be used to iteratively ﬁll the target image. g Time series of ALEXI ET (4 km), Landsat ET retrieved on Landsat overpass dates and Landsat–MODIS fused ET (both at 30 m resolution) are compared in Fig. 8 with ET observed at both the NC2 and NC3 sites from DOY 50 to 330. In addi- tion, daily ET values generated using a simple Landsat-only interpolation scheme are shown for comparison. These were generated using the MODIS and ALEXI gap-ﬁlling tech- nique described in Sect. 2.2, conserving the ratio of actual-to- reference ET between Landsat overpass dates. Metrics of sta- tistical performance at daily to seasonal timescales are listed in Table 3. 4.1 Performance of the Landsat gap-ﬁlling algorithm Examples of results from the Landsat gap-ﬁlling method are shown in Figs. 4 and 5. For DOY 96 (Fig. 4), L7 SLC stripes and also a few cloudy areas were ﬁlled by combining the direct Landsat retrieval (left panel) with the STARFM ET prediction for DOY 96 generated using a Landsat–MODIS image pair from DOY 104. The cloudy areas in DOY 200 (Fig. 5) were ﬁlled using a Landsat–MODIS pair from DOY 152. In each case, the size of the overlapped mov- ing window was 420 m by 420 m. This means that contigu- ous gaps larger than the window were not ﬁlled since there were not enough candidate pixels to create the statistical re- lationship needed between the direct Landsat retrieval and Figure 6 shows a synthetic study used to assess the ac- curacy of the gap-ﬁlling procedure. Here, a direct Land- sat ET retrieval for DOY 152 was artiﬁcially masked using SLC stripes from DOY 96. The ﬁnal panel shows the gap- ﬁlled image. Comparing the original values with the gap- ﬁlled values yields an R2 of 0.89 and a mean absolute er- ror (MAE) of −0.01 mm day−1, with the average of original values as 5.81 mm day−1 and the average of gap-ﬁlled values as 5.80 mm day−1. Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1025 Figure 4. Example of gap-ﬁlling SLC-off stripes in a Landsat 7 ET image for DOY 96, 2013. The left image is Landsat-retrieved ET with stripes, while the right image has been gap ﬁlled using the method described in Sect. 2.2.3. Figure 4. Example of gap-ﬁlling SLC-off stripes in a Landsat 7 ET image for DOY 96, 2013. The left image is Landsat-retrieved ET with stripes, while the right image has been gap ﬁlled using the method described in Sect. 2.2.3. Figure 5. Example of gap-ﬁlling cloudy regions in a Landsat 8 ET image for DOY 200. The left image is Landsat-retrieved ET with clouds masked using the Fmask data layer and the right image has been processed through the Landsat gap-ﬁlling method. Figure 5. Example of gap-ﬁlling cloudy regions in a Landsat 8 ET image for DOY 200. 4.2 Evaluation of daily ET retrievals from DisALEXI at the ﬂux tower sites Modeled and measured instantaneous and daytime integrated surface energy ﬂuxes on Landsat overpass dates are com- pared in Fig. 7, demonstrating good correspondence. Statis- tical performance metrics for each ﬂux component for both sites are shown in Table 2, including MAE, root mean square error (RMSE) and mean bias error (MBE). The model perfor- mance for each ﬂux is similar between sites, with somewhat lower errors obtained for the clear-cut site (NC3). The la- tent heat observed at the NC2 site is higher than that at NC3 Figure 8 highlights the value of disaggregation to the tower footprint scale for the purposes of model validation. For NC2, ALEXI 4 km ﬂuxes agree well with tower observations, suggesting that the tower footprint at NC2 is reasonably rep- www.hydrol-earth-syst-sci.net/21/1017/2017/ 2Note: an updated NLCD map for 2011 was published after the study was implemented, but there was no notable change in land cover types over the study area in comparison with NLCD2006. www.hydrol-earth-syst-sci.net/21/1017/2017/ Comparison of time series of ALEXI ET (4 km), observed ET, Landsat ET retrieved on Landsat overpass dates, Landsat-only interpolated ET and Landsat–MODIS fused ET for the NC2 site (and NC3, bottom panel) sites in 2013. The seasonal cumulative ET at NC2 and NC3, calcu- lated for DOY 90–330 from both the observed ET and the Landsat–MODIS fused time series, is shown in Fig. 9. For NC3, the accumulation does not include values of modeled or measured ET during the period DOY 232–248 when ﬂux tower data were not available, so these values do not repre- sent the total seasonal water use at this site. The modeled cumulative water use on DOY 330 agrees with the observed values to within −0.9 % at NC2 and 0.4 % at NC3. Overall, the modeled and measured cumulative ET curves agree well throughout the growing season, indicating the remote sensing method has utility for water use management and assessment at subseasonal timescales. of land where the NC3 tower is located. This underscores the need for appropriate spatial resolution when comparing modeled with observed ﬂuxes, especially for the more het- erogeneous land surfaces (e.g., Anderson et al., 2004). Overall, the performance of the two Landsat retrievals (STARFM and Landsat only) are comparable between sites, with RMSE at daily time steps of ∼0.8 to 1.0 mm day−1 and MAE of 0.6 to 0.8 mm day−1 (19–30 % of the mean observed ET). At monthly time steps, performance improves to 11– 14 %, due to averaging of random errors – including errors in daily insolation forcings from the NARR meteorological dataset. Fluxes are somewhat underestimated at the end of the growing season at each site due largely to the Landsat re- trieval on DOY 312. This highlights the importance of good temporal sampling at the Landsat scale – an additional Land- sat scene around DOY 270 during the prolonged gap in cov- erage may have improved the seasonal water use estimates. A small negative mean model bias is observed for both sites, due primarily to underestimation at the end of growing sea- son of the Landsat retrieval on DOY 312. This is also the reason that Landsat-only interpolated ET performs slightly better than STARFM. More details about the comparison of the two Landsat retrievals can be found in the Discussion section. www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1026 Figure 6. Comparison between the original Landsat ET retrieval for DOY 152 (left panel), an artiﬁcially gapped version, imposing SLC gaps from DOY 96 (middle panel), and the gap-ﬁlled map (right panel). Figure 6. Comparison between the original Landsat ET retrieval for DOY 152 (left panel), an artiﬁcially gapped version, imposing SLC gaps from DOY 96 (middle panel), and the gap-ﬁlled map (right panel). Figure 7. Scatterplot of modeled and measured instantaneous (top row panels) and daily surface ﬂuxes (bottom row panels) on Landsat overpass dates for NC2 (left column panels) and NC3 (right column panels) ﬂux tower sites. Figure 7. Scatterplot of modeled and measured instantaneous (top row panels) and daily surface ﬂuxes (bottom row panels) on Landsat overpass dates for NC2 (left column panels) and NC3 (right column panels) ﬂux tower sites. resentative of the surrounding 4 km ALEXI pixel area. The disaggregated 30 m ﬂuxes are also similar to both ALEXI and observations at this site. At NC3, however, the 4 km ALEXI ﬂuxes are notably higher than the observed ET, while the dis- aggregated ﬂuxes are comparable. The NC3 tower site is not representative at the ALEXI pixel scale, and disaggregation to the tower footprint scale is required to account for local sub-pixel heterogeneity. Even at 1 km resolution, the MODIS retrieval accuracy was degraded at NC3 in comparison with the Landsat-scale retrievals (Table 3). Recall that NC3 was recently clear-cut with surrounding areas still comprised by more mature forest stands. With the 4 km spatial resolution, ALEXI ET is able to capture the ET status of the major land cover type (i.e., mature forest), but not the particular patch www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1027 Figure 8. Comparison of time series of ALEXI ET (4 km), observed ET, Landsat ET retrieved on Landsat overpass dates, Landsat-only interpolated ET and Landsat–MODIS fused ET for the NC2 site (and NC3, bottom panel) sites in 2013. 1027 Figure 8. 4.3.1 ET variations with land cover Water use in the woody wetland areas was the highest among all the different land cover types but was similar to natural forest and mature man- aged forest during the peak growing season. The seasonal cumulative ET from these ﬁve land cover types is shown in Fig. 13. The woody wetlands tended to have higher seasonal ET than the other three classes, slightly exceeding that of nat- ural forest and mature pine plantations. Modeled water use in crop lands exceeded that in young plantation stands, result- ing from relatively higher LAI and lower LST observed over the cropped areas. Seasonal ET time series were developed for ﬁve general- ized land cover classes (crop land, natural forest, woody wet- land, mature plantation and young plantation) to assess vari- ability in water use with land use/land cover type in the study area. The term “natural forest” is used to describe unmanaged mixed forested areas within the study domain. “Mature plan- tation” refers to managed stands of loblolly pine within the Parker Tract with ages ranging from 10 to 20 years, while “young plantation” indicates stand ages less than 3 years. Figure 12 shows the time series of modeled ﬁeld-scale ET averaged from 10 randomly sampled pixels associated with each generalized land cover class for 2013. For the woody wetland class, care was taken to select pixels that were cor- rectly classiﬁed by visual inspection of Google Earth im- agery. A 7-day moving average was applied to the modeled daily ET to reduce noise and facilitate visual comparison. Figure 14 shows the average cumulative ET between DOY 50 and 330 associated with the ﬁve different land cover types, computed from the 10 random samples per class, and the black bars represent the standard deviation among the samples. Natural forests showed the lowest variability in ET (30 mm), while the woody wetland and mature plantation pixels had the highest standard deviations (74 and 73 mm, respectively). The high variability in the latter classes may reﬂect both management effects and misclassiﬁcation. Crop lands and young forest plantations showed moderate variabil- ity in water use, with standard deviations of 64 and 50 mm, respectively. In terms of coefﬁcient of variation in water use across the modeling domain, the crops and young plantation classes are relatively high at 7.1 %, compared to natural for- est at 2.8 %. 4.3.1 ET variations with land cover Figure 10 shows the land cover types over the study area as described in the NLCD from 20062. The major classes rep- resented in the NLCD over the study area include crop land Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 28 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1028 Figure 10. Land cover types over the study area from NLCD 2006. Area in the black outline is the plantation area. Figure 9. Comparison between the modeled and observed seasonal cumulative ET at NC2 and NC3 during 2013. Figure 9. Comparison between the modeled and observed seasonal cumulative ET at NC2 and NC3 during 2013. Figure 9. Comparison between the modeled and observed seasonal cumulative ET at NC2 and NC3 during 2013. Figure 9. Comparison between the modeled and observed seasonal cumulative ET at NC2 and NC3 during 2013. (including corn, cotton and soybeans), forest and woody wet- land. The land cover in many plots within the Parker Tract plantation, including the NC2 site, was classiﬁed as woody wetland rather than evergreen forest. This misclassiﬁcation, however, had little impact on the model ET estimates at NC2 due in part to the normalization constraint imposed by the 4 km ALEXI ET output. Figure 10. Land cover types over the study area from NLCD 2006. Area in the black outline is the plantation area. Time series maps of monthly and cumulative ET in Fig. 11 over the study area exhibit spatiotemporal water use patterns that are related to land cover type (Fig. 10). The relatively high rates of ET during midseason in the riparian and more densely forested regions are readily apparent. Water use pat- terns in the cultivated agricultural areas reﬂect the diversity of crops and water management strategies. Within the Parker Tract plantation, a few ﬁelds with persistently low ET may be fresh clear cuts, possibly with a layer of slash to inhibit emergence of new vegetation. In the summer of 2014, after the slash has been collected and piled, these plots may appear more like the recent clear cut near NC3. than did the young plantation pixels. 4.3.1 ET variations with land cover Crops and young plantations also have higher coefﬁcients of variation in LAI than other land cover types, Pixels classiﬁed as woody wetland, natural forest, agri- culture and mature plantation generally showed higher ET Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1029 leading to larger variability in water use demand through transpiration. Because the two-source land-surface representation in DisALEXI also provides estimates of the evaporation (E) and transpiration (T ) components of ET, the model output can also be used to assess variability in E / T partitioning be- tween land cover types and through the season (Fig. 15). In general, soil evaporation losses account for a higher percent- age of total ET early in the season, after the spring rains but before the canopies have completely leafed out. On average through the season, the E / T ratio is highest for crop lands, young plantations and woody wetlands. This is reasonable given the lower leaf area characteristic of these classes and the abundant substrate moisture in the case of woody wet- lands. Partitioning to T is maximized during the peak grow- ing season (DOY 152, 200 and 248) for all the land cover types. Natural forests and mature plantations tend to have higher rates of transpiration than other land cover types. Table 2. Summary of the statistical indices quantifying model performance for instantaneous and daytime integrated surface energy ﬂuxes on Landsat overpass dates. 4.3.2 ET variations with LAI Many forest hydrology models (Lu et al., 2003; Scott et al., 2006; Sun et al., 2011a) assume seasonal ET is well correlated with LAI. This assumption was tested over the set of points chosen randomly from different land cover types. Sample points from mature plantations, young planta- tions and crop lands were all located in drained areas, while sample points from natural forest and woody wetland were in undrained areas. However, LAI values from the sample points were not affected by drainage conditions. High LAI values were obtained from both drained mature plantations and undrained natural forest, while relatively low LAI val- ues were from both drained young plantations and undrained woody wetlands. Figure 16 shows that some (but not all) variability in ET, as predicted by the fusion estimates, is ex- plained by variability in average LAI over the prediction time period (R2 = 0.59). These results indicate that, in 2013, in- creasing LAI added up to 350 mm of seasonal water use on top of nearly 800 mm from the soil evaporation contribution. The high rates of ET at low LAI are reasonable, given that the study area was fairly wet during 2013 due to plenty of precipitation and shallow water tables. 4.3.1 ET variations with land cover Daily ﬂuxes Instantaneous ﬂuxes Variable Rs,d Rn,d Gd Hd LEd LEd Rs Rn G0 H LE LE (closed) (unclosed) (closed) (unclosed) Unit MJ m−2 day−1 MJ m−2 day−1 MJ m−2 day−1 MJ m−2 day−1 MJ m−2 day−1 MJ m−2 day−1 W m−2 W m−2 W m−2 W m−2 W m−2 W m−2 NC2 site n 8 8 8 8 8 8 8 8 8 8 8 8 Ø 20.70 14.60 0.10 4.80 9.70 9.40 714 540 1 214 326 288 MAE 1.10 1.70 0.50 2.80 2.30 2.90 63 48 34 78 68 113 RMSE 4.20 2.80 0.20 4.80 3.00 5.70 78 51 36 89 89 125 MBE 1.60 −0.30 0.50 −1.90 1.10 1.40 63 21 34 −55 41 91 NC3 site n 8 8 8 8 8 8 8 8 0 8 0 8 Ø 22.30 13.80 NA 5.20 NA 6.50 776 507 NA 205 NA 209 MAE 1.50 1.90 NA 2.00 NA 1.70 28 41 NA 64 NA 60 RMSE 2.30 2.20 NA 2.70 NA 2.20 31 44 NA 75 NA 72 MBE 0.70 0.90 NA −0.10 NA 0.50 2 29 NA −11 NA 10 Rs,d is daytime integrated solar radiation; Rn,d is daytime integrated net radiation; Gd is daytime integrated soil ﬂux; Hd is daytime integrated sensible heat; LEd is daytime integrated latent heat; Rs, Rn, G0, H and LE are instantaneous ﬂuxes; “closed” indicates energy balance closure by residual, while “unclosed” indicates that energy balance closure was not imposed on the EC measurements. In addition: n is the number of observations; ¯O is mean measured ﬂux; MAE is mean absolute error between the modeled and measured quantities; RMSE is root mean square error; MBE is mean bias error. NA means not available. 4.3.3 ET variations with plantation stand age Within the managed pine plantation at Parker Tract, we also examined variations in seasonal water use with stand age (Fig. 17). This has relevance to forest management prac- tices and their impacts on the water yield of the watershed, which is the difference between precipitation and evapotran- spiration over the long term. Higher water yield translates to higher streamﬂow available for downstream use. Many for- est management practices, for example, thinning and refor- estation, need to consider the inﬂuence of stand age on the hydrological response. Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1031 Figure 12. Time series of modeled plot-scale ET (daily values smoothed with a 7-day moving average) associated with different land cover types. Figure 12. Time series of modeled plot-scale ET (daily values smoothed with a 7-day moving average) associated with different land cover types. Figure 13. Seasonal cumulative ET for different land cover types. Figure 13. Seasonal cumulative ET for different land cover types. Figure 14. Average cumulative ET at DOY 330 in 2013 over dif- ferent land cover types, and standard deviations within the sample populations (the black bar). Plotting cumulative ET at DOY 330 from various sites against stand age (Fig. 17), there is a clearly positive linear relationship between water use and stand age for the younger stands, between a few years old and around 20 years old (R2 is 0 82) As the stand age increases more water is used as ex- Figure 15. Average evaporation (E) and transpiration (T ) compo- nents of ET for ﬁve land cover types on Landsat overpass days. pected to sustain larger amounts of biomass. Differences in seasonal cumulative ET curves for different stand ages begin to signiﬁcantly diverge after DOY 130 – around the middle of May (not shown) When the stand matures beyond 20 years Figure 13. Seasonal cumulative ET for different land cover types. Figure 13. Seasonal cumulative ET for different land cover types. Figure 15. Average evaporation (E) and transpiration (T ) compo- nents of ET for ﬁve land cover types on Landsat overpass days. Figure 15. Average evaporation (E) and transpiration (T ) compo- nents of ET for ﬁve land cover types on Landsat overpass days. Figure 14. Average cumulative ET at DOY 330 in 2013 over dif- ferent land cover types, and standard deviations within the sample populations (the black bar). Figure 14. Average cumulative ET at DOY 330 in 2013 over dif- ferent land cover types, and standard deviations within the sample populations (the black bar). Figure 15. Average evaporation (E) and transpiration (T ) compo- nents of ET for ﬁve land cover types on Landsat overpass days. Plotting cumulative ET at DOY 330 from various sites against stand age (Fig. Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 17), there is a clearly positive linear relationship between water use and stand age for the younger stands, between a few years old and around 20 years old (R2 is 0.82). As the stand age increases, more water is used as ex- pected to sustain larger amounts of biomass. Differences in seasonal cumulative ET curves for different stand ages begin to signiﬁcantly diverge after DOY 130 – around the middle of May (not shown). When the stand matures beyond 20 years, www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1030 patial patterns of monthly cumulative ET (left column panels) from April to October, and cumulative ET on the end day he study area. rth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/101 Figure 11. Spatial patterns of monthly cumulative ET (left column panels) from April to October, and cumulative ET on the end day of each month over the study area. Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 32 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina ble 3. Statistical metrics describing comparison of retrieved ET time series with tower observations at NC2 and NC3 at daily and monthly e steps, as well as cumulative values over the study period (DOY 50–330). Site NC2 site NC3 site Index STARFM Landsat MODIS ALEXI STARFM Landsat MODIS ALEXI only smoothed only smoothed Daily Daily n 281 216 281 281 223 199 223 223 MAE (mm day−1) 0.80 0.64 0.66 0.85 0.83 0.70 2.00 1.45 RMSE (mm day−1) 0.99 0.83 0.85 1.04 1.02 0.88 2.27 1.84 MBE (mm day−1) −0.03 −0.29 −0.10 −0.11 −0.05 0.10 1.96 1.31 RE (%) 27.9 19.2 22.0 28.0 30.6 25.1 85.0 56.0 Monthly Monthly n 12 8 12 12 10 8 10 10 MAE (mm day−1) 0.41 0.36 0.48 0.44 0.23 0.33 1.61 1.06 RMSE (mm day−1) 0.53 0.39 0.58 0.54 0.29 0.36 1.82 1.33 MBE (mm day−1) 0.00 −0.25 −0.11 −0.15 −0.01 0.16 1.61 1.06 RE (%) 13.6 12.1 16.1 14.8 10.9 15.5 76.3 50.1 Study period Study period n 1 1 1 1 1 1 1 1 MAE (mm day−1) 0.03 0.04 0.10 0.11 0.01 0.12 1.88 1.26 MBE (mm day−1) −0.03 0.04 −0.10 −0.11 0.01 0.12 1.88 1.26 RE (%) 0.9 1.3 2.7 3.2 0.4 5.2 81.0 54.3 RE is relative error, which is calculated by dividing MAE by observed average ET. ure 16. Modeled cumulative ET from DOY 50 to DOY 330 as a ction of LAI. water usage tends to plateau and may actually decrease ghtly for stands with trees older than 75 years. Discussion Utility of TIR-based data fusion as a daily ET ti ti th d Figure 17. Modeled cumulative ET at DOY 330 as a function of stand age. daily time step of 28 % for the mid-rotation pine plantation site and 31 % for the clear-cut site, and errors of 13.6 % at the monthly time step. The accuracy of these results is com- parable with earlier studies applying the STARFM ET data fusion approach. Over rain-fed and irrigated corn and soy- bean sites in central Io a (IA) STARFM ielded a relati e 032 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carol able 3. www.hydrol-earth-syst-sci.net/21/1017/2017/ Over rain-fed and irrigated corn and soy- bean sites in central Iowa (IA), STARFM yielded a relative error of about 11 % over eight ﬂux towers (Cammalleri et Figure 16. Modeled cumulative ET from DOY 50 to DOY 330 as a function of LAI. Figure 17. Modeled cumulative ET at DOY 330 as a function of stand age Figure 17. Modeled cumulative ET at DOY 330 as a function of stand age. Figure 16. Modeled cumulative ET from DOY 50 to DOY 330 as a function of LAI. Figure 17. Modeled cumulative ET at DOY 330 as a function of stand age. the water usage tends to plateau and may actually decrease slightly for stands with trees older than 75 years. daily time step of 28 % for the mid-rotation pine plantation site and 31 % for the clear-cut site, and errors of 13.6 % at the monthly time step. The accuracy of these results is com- parable with earlier studies applying the STARFM ET data fusion approach. Over rain-fed and irrigated corn and soy- bean sites in central Iowa (IA), STARFM yielded a relative error of about 11 % over eight ﬂux towers (Cammalleri et al., 2013). When STARFM was applied to the Bushland, TX, the daily ET estimation had a relative error of 26.6 % for ir- daily time step of 28 % for the mid-rotation pine plantation site and 31 % for the clear-cut site, and errors of 13.6 % at the monthly time step. The accuracy of these results is com- parable with earlier studies applying the STARFM ET data fusion approach. Over rain-fed and irrigated corn and soy- bean sites in central Iowa (IA), STARFM yielded a relative error of about 11 % over eight ﬂux towers (Cammalleri et al., 2013). When STARFM was applied to the Bushland, TX, the daily ET estimation had a relative error of 26.6 % for ir- 5 Discussion www.hydrol-earth-syst-sci.net/21/1017/2017/ Site NC2 site NC3 site Index STARFM Landsat MODIS ALEXI STARFM Landsat MODIS ALEXI only smoothed only smoothed Daily Daily n 281 216 281 281 223 199 223 223 MAE (mm day−1) 0.80 0.64 0.66 0.85 0.83 0.70 2.00 1.45 RMSE (mm day−1) 0.99 0.83 0.85 1.04 1.02 0.88 2.27 1.84 MBE (mm day−1) −0.03 −0.29 −0.10 −0.11 −0.05 0.10 1.96 1.31 RE (%) 27.9 19.2 22.0 28.0 30.6 25.1 85.0 56.0 Monthly Monthly n 12 8 12 12 10 8 10 10 MAE (mm day−1) 0.41 0.36 0.48 0.44 0.23 0.33 1.61 1.06 RMSE (mm day−1) 0.53 0.39 0.58 0.54 0.29 0.36 1.82 1.33 MBE (mm day−1) 0.00 −0.25 −0.11 −0.15 −0.01 0.16 1.61 1.06 RE (%) 13.6 12.1 16.1 14.8 10.9 15.5 76.3 50.1 Study period Study period n 1 1 1 1 1 1 1 1 MAE (mm day−1) 0.03 0.04 0.10 0.11 0.01 0.12 1.88 1.26 MBE (mm day−1) −0.03 0.04 −0.10 −0.11 0.01 0.12 1.88 1.26 RE (%) 0.9 1.3 2.7 3.2 0.4 5.2 81.0 54.3 RE is relative error, which is calculated by dividing MAE by observed average ET. gure 16. Modeled cumulative ET from DOY 50 to DOY 330 as a nction of LAI. e water usage tends to plateau and may actually decrease ightly for stands with trees older than 75 years Figure 17. Modeled cumulative ET at DOY 330 as a function of stand age. RE is relative error, which is calculated by dividing MAE by observed average ET. RE is relative error, which is calculated by dividing MAE by observed average ET. Figure 16. Modeled cumulative ET from DOY 50 to DOY 330 as a function of LAI. the water usage tends to plateau and may actually decrease slightly for stands with trees older than 75 years. 5 Discussion 5.1 Utility of TIR-based data fusion as a daily ET estimation method Figure 17. Modeled cumulative ET at DOY 330 as a function of stand age. daily time step of 28 % for the mid-rotation pine plantation site and 31 % for the clear-cut site, and errors of 13.6 % at the monthly time step. The accuracy of these results is com- parable with earlier studies applying the STARFM ET data fusion approach. www.hydrol-earth-syst-sci.net/21/1017/2017/ Statistical metrics describing comparison of retrieved ET time series with tower observations at NC2 and NC3 at daily and mon me steps, as well as cumulative values over the study period (DOY 50–330). Site NC2 site NC3 site Index STARFM Landsat MODIS ALEXI STARFM Landsat MODIS ALEXI only smoothed only smoothed Daily Daily n 281 216 281 281 223 199 223 223 MAE (mm day−1) 0.80 0.64 0.66 0.85 0.83 0.70 2.00 1.45 RMSE (mm day−1) 0.99 0.83 0.85 1.04 1.02 0.88 2.27 1.84 MBE (mm day−1) −0.03 −0.29 −0.10 −0.11 −0.05 0.10 1.96 1.31 RE (%) 27.9 19.2 22.0 28.0 30.6 25.1 85.0 56.0 Monthly Monthly n 12 8 12 12 10 8 10 10 MAE (mm day−1) 0.41 0.36 0.48 0.44 0.23 0.33 1.61 1.06 RMSE (mm day−1) 0.53 0.39 0.58 0.54 0.29 0.36 1.82 1.33 MBE (mm day−1) 0.00 −0.25 −0.11 −0.15 −0.01 0.16 1.61 1.06 RE (%) 13.6 12.1 16.1 14.8 10.9 15.5 76.3 50.1 Study period Study period n 1 1 1 1 1 1 1 1 MAE (mm day−1) 0.03 0.04 0.10 0.11 0.01 0.12 1.88 1.26 MBE (mm day−1) −0.03 0.04 −0.10 −0.11 0.01 0.12 1.88 1.26 RE (%) 0.9 1.3 2.7 3.2 0.4 5.2 81.0 54.3 RE is relative error, which is calculated by dividing MAE by observed average ET. Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscap Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1032 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 032 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1032 1032 Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Table 3. Statistical metrics describing comparison of retrieved ET time series with tower observations at NC2 and NC3 at daily and monthly time steps, as well as cumulative values over the study period (DOY 50–330). 032 Y. Yang et al.: Daily Landsat scale evapotranspiration estimation over a forested landscape in North Carolina able 3. Statistical metrics describing comparison of retrieved ET time series with tower observations at NC2 and NC3 at daily and monthly me steps, as well as cumulative values over the study period (DOY 50–330). Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 1033 rigated area and 27 % for rain-fed area (Cammalleri et al., 2014). For a study area near Mead, NE, the relative error was 20.8 % for irrigated crops and 25.4 % for rain-fed crops (Cammalleri et al., 2014). Semmens et al. (2015) estimated daily Landsat-scale ET over California vineyards with rela- tive error of 18 % for an 8-year-old vineyard and 23 % for a 5-year-old vineyard. For real-time applications in water management, STARFM can be used to project water use information beyond the date of the last Landsat overpass within some limited time range, assuming MODIS data are available with low time latency. Practical limits to a viable projection time range may vary with site and season and will depend on the rate of change in weighting factors governing the STARFM fusion process. y y In the current study, we found that STARFM yielded sim- ilar or marginally lower accuracy than did a simple Landsat- only ET interpolation scheme in estimating ﬂuxes between Landsat overpasses. This is in contrast to results from Cam- malleri et al. (2013), who found that STARFM signiﬁcantly outperformed the Landsat-only approach in comparison with ﬂux measurements acquired over rain-fed crops in central IA during the soil moisture experiments in 2002 (SMEX02). There may be several factors that inﬂuence the potential value added by STARFM that can be deduced from this comparison. One factor may be climate and moisture sta- tus within the target modeling domain. During the ﬁrst part of SMEX02 experiment, conditions were becoming quite dry and the crops – particularly the corn ﬁelds – were be- coming notably stressed, exhibiting leaf curl near the ﬁeld edges. A rainfall event near the beginning of July (between Landsat overpasses) signiﬁcantly relieved the crop stress and greatly impacted soil moisture conditions over the SMEX02 study area (Anderson et al., 2013). In contrast, the climate of the Parker Tract study site studied here was very wet dur- ing 2013, and vegetation condition was not water limited. Consequently, as long as other factors do not affect the health of the vegetation (e.g., pest/disease infestation) or large vari- ations do not occur in atmospheric demand (large oscillations in radiation, wind and temperature), the evapotranspiration process will remain near a constant fraction of potential and may be reasonably captured by a simple daily interpolation scheme. 5.2 Comparison with prior ET studies over the Parker Tract Direct measurements of ET within the Parker Tract study area are only available for pine plantations (NC2 and NC3 AmeriFlux sites) (Sun et al., 2010; Domec et al., 2012). An- nual ET rates for the NC2 site reported by Sun et al. (2010) vary from 892 mm (a dry year, 2007) to 1226 mm in a normal year (2006). A process-based forest hydrological model (DRAINMOD-FOREST) has been calibrated for the NC2 eddy ﬂux site using drainage and groundwater ta- ble data for the period of 2005–2012. ET estimates by the calibrated model vary from 903 mm yr−1 in 2008 to 1170 mm yr−1 in 2006. These reported annual ET rates are consistent with results from the present study, which indicate ET values of 1000–1200 mm yr−1 for pine plantations estab- lished in the 1990s. Empirical models of ET, operative at the monthly timescale, have been developed for the Parker Tract study area (Sun et al., 2011a) and the accuracy is generally un- certain due to the large variability of climate and ecosys- tem structure and ET processes in general. For example, Sun et al. (2011a) proposed a general predictive model to esti- mate monthly ET using reference ET, precipitation and LAI over 13 ecosystems, which included the mid-rotation NC2 site and another clear-cut site (NC1 in AmeriFlux database) also in our study area. The monthly ET estimates from that approach had a relative error of 23 % and a RMSE of 15.1 mm month−1, while STARFM relative errors from the current study were 13.6 % at the monthly time step – a sub- stantial improvement in accuracy. Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina At the NC sites studied here, there were no major changes in soil moisture status that were additionally cap- tured by the MODIS ET retrievals. This might not be the case in severe drought years. Another factor to be considered is predominant land cover type. The NC sites are dominated by trees, with deeper rooting systems than perennial crops, and are able to extract water that is available much deeper in the soil proﬁle. This further reduces variability in vegeta- tion response during the study period in comparison with the SMEX02 study. 5.1 Utility of TIR-based data fusion as a daily ET estimation method 5.1 Utility of TIR-based data fusion as a daily ET estimation method In this study, the STARFM modeling approach applied over a full growing season resulted in a relative errors in ET at the Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 www.hydrol-earth-syst-sci.net/21/1017/2017/ Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina Y. Yang et al.: Daily Landsat-scale evapotranspiration estimation over a forested landscape in North Carolina 20 years. An investigation of mountain ash forests in Victo- ria, Australia, found that annual ET from a nearly 35-year stand was 245 mm more than that from a 215-year stand (Vertessy et al., 2001). Similarly, another study of three for- est stands, aged 14, 45 and 160 years, found plot transpira- tion declined from 2.2 mm per day in the 14-year stand and 1.4 mm per day in the 45-year stand to 0.8 mm per day in the 160-year-old forest (Roberts et al., 2001). In another study, Kuczera (1987) found a rapid decrease of mean annual water yield from a mountain ash forest watershed when stand age increased from 1 year to around 25 years, suggesting greater water use by the older forest stands. Murakami et al. (2000) used a Penman–Monteith-based model to simulate an ET– stand age relationship, which also showed a clear upward trend for young forests and a peak in ET at 20 years. The sharp increase of seasonal ET with the increase of stand age from 1 year to around 20 years illustrated in Fig. 17 is consis- tent with these earlier studies. Plant LAI is closely related to ET (Sun et al., 2011a) and is also an important input in plant physiological and hydrologic/land surface models as well as crop models (Duchemin et al., 2006; Nemani et al., 1993; Tague et al., 2013). As shown in Fig. 16, we also ﬁnd corre- lation between cumulative ET and season-average LAI over different land cover types, although LAI explained only 59 % of the modeled variability in ET. potentially real-time remote sensing method for estimating landscape-level ET which is suitable for operational applica- tions. 7 Data availability The MODIS data were downloaded from https://reverb.echo. nasa.gov/. The Landsat data were obtained from USGS (https://earthexplorer.usgs.gov/). The 30 m land cover map was available at https://www.mrlc.gov/nlcd2006.php. The ﬂux tower data were obtained from Dr. Noormets and can also be downloaded from Ameriﬂux (http://ameriﬂux-data. lbl.gov:8080/SitePages/siteInfo.aspx?US-NC2). The pine plantation stand age maps and tree planting history were ob- tained from the Weyerhaeuser Company. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. This work was funded in part by a grant from NASA (NNH14AX36I). We thank the Weyerhaeuser Company for providing stand age data. The US Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal or because all or part of an individual’s income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and TDD). To ﬁle a complaint of discrimination, write to USDA, Director, Ofﬁce of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720- 6382 (TDD). USDA is an equal opportunity provider and employer. 6 Conclusions This study demonstrates the capability of a multiscale data fusion ET model to estimate daily ﬁeld-scale ET over a forested landscape. Daily ET retrievals over the growing sea- son of 2013, generated at 30 m spatial resolution, compared well with observed ﬂuxes at AmeriFlux tower sites in a ma- ture pine stand and a recent clear-cut site, demonstrating ca- pability to reasonably capture a range in land-surface condi- tions within a managed pine plantation. Errors were 29 % at daily time step, 12 % at monthly time step and 0.7 % over the study period. Edited by: Y. Chen Reviewed by: two anonymous referees Edited by: Y. Chen Reviewed by: two anonymous referees A new scene gap-ﬁlling method was described to maxi- mize the number of Landsat images used for ET retrieval at Landsat scale and will be of beneﬁt in areas with persis- tent partial cloud cover and for recovering scenes from the Landsat 7 archive that are impacted by the SLC failure. The STARFM data fusion method can help to mitigate the dearth of high spatial–temporal resolution land surface temperature data from currently available satellite systems. 5.3 Water use variations with stand age and land cover type Previous studies investigating the relationship between land cover and age of forest stands on water usage mainly focused on the resulting impact on the water balance via impacts on streamﬂow at the watershed scale (Matheussen et al., 2000; Vertessy et al., 2001; Williams et al., 2012). Matheussen et al. (2000) analyzed the hydrological effects of land cover change in the Columbia River basin and found a signiﬁcant correlation between hydrological change and the tree matu- rity in the forested areas. In the current study, we found that water use increased linearly with stand age between 1 and 20 years, then plateaued or decreased with age after about These two factors are consistent with the assumptions in the Landsat-only interpolation, which assumes the ratio be- tween actual ET and reference ET is smoothly varying over time. However, for rain-fed crop land areas (like the central IA sites) that occasionally go through relatively dry and wet periods, STARFM may be better able to capture time vari- ability in fRET in response to changing moisture conditions. Despite the limited value added by STARFM over standard methods in this case, Fig. 8 shows that both methods per- formed very well. www.hydrol-earth-syst-sci.net/21/1017/2017/ Hydrol. Earth Syst. Sci., 21, 1017–1037, 2017 1034 Y. 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https://openalex.org/W1991869892	https://link.springer.com/content/pdf/10.1007/s11115-015-0303-5.pdf	English	null	Learning in Public Sector Organizations: A Theory of Action Approach	Public organization review	2,015	cc-by	8,552	M. Visser (*): K. Van der Togt Institute for Management Research, Radboud University, P.O. Box 9108, 6500 HK Nijmegen, The Netherlands e-mail: m.visser@fm.ru.nl URL: http://www.ru.nl/fm/visser Public Organiz Rev (2016) 16:235–249 DOI 10.1007/s11115-015-0303-5 Public Organiz Rev (2016) 16:235–249 DOI 10.1007/s11115-015-0303-5 Learning in Public Sector Organizations: A Theory of Action Approach Max Visser & Kim Van der Togt Published online: 15 March 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Published online: 15 March 2015 Abstract The importance of learning has since long been acknowledged for both business and public sector organizations. However, learning theory and research have tended to neglect the differences between these organizations, and to develop in separate and unrelated directions. To address these developments, this paper purports, first, to develop a theoretical framework that integrates various concepts from the fields of public policy learning and organizational learning, and that is specifically directed at public sector organizations. Second, the paper purports to illustrate the utility of this framework by applying it to policy-making in a Dutch municipality, the Pegasustown prostitution case. Keywords Organizationallearning.Publicpolicylearning.Municipaldecision-making . Theory of action Introduction The importance of learning in and by organizations has since long been recognized by organization scientists. In particular in the last two decades interest in organizational learning has been growing, as evidenced by a continuously increasing output in journals and books and an increasing number of reviews of the field (e.g., Argote 2011; Rashman et al. 2009; Shipton 2006). While in general the main thrust of organizational learning research has been directed at business organizations, in the last 15 years learning in public sector organizations (hereafter PSOs) increasingly has received attention (Gilardi and Radaelli 2012; Maden 2012; Rashman et al. 2009). Following trends towards BReinventing government^ and BNew public management,^ PSOs increasingly face public and political pressures to become more efficient and effective in their public services 236 M. Visser, K. Van der Togt delivery and more transparent and accountable in their administrative processes (Dent et al. 2007; Dilworth 1996; Osborne and Gaebler 1992). To achieve all this, learning is more and more being considered as an important requirement for PSOs (Betts and Holden 2003; Bovens et al. 2008; Brown and Brudney 2003; Greiling and Halachmi 2013; Schofield and Sausman 2004). However, there are important differences between business and public sector orga- nizations that impact the ways in which these organizations can learn. Business organizations generally work under market conditions, in which profit, sales and expenditure figures constitute a fairly small set of relatively simple and straight- forward indicators guiding decision-making and learning. PSOs generally work under bureaucratic or hybrid conditions, in which departmental governance, political rules, regulations and conflicting pressures, sudden public events, annual budgets and private- public interdependencies constitute a fairly large set of relatively complex and ambig- uous indicators guiding decision-making and learning (Busenberg 2001; Dekker and Hansen 2004; Mintzberg 1996; Rashman et al. 2009). Following these differences, scientists in the field of public administration have been more interested in public policy learning than in organizational learning (Bennett and Howlett 1992; Hall 1993; Browne and Wildavsky 1984; Howlett 2012). Although theory and research on public policy and organizational learning thus developed along separate lines, policy researchers generally were aware of the importance of organiza- tions for policy learning, as is, for example, evidenced in the concepts of the Bself evaluating organization^ (Wildavsky 1972; Boyne et al. 2004), and more recently the neo-Pragmatist Bexperimentalist organization^ (Evans 2000; Sabel and Simon 2011; Sabel and Zeitlin 2012; Zeitlin 2011). Learning in Public Sector Organizations: A Framework Because the fields of public policy and organizational learning often have been characterized as having a high amount of conceptual diversity and proliferation (Shipton 2006; Tosey et al. 2012; Visser 2007), we have decided to adopt an existing theoretical framework, rather than invent a new one. For several reasons, we have selected the theory of action approach by Argyris and Schön (1974, 1978, 1996). First, Argyris and Schön were among the first organization scientists to emphasize the necessity of learning for PSOs, to provide theoretical guidelines for this and to work in practice with PSOs (Argyris 1976, 1980; Rein and Schön 1991; Schön 1971, 1975, 1979; 1983). Consequently, their approach appears to accommodate the differences between business organizations and PSOs in the nature and number of values that have to be attended to, and in the contested, political nature of the learning that takes place. Following this, their approach seems among the few to be in fairly good currency in business administration (Argote 2011; Easterby-Smith and Lyles 2003, 2011; Easterby- Smith et al. 2004; Visser 2007), and in public administration (Browne and Wildavsky 1984; Duijn et al. 2010; Grin and Loeber 2007; In ‘t Veld 1991; Kim et al. 2013; Kolibra and Lathrop 2007; Maden 2012; Leeuw and Sonnichsen 1994; Van Dooren 2011). Central to Argyris and Schön’s approach is the relationship between knowledge and action. They consider integrating thought with action as the key challenge facing Ball human beings…[who] need to become more competent in taking action and simulta- neously reflecting on this action to learn from it^ (Argyris and Schön 1974, p. 4). Central to this endeavor are theories of action, which have the general form: Bin situation S, if you want to achieve consequence C, under assumptions a1…an, do A^ (Argyris and Schön 1974, p. 6). Assumptions a1…an constitute a model of the world, in which it is probable that action strategy A will lead to consequence C in situation S. Consequence C refers to all kinds of purposes and goals which an actor may aspire to achieve. Besides these elements, theories of action contain governing variables. These are deeper lying norms, values and core beliefs of persons and organizations that make consequence C desirable or worthwhile to achieve (Fig. 1). Different from business organizations, for PSOs the consequences they intend to achieve are primarily policy goals and purposes. Introduction This division between organizational and public policy learning theory and research reflects wider differences in the social science community at large. While much of the pioneering work in the fields of business administration and organization theory originated in public organizations (e.g., Simon, Selznick, Crozier), in the past three decades scientists in these fields have tended to lose interest in public organizations and public problems. At the same time scientists in the field of public administration have tended to isolate themselves from the larger world of mainstream organization theory (Kelman 2005, 2007; Kochan et al. 2009; Schofield 2001, 2004). ( ) This division has had various negative consequences for the field of learning. First, it has sometimes led to an uniform and uncritical application of organization learning theories to business and public organizations, without taking into account the profound differences between the two (Dekker and Hansen 2004; Mintzberg 1996). Further, it has sometimes led to separate but comparable developments in theory building, where scientists at one side of the divide declined to build on theoretical advances at the other side (Busenberg 2001; Kelman 2005, 2007; Schofield 2004). Finally, with a few exceptions theories of policy learning and organizational learning seldom have been related to one another or analyzed from a common perspective (Common 2004; Gilardi and Radaelli 2012; Grin and Loeber 2007). To address some of these negative consequences and to contribute to some degree of closure of the observed public-business division, the first purpose of this paper is to develop a theoretical framework that appears capable of incorporating and accommo- dating various concepts from the fields of public policy learning and organizational learning and that is specifically directed at public sector organizations. The second Learning in Public Sector Organizations 237 purpose of this paper is to illustrate the applicability and utility of this framework by applying it to an instance of policy-making in a Dutch municipality, the Pegasustown prostitution case. Finally, the paper ends with discussion and conclusions. Learning in Public Sector Organizations: A Framework We define a policy as an elaborate theory of action at the organizational level of a PSO. Such a policy theory of action is Situation Action strategy & assumptions Consequences Governing vari- ables Fig. 1 Theory of action Situation Action strategy & assumptions Consequences Governing vari- ables y of action Situation Action strategy & assumptions Consequences Governing vari- ables of action Governing vari- ables Fig. 1 Theory of action Fig. 1 Theory of action 238 M. Visser, K. Van der Togt comparable to a policy belief system (Grin and Loeber 2007; Jenkins-Smith and Sabatier 1993; Koppenjan and Hufen 1991), policy paradigm (Bennett and Howlett 1992; Hall 1993), and policy frame (In ‘t Veld 1991; Rein and Schön 1991). comparable to a policy belief system (Grin and Loeber 2007; Jenkins-Smith and Sabatier 1993; Koppenjan and Hufen 1991), policy paradigm (Bennett and Howlett 1992; Hall 1993), and policy frame (In ‘t Veld 1991; Rein and Schön 1991). Argyris and Schön distinguish between espoused theory of action and theory in use. For persons espoused theory refers to the theory they say they will follow when queried about it. For organizations espoused theory refers to the theory that persons, acting for the organization, formally announce the organization will follow, and also to the theory that the organization formally states it will follow, for example in policy plans, strategic documents, rules and procedures. Theory in use, on the other hand, refers to the theory that persons and organizations actually follow in their daily actions, and which may reconstructed on the basis of observation of those actions. These two theories may or may not coincide, and persons or organizations may or may not be aware of possible inconsistencies between the two. The development of espoused policy theory starts in the political domain, i.e., as the formal promulgation of laws and regulatory rules by acts of legislative bodies. These laws and rules contain the aspired policy consequences, assumptions, instruments and action strategies, tied together in more or less coherent ways with governing variables, the deeper lying norms, values and core beliefs (Hoogerwerf 1990; Sabatier and Jenkins-Smith 1993). When PSOs are charged with the execution of these formal laws and regulations, they first develop their own espoused policy theory on the basis of the policy theory they view as underlying these formal laws and regulations. Learning in Public Sector Organizations: A Framework the governing variables, the deeper lying norms, values and core beliefs that make consequences important to attain (Fig. 2). This type of learning is comparable to first order learning (Hall 1993; In ‘t Veld 1991), technical learning (Fiorino 2001), instru- mental learning (Gilardi and Radaelli 2012; May 1992), policy-oriented learning (Howlett 2012; Jenkins-Smith and Sabatier 1993), and improvement learning (Van der Knaap 2004). p ) However, if after several cycles of single loop learning errors still persist, a more comprehensive and incisive form of error detection and correction may be necessary. This is accomplished through double loop learning. Here the governing variables, the deeper lying norms, values and core beliefs that make consequences important to attain, are subject to joint reflection and inquiry and, if necessary, changed. When these norms, values and core beliefs change, a whole set of new consequences may come into being, leading to all kinds of new relationships between situations, action strategies, assump- tions and consequences (Fig. 3). This type of learning is comparable to second order learning (Hall 1993; In ‘t Veld 1991), conceptual learning (Fiorino 2001), social learning (Gilardi and Radaelli 2012; May 1992), substantive learning (Ventriss and Luke 1988), and innovative learning (Van der Knaap 2004). Organizations differ in the degree to which errors are surfaced and put to reflection and inquiry. To account for these differences, Argyris and Schön (1978, 1996) have inquired into the ways in which the learning climate (or behavioral world) of an organization inhibits or promotes reflection and inquiry, and thus learning. Based on their action research and intervention experiences, Argyris and Schön assert that most organizations are driven by a Model O-I learning climate. This model is characterized by a closed attitude among individuals and defensive routines in the organization as a whole, exemplified in a general atmosphere of distrust and lack of respect between managers and employees, blocked communication, contested problem definitions, and Bnaming and blaming^ in the case of errors (Bovens et al. 2008; Dilworth 1996; Howlett 2012; Jenkins-Smith and Sabatier 1993). All this conspires to make collaborative reflection on and inquiry into the causes of error, and hence learning, hard to achieve. As an alternative to Model O-I and to enable collaborative reflection and inquiry, Argyris and Schön (1978, 1996) advocate a Model O-II learning climate. Learning in Public Sector Organizations: A Framework Gradually however, their espoused policy theory acquires an in use character, as it moves forward in the direction of concrete execution. We define policy implementation as the final phase in this transition. The policy theory in use guides the ways in which concrete actions are devised for concrete situations and carried out in ways that lead to tangible consequences among policy subjects. Such consequences become most visible at the street-level of bureaucracy (Evans and Harris 2004; Lipsky 1980), when behavior of policy subjects is changed in accordance with policy goals or not. Policy theory in use may or may not differ from espoused policy theory, but the way in which it is implemented ultimately is decisive for the success or failure of the policy theory involved (Browne and Wildavsky 1984; Schofield 2001; Schön 1979; Wildavsky 1972). Learning starts when actual consequences of an action strategy do not correspond to expected consequences. This discrepancy between expectation and result is considered an error and leads to a problematic situation, which calls for a period of joint and collaborative reflection and inquiry by the acting organizational members (Argyris and Schön 1978, 1996; Schön 1983). Such joint reflection and inquiry may proceed on a more informal or a more formal basis. For PSOs the latter case often involves policy evaluation, defined here as a formal, prolonged and structured form of inquiry and reflection on the ways in which action strategies in policy theory in use have led to expected, or more often to unexpected, consequences (Duijn et al. 2010; Leeuw and Sonnichsen 1994; Minnett 1999; Rist 1994; Van der Knaap 2004). We define learning as the Bdetection and correction of error,^ on the basis of (formal and informal) joint reflection and inquiry (Argyris and Schön 1978, p. 2). It may first be accomplished through single-loop learning. Here organization members mitigate the discrepancy between expected and actual consequences by adjusting the relationships between situation, action strategy, assumptions and consequences, without questioning Learning in Public Sector Organizations 239 the governing variables, the deeper lying norms, values and core beliefs that make consequences important to attain (Fig. 2). This type of learning is comparable to first order learning (Hall 1993; In ‘t Veld 1991), technical learning (Fiorino 2001), instru- mental learning (Gilardi and Radaelli 2012; May 1992), policy-oriented learning (Howlett 2012; Jenkins-Smith and Sabatier 1993), and improvement learning (Van der Knaap 2004). Learning in Public Sector Organizations: A Framework This model is characterized by an open attitude among individuals and a productive learning climate in the organization as a whole, exemplified in a general atmosphere of trust and respect between managers and employees, open communication, fact-based problem defini- tions, and openness towards errors (Bovens et al. 2008; Dilworth 1996; Jenkins-Smith and Sabatier 1993; Wildavsky 1972). Argyris and Schön regard Model O-II as a crucial Expected Unexpected Situation Action strategy & assumptions Consequences Governing vari- ables Fig. 2 Single loop Learning Governing vari- ables Fig. 2 Single loop Learning 240 M. Visser, K. Van der Togt Expected Unexpected Situation Action strategy & assumptions Consequences Governing vari- ables Fig 3 Double loop Learning condition for double-loop learning, and double loop learning in its turn as a crucial condition for organizational survival in the long run. Model O-II organizations, how- ever, are rarely found in practice, and often external interventions are necessary to create a productive learning climate. Expected Unexpected Situation Action strategy & assumptions Consequences Governing vari- ables Fig. 3 Double loop Learning Unexpected Fig. 3 Double loop Learning condition for double-loop learning, and double loop learning in its turn as a crucial condition for organizational survival in the long run. Model O-II organizations, how- ever, are rarely found in practice, and often external interventions are necessary to create a productive learning climate. Finally, Argyris and Schön (1978) link individual learning to organizational learning by pointing out that, by virtue of delegated responsibilities, some employees are empowered to speak and act on behalf of the whole organization, not only in top positions, but in every role or function dealing with external relations or subjects. Those employees learn when they experience the discrepancy between consequences of an action, expected on the basis of organizational theory of action, and actual consequences. If the organiza- tion as a whole is to learn, then the corrective actions these employees undertake on the basis of reflection and inquiry into that discrepancy should become embedded in the espoused organizational theory of action (e.g., policy plans, strategic documents, rules and procedures), and then in the organizational theory in use guiding daily actions. For PSOs the room for empowerment, and thus for learning at both individual and organizational levels, may be more circumscribed than for business organizations. 1 The empirical data for this case were collected in Spring 2008. The data collection involved a detailed content analysis of relevant municipal policy documents, news paper articles and court verdicts, and a round of semi-structured interviews with municipal council members and aldermen from different political back- grounds, who were all actively involved in the policy changes in the case. Further background information was found in a dissertation by Gorgels (1993), who researched prostitution policy-making and implementation in Pegasustown and five other Dutch municipalities. Public Sector Organizational Learning: The Pegasustown Prostitution Case The municipality of Pegasustown includes the city of Pegasustown and a few suburbs, numbers a total of 141.000 inhabitants, and is located in the Eastern part of the Netherlands. Dutch municipalities like Pegasustown are governed by a Board of Mayor and Aldermen (hereafter the Board), who in the formulation and implementation of policies are supported by the municipal administrative service. The Board is politically accountable and responsible to the Municipal Council (hereafter the Council), elected every 4 years by the eligible electorate of the municipality. Prostitution was officially illegal in the Netherlands since 1911, but it was tolerated as long as prostitution business owners and prostitutes adhered to the rules and regulations set by local authorities. In the 1960s prostitution became more openly tolerated than before, until it finally became legalized in 2000. The city of Pegasustown housed a thriving prostitution business in its so-called Red Quarter since the mid 1960s. Although this business caused much annoyance and trouble for the Red Quarter inhabitants not involved in it, the number of prostitution windows and houses was allowed to expand during the early 1970s, in line with the permissive atmosphere of that time. However, from the late 1970s onwards the amount of trouble and nuisance associated with prostitution reached unprecedented heights, due to increasing drug abuse, drugs-related crime, street soliciting and intensive trafficking. Activist Red Quarter inhabitants organized themselves in a Work Group Red Quarter and staged various large-scale protest meetings, which received broad media coverage and gradually propelled Board and Council into action. As a first step, in the late 1970s the prostitution windows were concentrated in only one part of the Red Quarter. This policy measure had some effect, but after the protest meetings, the Working Group Red Quarter continued to pressure the Council to put an end to window prostitution altogether, and the Council made implicit promises to do so. In response to those pressures and also in line with the less permissive atmosphere of the 1980s, consensus grew in the Council that Bprostitution does not belong in a residential area.^ However, Board and Council sensed that they could not close down the prostitution business in a fortnight, lest they be confronted with financial claims by the prostitution business owners. Therefore they anticipated future developments by promising these owners an alternative location at an BEros Center,^ to be developed in the industrial area of Pegasustown. Learning in Public Sector Organizations: A Framework When policies as implemented deviate too much from policies as espoused by the political authorities, issues of democratic responsiveness and accountability arise (Bovens et al. 2008; Mintzberg 1996). While Bstreet-level bureaucrats^ may want empowerment and administrative discretion to adapt policy rules to concrete client needs and field developments, political authorities and the public-at-large may want predictability and uniformity in the provision of public services, thus curtailing discre- tion (Bovens and Zouridis 2002). In summary, the framework for public sector organizational learning centers on the concepts of policy theory of action, espoused policy theory, policy theory in use, single loop learning, double loop learning, Model O-I and Model O-II learning climate. In the next section we will attempt to illustrate the applicability and utility of this framework by applying it to a case of municipal policy-making, the Pegasustown prostitution case. 1 Learning in Public Sector Organizations 241 Public Sector Organizational Learning: The Pegasustown Prostitution Case Public Sector Organizational Learning: The Pegasustown Prostitution Case As a consequence, in the 1980s and early 1990s further policy measures were taken. Spots for street soliciting were relocated from the Red Quarter to an isolated zone in the industrial area of Pegasustown, where authorities could exert more control over the situation. Further several hotels and cafes, suspected of involvement in drugs related crime, were closed and drugs addicted junks were taken off the streets. Trafficking was regulated by installing one-way direction streets and closing off the whole quarter for cars at night. These policy measures went a long way in relieving the problems of the inhabitants in the Red Quarter, although the Work Group Red Quarter remained adamant in battling prostitution. Not unexpectedly, other groups in and outside the Red Quarter were less content with these developments. The prostitutes feared the loss of their familiar work places and the loss of anonymity that relocation would bring. Many of them were involved in formally illegal activities, such as the use of hard drugs and illegal residence in the Netherlands, and consequently were not very keen on too much municipal involvement in 242 M. Visser, K. Van der Togt their work area. They did, however, organize themselves in an interest group, but since they were never involved in municipal policy-making, they remained relatively politically powerless. The visitors of the prostitutes also were not happy with these developments, but for understandable reasons they could not raise their voices in an organized manner. One group that did organize itself effectively was the prostitution business owners. They brought several law suits against the Pegasustown municipality, but they lost all of them and early 2006 the last prostitution house in the Red Quarter was closed down. A group outside the Red Quarter showing discontent with the process was the industrial business owners in Pegasustown’s industrial area. Not only was the new spot for street soliciting practically put on their doorstep, their area also already housed a drugs rehabilitation center. Initially the industry owners were promised by the Council that the new Eros Center would not be planned in their area, but this promise could not be formally held. In the meantime, however, this Eros Center still has not materialized. Public Sector Organizational Learning: The Pegasustown Prostitution Case The prostitute business owners were invited by Board and Council to propose their own ideas for that Center, be it within several constraints regarding the visibility of the Center, the number of windows and the regulation of traffic. Until today they have not been able to come up with a proposal, and, given the resistance of the industrial business owners, the Board has not been particularly anxious to receive one, according to one respondent. In terms of the learning framework developed in this paper, it may be argued that in the early 1970s the Pegasustown policy theory in use was one of Blaissez-faire^ and tacit recognition of prostitution, although its espoused policy theory, as laid down in formal municipal rules and regulations, was stricter and less permissive on this subject. When in the course of that decade the situation escalated and provoked large-scale protests, municipal policy theory in use changed more in the direction of the espoused policy theory. The desired consequence of policy theory in use now became the reduction of the problems and nuisance associated with prostitution in the Red Quarter, on the basis of a deeper-lying and increasingly shared governing variable that Bprostitution does not belong in a residential area.^ The various promises the Council made to different groups constitute a less formal espoused policy theory, but here the different groups saw to it that this particular espoused theory was put to use. These changes in policy theory in use seem to have been the result of single loop learning, rather than double loop learning. The first policy measures of the Board and Council only partly achieved the expected consequence of reducing the prostitution- related problems in the Red Quarter. The continuing pressures of the Working Group Red Quarter made it clear to Board and Council that this consequence only could be achieved by taking more drastic policy measures, i.e., by changing action strategies and assumptions in the direction of full abolishment of window prostitution in the Red Quarter and its relocation to a less populated area of the city. With the closure of the last prostitution window in 2006, this consequence at last has been achieved. The respondents hold somewhat different views on this course of events, though. Public Sector Organizational Learning: The Pegasustown Prostitution Case Two respondents indicate that the first policy measures in fact went a long way in reducing nuisance and problems in the Red Quarter, almost to the extent that relocation of window prostitution probably would not have been necessary. One of these respon- dents even argues that the Working Group Red Quarter was at least partly motivated by financial motives. All active Working Group members owned one or more houses in the Red Quarter, and they all stood to gain much by the total abolishment of prostitution in their neighborhood. Another respondent disagrees with this view and argues that the Learning in Public Sector Organizations 243 amount of problems and nuisance was still high enough to legitimize the total reloca- tion of the prostitution business. Double loop learning does not appear to have taken place. For this type of learning it is necessary that the governing variables that make consequences worthwhile to achieve are subject to inquiry and changed. However, the governing variable that Bprostitution does not belong in a residential area^ became generally accepted by the Council in the early 1980s and continues to do so in the present. In the beginning, however, Board and Council were not ready to accept the ultimate consequences of this variable, because, as one respondent puts it, Beverybody knew that relocation of the whole prostitution business certainly would mean lots of problems, legal procedures, financial risks, and media attention.^ Only when the Working Group Red Quarter increased the pressure on the Council did it change its policy towards relocation. At the same time the Council attempted to avoid too many frictions with the prostitution business owners, in which it did not succeed. The external pressures in this case thus reinforced existing governing variables and in this way accelerated single loop learning by Board and Council. The learning climate in this case may be characterized as predominantly Model O-I, although a few Model O-II characteristics can be discerned. Regarding Model O-II, one respondent observes that in the whole period between the early 1980s and today Board and Council Blooked after each other,^ because of the tenacity of the problems and the toughness of the decisions involved. Although some political games were played once in a while, on the whole decision-making occurred in an atmosphere of mutual regard and relative openness between Board and Council. Conclusions and Discussion Three conclusions may be drawn on the basis of this illustrative application of the learning framework to the Pegasustown prostitution case. First, for PSOs the different elements of this framework more often constitute contested areas of public debate than neatly ordered and detectable action and performance indicators. The degree to which the municipal authorities succeeded in achieving the consequence of reducing prostitution-related problems in the Red Quarter, and the question which action strat- egies and assumptions were necessary for that achievement were subject to different, intensely held views inside and outside the Council. Second, depending upon the amount of public attention to a certain policy domain, PSOs can allow espoused policy theory to deviate only to a small extent from policy theory in use in that domain. Commitments and promises made in public that cannot be put into policy theory in use are likely to raise questions about democratic account- ability and reliability in the media and the public, and they are also likely to be used against the policy-making authorities in court proceedings. Third, the learning in this case appears to have been more single than double loop, in a learning climate that appears to have been more Model O-I than O-II. The broad media coverage and attention in this case, together with the pressures of the various groups involved and publicly announced promises and commitments, generally militated against a productive reflection on the wisdom of existing policy measures, and an open inquiry into possible alternatives. The governing variable underlying municipal prosti- tution policy measures was not questioned, at any rate not by the dominant actors in this case. The prostitutes and the owners of prostitution businesses might have held different governing variables in this respect, but they were not politically able to turn the tables. g g p y p y Regarding the applicability and utility of this framework, three points of discussion emanate from this paper. First, learning in PSOs cannot be separated from questions of power and influence (Betts and Holden 2003; Jenkins-Smith and Sabatier 1993). In any given policy domain different groups with different interests and different norms and values will attempt to influence the development and implementation of policy theories. They will do so by contesting existing action strategies, assumptions and consequences, and by emphasizing different governing variables than the ones apparently underlying the policy involved. Public Sector Organizational Learning: The Pegasustown Prostitution Case Another respondent notes that the different political backgrounds of the Council members never caused much tension because all members highly valued consensus. However, he thinks that some tension would have spurred more debate between the members, which in turn might have led to better decisions in some of the events described in this paper. Regarding Model O-I, two factors seem to have been influential, according to all respondents. First, they all point at the pervasive influence of media attention in the whole case. The broad media coverage of the protests and grievances against prostitution-related problems in the Red Quarter put decision-making in the Council under severe pressure. Many Council members saw the media attention as a fine opportunity to have their Bone minute of fame,^ and, not wanting to appear unsympa- thetic to the inhabitants’ complaints in public, made implicit promises to remove prostitution from the Red Quarter. When later doubts arose in the Council about the wisdom of a wholesale relocation of the prostitution business in the light of expected damage claims and other financial consequences, many Council members were reluc- tant to rescind their promise, because they feared to be exposed in the media as untrustworthy. Defensive Bface saving^ became more important than an open-minded inquiry into new possible ways of action. Second and related to the first factor, the three implicit and explicit promises made by the Council to Red Quarter inhabitants, prostitution business owners and industry owners made it difficult for Board and Council to act on new information or to reflect on the wisdom of adopted policy measures on the basis of open-minded inquiry. The Working Group Red Quarter used the implicit promise to remove prostitution to keep pressure on the Council, aided by media attention. The prostitution business owners used the promise of a new Eros Center in several court proceedings to argue for a moratorium on their forced relocation from the Red Quarter. Since Pegasustown had not arranged for a suitable 244 M. Visser, K. Van der Togt new prostitution location, their forced relocation was unlawful, an argument which the Court declined to accept, however. Finally, the industry owners intended to keep the Council to its initial promise of Bno Eros Center in their area,^ which paradoxically helped the prostitution business owners in building their case in Court. 2 While the neo-Pragmatist arguments appear quite new, it may be noted here that the Prussian-German Army in the early 19th century already adopted a similar philosophy of command and control, which later became known as ‘Auftragstaktik’ (Visser 2008, 2010; Wilson 1989). A century later the Israeli Defense Forces came to embrace an even more decentralized form of command and control (Horowitz 1970; Van Creveld 2002). These army examples seem to suggest that hierarchical organization does not necessarily preclude learning, provided that senior commanders set broadly specified goals, give their subordinate commanders considerable discretion for independent decision-making in the pursuance of those goals, assure that they are sufficiently trained and equipped to do so, and provided that these senior commanders use the errors that invariably occur in independent decision-making as learning opportunities in an atmosphere of trust and respect (Visser 2008, 2010; Wilson 1989). Conclusions and Discussion Many learning theorists do not consider these conditions very conducive to the collaborative inquiry and reflection, necessary for double loop learning or even solid single loop learning (e.g., Argyris 1976, 1980; Argyris and Schön 1978). Others, however, argue that this really depends upon the quality of the political decision-making process and the commitment of politicians towards policy theories in use (e.g., Dekker and Hansen 2004). The prostitution case seems to support the latter view: the more politicians give in to and honor competing demands by different interest groups, the less room they create for themselves for open-minded reflection and inquiry, and hence for double loop learning. 245 Learning in Public Sector Organizations Second, the depth of change in policy theories of action as a result of single and double loop learning is a point of debate in the literature. Some argue for a rather sharp binary division between both forms of learning, in which changes in governing variables appear as paradigm shifts that occur fairly independent of single loop learning results (Gilardi and Radaelli 2012; Hall 1993). Others argue that such paradigm shifts may be more characteristic of majoritarian democratic systems (like the Anglo-Saxon countries) than consociational democratic systems (like the Netherlands and most Continental European countries). In the latter systems conflicting sets of governing policy variables are less likely to gain majority support than in the former, and consequently, these conflicting sets will tend to coexist rather than dominate one another. Given this coexistence, in consociational democracies single and double loop learning results are expected to be less sharply divided and more accumulative (Grin and Loeber 2007; Helderman et al. 2005; Hemerijck and Visser 1997; Van Gestel et al. 2008). The prostitution case seems to modify the latter position in that some sets of governing variables may actually become dominant even in consociational systems, because competing sets of governing variables are only supported by politically powerless or unpopular minorities, like the prostitutes and their business owners in this case. Learning here will then be by definition of the single loop kind, since the broad agreement on the underlying governing variables is not disturbed. Third, the nature of the relationship between political authorities and PSOs has recently been questioned on neo-Pragmatist grounds (Evans 2000; Sabel and Simon 2011; Sabel and Zeitlin 2012; Zeitlin 2011). Conclusions and Discussion In the face of current environmental turbulence, complexity and change, political authorities can no longer be held capable of predicting and steering environmental events on the basis of detailed hierarchical top-down command and control. Instead these authorities should set general goals, and monitor the efforts of local actors to achieve those goals by means of their own devising. The authorities should intervene only when the efforts of the latter fall short or are inadequately accounted for. PSOs should be given room to develop policies, experiment with their implementation, and to learn from one another’s problem solving in the pursuance of these general goals. Although in this paper we have not differen- tiated between municipal political authorities and administrative services, the prostitu- tion case seems to suggest that this neo-Pragmatist view may be unduly optimistic about the ways in which political authorities can shield themselves from public demands and emotions. Further, this view may be unduly optimistic about the amount of trust and administrative discretion politicians are inclined to lend to PSOs when it comes to implementation of publicly intensively debated and contested policies.2 Finally, this paper has some clear limitations. First of all, its empirical base is modest, which precludes far reaching conclusions and recommendations. Besides 246 M. Visser, K. Van der Togt document content analyses, the findings are based on a set of interviews among political actors only, where a more comprehensive design should have included Red Quarter inhabitants, prostitutes, business owners, etc.. Second, the municipality of Pegasustown has been treated as a unitary PSO, where a subdivision between political authorities and administrative services would in all probability have provided a more detailed and nuanced picture of the events in this case. Finally, the relations between the municipality and other important government agencies, like the local police force, public health and welfare agencies, have not been researched in depth, although it is highly probable that these agencies have played an important role in the implementa- tion of prostitution-related policies. In spite of these empirical limitations, it has proved possible to develop a framework for learning in public sector organizations that appeared capable of incorporating and accommodating various concepts from the fields of public policy learning and organi- zational learning and that could be illustrated by an empirical case in a meaningful way. Conclusions and Discussion It is hoped that in this way this paper contributes to closing some of the distance between the fields of business and public administration and to improving the effec- tiveness of public service organizations. 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Max Visser is currently an associate professor at the Institute for Management Research, Radboud University, the Netherlands. His research interests include the relation between organizational learning, management control and performance, in particular in public and governmental organizations. References Further, he is interested in the relations between governance, performance and behavior in organizations, increasingly from a Critical perspective. Kim Van der Togt is currently an attorney at law at DLA Piper, Amsterdam, the Netherlands. She graduated in Criminal Law and in Business Administration (Radboud University), and in Criminology and Criminal Justice (University of Oxford, UK).
https://openalex.org/W2046145002	https://europepmc.org/articles/pmc3701149?pdf=render	English	null	Action simulation in hallucination-prone adolescents	Frontiers in human neuroscience	2,013	cc-by	13,399	Tarik Dahoun1, Stephan Eliez 1,2, Fei Chen 1, Deborah Badoud1,3, Maude Schneider 1,3, Frank Larøi 4 and Martin Debbane1,3 1 Ofﬁce Médico-Pédagogique Research Unit, Department of Psychiatry, University of Geneva School of Medicine, Geneva, Switzerland 2 Department of Genetic Medicine and Development, University of Geneva School of Medicine, Geneva, Switzerland 3 Adolescence Clinical Psychology Research Unit, Faculty of Psychology and Educational Sciences, University of Geneva, Geneva, Switzerland 4 Department of Psychology University of Liège Liège Belgium 1 Ofﬁce Médico-Pédagogique Research Unit, Department of Psychiatry, University of Geneva School of Medicine, Geneva, Switzerland 2 Department of Genetic Medicine and Development, University of Geneva School of Medicine, Geneva, Switzerland 3 Adolescence Clinical Psychology Research Unit, Faculty of Psychology and Educational Sciences, University of Geneva, Geneva, Switzerland 4 Department of Psychology, University of Liège, Liège, Belgium Theoretical and empirical accounts suggest that impairments in self-other discrimination processes are likely to promote the expression of hallucinations. Studies using a variety of paradigms involving self-performed actions argue in favor of perspective taking confusion in hallucination-prone subjects. However, our understanding of such processes during adolescence is still at an early stage. The present study thus aims (1) to delineate the neural correlates sustaining mental simulation of actions involving self-performed actions (ﬁrst-person perspective; 1PP) and other-performed actions (third-person perspective; 3PP) during adolescence (2) to identify atypical activation patterns during 1PP/3PP mental simulation of actions in hallucination-prone adolescents (3) to examine whether differential risk for schizophrenia (clinical vs. genetic) is also associated with differential impairments in the 1PP/3PP mental simulation of actions during adolescence. Twenty-two typically developing controls (Control group; 6 females), 12 hallucination-prone adolescents [auditory hallucination (AH) group; 7 females] and 13 adolescents with 22q11.2 Deletion Syndrome (22q11.2DS group; 4 females) were included in the study. During the fMRI task, subjects were presented with a cue (self-other priming cues) indicating to perform the task using either a ﬁrst person perspective (“you”-1PP) or a third person perspective (“best friend”-3PP) and then they were asked to mentally simulate actions based on the type of cue. Hallucination-proneness was assessed using a self-report questionnaire [Cardiff Anomalous Perception Scale (CAPS)]. Our results indicated that atypical patterns of cerebral activation, particularly in the key areas of self-other distinction, were found in both groups at risk for auditory hallucinations (AHs and 22q11.2DS). More precisely, adolescents in the AH group presented decreased activations in the right middle occipital gyrus BA19, left cingulate gyrus BA31, and right precuneus BA31 for the 3PP > 1PP contrast. Adolescents in the 22q11.2DS group presented decreased activations in the right superior occipital gyrus BA19, left caudate tail and left precuneus BA7 for the 3PP > 1PP contrast. In comparison to the Control group, only the 22q11.2DS adolescents showed a decreased activation for other-related cues (prime other > prime self contrast) in areas of visual imagery, episodic memory and social cognition. Tarik Dahoun1, Stephan Eliez 1,2, Fei Chen 1, Deborah Badoud1,3, Maude Schneider 1,3, Frank Larøi 4 and Martin Debbane1,3 1 Ofﬁce Médico-Pédagogique Research Unit, Department of Psychiatry, University of Geneva School of Medicine, Geneva, Switzerland 2 Department of Genetic Medicine and Development, University of Geneva School of Medicine, Geneva, Switzerland 3 Adolescence Clinical Psychology Research Unit, Faculty of Psychology and Educational Sciences, University of Geneva, Geneva, Switzerland 4 Department of Psychology University of Liège Liège Belgium This study characterizes the neural correlates of mental imagery for actions during adolescence, and suggests that a differential risk for hallucination-proneness (clinical vs. genetic) is associated to similar patterns of atypical activations in key areas sustaining self-other discrimination processes. These observations may provide relevant information for future research and prevention strategies with regards to hallucination-proneness during adolescence. Correspondence: Correspondence: Tarik Dahoun, Ofﬁce Médico-Pédagogique Research Unit, Department of Psychiatry, University of Geneva School of Medicine, 1 David Dufour, CP 50, 1211, Geneva 8, Switzerland e-mail: tarik dahoun@unige ch ORIGINAL RESEARCH ARTICLE bli h d 04 J l 2013 ORIGINAL RESEARCH ARTICLE bli h d 04 J l 2013 Keywords: auditory hallucinations, 22q11.2, action simulation, perspective-taking HUMAN NEUROSCIENCE published: 04 July 2013 doi: 10.3389/fnhum.2013.00329 Frontiers in Human Neuroscience Edited by: Edited by: Kelly M. J. Diederen, University of Cambridge, UK Edited by: Kelly M. J. Diederen, University of Cambridge, UK Cambridge, UK Reviewed by: Carrie E. Bearden, UCLA School of Medicine, USA Emma Barkus, University of Wollongong, Australia *Correspondence: Tarik Dahoun, Ofﬁce Médico-Pédagogique Research Unit, Department of Psychiatry, University of Geneva School of Medicine, 1 David Dufour, CP 50, 1211, Geneva 8, Switzerland e-mail: tarik.dahoun@unige.ch Reviewed by: Carrie E. Bearden, UCLA School of Medicine, USA Emma Barkus, University of Wollongong, Australia Reviewed by: Carrie E. Bearden, UCLA School of Medicine, USA INTRODUCTION However Larøi and collaborators used a SM task for actions and found evi- dence for misattribution of imagined actions in hallucinations- prone adults (Larøi et al., 2005). In this study, subjects were asked to (1) imagine themselves or (2) the experimenter performing an action (3) repeat the action statement without imaging the action or (4) simply observe the experimenter carrying out the action. The results revealed that hallucination-prone subjects more often remembered self-performed imagined actions as being imagined actions performed by the experimenter. A subsequent study employed a similar action-monitoring paradigm with adolescents affected by a 22q11.2 deletion syn- drome (22q11.2DS) (Debbane et al., 2008). 22q11.2DS is a neurogenetic disorder with an ultra-high risk for developing schizophrenia (Murphy et al., 1999; Karayiorgou et al., 2010). Transient psychotic experiences are characteristic of more than half of the adolescents with this syndrome (Baker and Skuse, 2005). Furthermore, AHs are the most commonly reported symp- toms in the sample of 22q11.2DS children and adolescents inves- tigated by our group (Debbane et al., 2006). The assessment of 22q11.2DS adolescents with a SM task adapted from Larøi et al. (2005), showed that adolescents with 22q11.2DS commit- ted more source confusions by recalling imagined-experimenter actions as actions they had mentally repeated (and vice versa), suggesting potential impairments in third person perspective (3PP) taking. Another study focused more speciﬁcally on the visuo-spatial aspects of perspective taking during action imagery (Jeannerod and Anquetil, 2008). The authors compared brain activity with PET while subjects imagined the same action (reaching and grasping a cylinder) from a 1PP and 3PP. This paradigm revealed increased activation in the parieto-occipital junction (BA19) speciﬁcally for the 3PP. The authors conclude that the right BA 19 is a key area for self-other differentiation by evaluating the difference in spatial localization between oneself and an other’s perspective. These two studies (Larøi et al., 2005; Debbane et al., 2008) highlight the impairments in ofﬂine SM for actions in two populations with hallucination-proneness. Their results might come from disturbances in how information is encoded between ﬁrst-person perspective (1PP) and 3PP. INTRODUCTION Theoretical explanations suggest that encoding processes during online representation of actions may promote subsequent confusion between self and other by two complementary aspects (1) increased salience of internal representations leading to exaggerated self-focused ori- entation (Ingram, 1990; Ensum and Morrison, 2003; Kapur, 2003; Perona-Garcelan et al., 2011) (2) impairments in the sense of agency, i.e., the ability to experience oneself as the agent of one’s own actions (Gallagher, 2000), as evoked by several authors (Schneider, 1959; Seal et al., 2004; Jones and Fernyhough, 2007; Asai and Tanno, 2012). Among the multiple neurocognitive mod- els of the sense of agency (David et al., 2008; Sperduti et al., 2011; Gallagher, 2012), Jeannerod and colleagues propose to dif- ferentiate between actions overtly executed and those that remain covert, i.e., internally represented (Jeannerod, 1994; Georgieff and Jeannerod, 1998). Self-other attribution of covert actions might be sustained by the activity of brain areas speciﬁcally devoted to self-other representations (Georgieff and Jeannerod, 1998; Jeannerod and Pacherie, 2004; Jeannerod, 2006). In summary key areas of self/other distinction for covert actions are thought to essentially engage the parietal cortex region for multi-modal integration and the parietal-temporal-occipital region, which underpins the shift to another location in space during perspective taking (Ruby and Decety, 2001, 2003, 2004; Vogeley and Fink, 2003; Jeannerod, 2004; David et al., 2007). In order to investigate the neural correlates underlying both self- and other-focused orientation and self-other perspective taking during action imagery, we used a functional magnetic reso- nance imagery paradigm adapted from Larøi et al. (2005). During this task subjects were ﬁrst primed with a self-other priming cues (namely “you” or “best friend”) and secondly were asked to men- tally simulate actions with either from a ﬁrst-person (1PP) or a third-person (best friend) perspective (3PP) in accordance with the priming cue. Typically developing adolescents and adoles- cents clinically prone to hallucinate (AH group) as well as with a 22q11.2 deletion syndrome (22q11.2DS group) underwent this task. This study has three aims: (1) delineate the neural correlates of action simulation in speciﬁc 1PP and 3PP during adolescence (2) identify potential impairments at a neurofunctional level in hallucination-prone adolescents (3) examine whether a differen- tial risk for schizophrenia (clinical vs. genetic) is also associated with differential impairments in the mental simulation of action during adolescence. INTRODUCTION investigating hallucination-proneness during these key devel- opmental windows may help better understand the onset of early AH. Auditory hallucinations (AHs) have been conceptualized as a neurodevelopmental phenomenon (Bentall et al., 2007) with a prevalence varying from 6 to 33% in adolescence (see review Larøi et al., 2006). A number of cognitive processes are thought to sus- tain the expression of AH, such as attention shift/enhancement, executive and inhibitory deﬁcits, and source monitoring (SM) (Hugdahl, 2009; Jones, 2010; Badcock and Hugdahl, 2012; Waters et al., 2012). The developmental course of these cogni- tive processes during childhood and adolescence suggests that SM is a processes associated with the development of AH. Impairments in SM are thought to lead to the misattribution of self-generated mental contents such as thoughts, memories or action to external sources in hallucination-prone adults (Bentall and Slade, 1985; Rankin and O’Carroll, 1995; Larøi et al., 2004, 2005), in adults with schizophrenia (Bentall et al., 1991; Rankin and O’Carroll, 1995; Blakemore et al., 2000; Brebion et al., 2000; Frontiers in Human Neuroscience July 2013 \| Volume 7 \| Article 329 \| 1 www.frontiersin.org www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. Brunelin et al., 2006) and in adolescents at high genetic risk for psychosis (22q11.2 Deletion Syndrome; Debbane et al., 2010). representations theory (Georgieff and Jeannerod, 1998; Grezes and Decety, 2001; Decety and Chaminade, 2003; Decety and Sommerville, 2003). However, speciﬁc regions were identiﬁed in the right inferior parietal, precuneus, posterior cingulate and frontopolar cortices for 3PP, and in the left inferior parietal and somatosensory cortices for 1PP. The authors concluded that the right inferior parietal, precuneus and somatosensory cortices are key areas involved in self/others action discrimination. The inferior parietal lobule is thought to be involved in body image, self-recognition and integration of information coming from sensory modalities and proprioceptive signals (Jeannerod and Pacherie, 2004; Torrey, 2007). Interestingly, increased activation in the inferior parietal lobule has been observed during conﬂict between a self-produced action and its consequences (Farrer et al., 2003). The anterior region of the precuneus is related to self-centered imagery and the posterior part to successful episodic memory retrieval (Cavanna and Trimble, 2006). According to the authors, the somatosensory cortex could play a role in self-representation (Ruby and Decety, 2001, 2003, 2004). Most of these studies used a verbal SM paradigm. Frontiers in Human Neuroscience www.frontiersin.org DESIGN AND PROCEDURE Before the scan session, the experimenter described the task to the participants (see Figure 1). The paradigm was adapted from Larøi et al. (2005) and included 60 actions to be mentally simu- lated (imagined) either from a 1PP or 3PP. Simple, universal and gender-neutral actions were chosen. All actions implied a move- ment and an object (for example take a picture, open a bottle, open a window, play the violin, brush your hair). 30 actions were tested with a 1PP and 30 with a 3PP, in the same randomized order for each participant. At the start of each session, the task’s instructions appeared on the screen as a reminder. Then, a cross appeared on the screen for 665 ms. Immediately after the self-other priming cue appeared for 1 s stating either “You” written in red or “Best friend” in blue. Then, the photo of an object accompanied by a written instruc- tion specifying the action to be imagined (i.e., play the violin, open a bottle, knock on a door) appeared for 1487 ms. Then the participants were reminded to either “Imagine yourself doing the action” or “Imagine your best friend doing the action” in accor- dance with the self-other priming cue. This instruction remained on the screen for a total of 4 s. An instruction in the center of the screen asked participants were asked to evaluate the difﬁculty of imagining the previous action, by pressing 1 (very easy) to 4 (very Out of the 22 subjects in the Control group (mean age: 16.00, SD = 2.04, 16 males), 6 were recruited within the siblings of 22q11.2DS participants and 16 from the Geneva state school system. In the AH group, 12 subjects with subclinical AHs (mean age: 15.97, SD = 2.12, 5 males) were recruited through patient associ- ations, by word of mouth or through the Child and Adolescents Outpatient Public Service (Ofﬁce Médico-Pédagogique). Subjects were selected on the basis of a positive answer (yes or no) on the Cardiff Anomalous Perceptions Scale (CAPS) items describ- ing AH items [i.e., items 3, 7, 11, 13, 28, or 32; (Bell et al., 2006; Debbané et al., 2011); see Table 1]. If they answered positively to Table 1 \| CAPS selected items for auditory hallucinations (Bell et al., 2006; Debbané et al., 2011). INTRODUCTION In order to identify the speciﬁc regions involved in the discrimination of self-other action simulation, Ruby and Decety (2001) employed positron emission tomography (PET) to com- pare the neural correlates of action simulation in a 1PP and a 3PP. Their results showed that both 1PP and 3PP involve overlapping areas of neural processing, in accordance with the shared neural We hypothesized that: (1) typically developing adolescents would activate speciﬁc regions devoted to 1PP and 3PP already observed in adult subjects (Ruby and Decety, 2001; Jeannerod July 2013 \| Volume 7 \| Article 329 \| 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. and Anquetil, 2008) (2) the AH group and the 22q11.2DS group would present atypical patterns of brain activation in regions sustaining self-other action simulation, along with confusion in self-other remembered actions. (3) Subjects with 22q11.2DS would present atypical patterns of activation in the parietal cor- tices due to functional and structural impairments (Simon et al., 2005b; Dufour et al., 2008; Bearden et al., 2009; Schaer et al., 2010; Debbane et al., 2012) whereas the AH group would exhibit atyp- ical activations in the prefrontal cortex, as suggested by previous SM studies (Vinogradov et al., 2008; Lagioia et al., 2011; Wang et al., 2011). an item, they were asked to rate their distress, the intrusiveness and the frequency of the experience by circling a number between 1 (not at all) and 5 (very). 1 In the 22q11.2DS group, all adolescents (mean age: 16.14, SD = 2.55, 9 males) were recruited through parent associations in France, Belgium and Switzerland. The 22q11.2 deletion was conﬁrmed using DNA polymorphism analysis based on short sequence repeats or by ﬂuorescence in situ hybridization per- formed on metaphase spreads spanning the deleted region. Written informed consent was accepted by all parents and/or subjects under protocols approved by the Institutional Review Board of the Geneva University School of Medicine. The three groups (Control, AH and 22q11.2DS) did not signiﬁcantly differ according to age and gender (p > 0.05). At the time of testing, no participants were receiving psychotropic medication (data for this was missing for one subjects in the 22q11.2DS group). All partic- ipants underwent the Block Design subtest (Kohs, 1920) in order to assess intellectual scores. PARTICIPANTS Eighty adolescents aged from 12 to 20 years participated in the study. Exclusion criteria included age, the presence of any neuro- logical problem or a diagnosis of schizophrenia or schizoaffective disorder according to DSM-IV-TR criteria. Thirty-two subjects were excluded for head movement exceeding 4.7 mm in any of the 6 directions during the scan sessions (Control group: N = 9, AH group: N = 6, 22q11.2DS group: N = 17). In the Control group, we excluded subjects with maladaptive functioning above the clinical cut-off of the Internalizing and Externalizing scales (t-score >64) in the Youth Self-Report and Adult Behavior Checklist (Achenbach, 1991, 1997) (N = 1). After excluding these 33 subjects, the 47 remaining youths were distributed in the following three groups: typically developing adolescents (Control group: N = 22), adolescents with transient AHs (AH group: N = 12) and adolescents with a 22q11.2 Deletion Syndrome (22q11.2DS group: N = 13). July 2013 \| Volume 7 \| Article 329 \| 3 DESIGN AND PROCEDURE Item 3: “Do you ever hear your own thoughts repeated or echoed?” Item 7: “Do you ever hear your own thoughts spoken aloud in your head, so that someone near might be able to hear them?” Item 11: “Do you ever hear voices commenting on what you are thinking or doing?” Item 13: “Do you ever hear voices saying words or sentences when there is no one around that might account for it?” Item 28: “Have you ever heard 2 or more unexplained voices talking with each other?” Item 32: “Do you ever hear sounds or music that people near you don’t hear?” CAPS mean sum selected items CAPS mean selected items distress CAPS mean selected items frequency CAPS mean selected items intrusiveness Control group 0 N/A N/A N/A AH group 2.25 (1.91) 2.66 (1.25) 1.77 (0.66) 2.78 1.10 22q11.2DS group 0.54 (0.47) 2.71 (1.89) 2.79 (1.81) 2.38 (1.60) Frontiers in Human Neuroscience www.frontiersin.org July 2013 \| Volume 7 \| Article 329 \| 3 Table 1 \| CAPS selected items for auditory hallucinations (Bell et al., 2006; Debbané et al., 2011). Action simulation, hallucinations, and adolescence Dahoun et al. FIGURE 1 \| Action simulation task adapted from Larøi et al. (2005). FIGURE 1 \| Action simulation task adapted from Larøi et al. (2005). We deﬁned two main conditions namely “self” and “other.” The “self” condition corresponds to the trials starting with the word “you” in the priming period and when imagining an action performed by oneself (action stimulation period). The “other” condition refers to the trials starting with the word “best friend” in the priming period and when imagin- ing an action performed by the best friend (action stimula- tion period). The return to baseline periods were set in the ITIs during which subjects saw a blank screen for 2990 ms to 5990 ms between each trial. In order to compare the speciﬁc areas devoted to the two different periods of the task (prime period and action simulation period) voxel value maps of t statis- tics were obtained for 4 contrasts: (1) prime self > prime other (2) prime other > prime self (3) 1PP > 3PP (4) 3PP > 1PP. These contrasts were performed for the following reasons. First, the prime self > prime other contrast will shield information on the neural correlates related to attention oriented to the self. Frontiers in Human Neuroscience fMRI DATA ACQUISITION A 3T Siemens TIM Trio system was used to acquire anatomical and functional images [TR (inter-trial between scan acquisition) = 2400 ms, Echo time (TE) = 30 ms, Slice thickness = 3.20 mm, Flip angle = 85◦, FOV 235 mm]. The functional scan session consisted of 380 volumes that comprised 38 slices oriented parallel to the AC-PC lines and collected in a descending sequence. High-resolution three-dimensional anatomical images were also obtained [TR (inter-trial between scan acquisition) = 2400 ms, TE = 30 ms, Slice thickness = 1.1 mm, Flip angle = 8◦, 192 coronal slices, FOV 220 mm]. DESIGN AND PROCEDURE In relation to our knowledge about overlapping activated brain regions for 1PP and 3PP-taking, some authors have sug- gested that 3PP requires “additional” areas in contrast to 1PP and vice-versa (Jeannerod, 2004, 2006; Jeannerod and Pacherie, 2004; Jeannerod and Anquetil, 2008). As such, it may be that hallucination-prone subjects fail to properly engage these areas, thereby increasing possible confusions between self and other. The two other contrasts (1PP > 3PP, 3PP > 1PP) follow the same logic, but when considering actual action imagery. T-maps were produced to identify atypical activation of the neural corre- lates sustaining self-other orientation and perspective taking for actions. hard) on a set of buttons on a console. This last step was used to make sure that the subjects performed the task. When a button was pressed, a blank screen appeared followed by the inter-trial interval (ITI) period, which lasted from 2990 to 5990 ms. fMRI DATA ANALYSIS We used Statistical Parametric Mapping (SPM) 8, (Welcome Department of Neuroscience, London, UK) to analyse the data. First of all images had to be spatially transformed during the pre-processing step in order to reduce movement effects or shape differences among a series of scans. We realigned every image with respect to the ﬁrst one. Then, slice timing correction was performed using the middle slice as a refer- ence. We co-registered structural images of each participant to the mean of the realigned functional images. Gray matter separation was established by segmentation of the anatomi- cal image. Thereafter, the normalization produced images that were warped to ﬁt to a standard Template brain. We normal- ized the realigned and slice-timed images into the Montreal Neurological Institute (MNI) template using 3 × 3 × 3 mm isotropic voxels. The images were spatially smoothed with an isotropic Gaussian smoothing Kernel of 6 mm full width half maximum (FWHM) to conform to inter-individual brain size variability. We ﬁrst performed a one-sample t-test to characterize typical activations in control adolescents, and then proceeded to group comparison analyses. Using a two-sample t-test (comparison between groups), we compared Control and AH groups, Control and 22q11.2DS groups, and ﬁnally 22q11.2DS and AH groups. S{T} maps were obtained with a threshold of p < 0.05 and an extend threshold k of 20 voxels. Cluster level peak functional activity at p < 0.05 (Family-Wise corrected) was then localized on a mean structural scan with approximate Brodmann areas esti- mated from the Talairach and Tournoux (1988) atlas after having After pre-processing, the brain responses of each subject were estimated at every voxel using a general linear mode. July 2013 \| Volume 7 \| Article 329 \| 4 www.frontiersin.org www.frontiersin.org www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. converted coordinates from MNI to Talairach templates (http:// www.bioimagesuite.org/Mni2Tal/index.html). Age was entered as a covariate in each analysis without any signiﬁcant effects on the results obtained. diagnosis [F(2, 44) = 1.875, p = 0.165], a signiﬁcant effect of con- dition [F(1, 44) = 13.315, p = 0.001∗∗∗] (mean evaluation for self condition = 1.6820, SD = 0.40360), (mean evaluation for other condition = 1.8504, SD = 0.47230) and a non-signiﬁcant interaction between diagnosis and conditions [F(2, 44) = 0.752, p = 0.477]. BEHAVIORAL RESULTS Differences regarding evaluation results between the three groups and the two different conditions were analysed using a repeated-measures ANOVA 3(groups) × 2(conditions) with post-hoc Tukey analyses. Action simulation period. The 3PP > 1PP contrast was associ- ated with activations in a ﬁrst cluster (4098 voxels, p = 0.000) Action simulation period. The 3PP > 1PP contrast was associ- ated with activations in a ﬁrst cluster (4098 voxels, p = 0.000) With regard to the evaluation of difﬁculty ratings (see Table 2), our 3 × 2 ANOVA yielded a non-signiﬁcant effect of Table 2 \| Evaluation, response time, and Block DESD in each group. Control group (N = 22) AH group (N = 12) 22q11.2DS group (N = 13) Evaluation other 1.72 (0.38) 2.06 (0.45) 1.87 (0.57) Evaluation self 1.59 (0.33) 1.79 (0.34) 1.74 (0.53) Evaluation total 1.67 (0.32) 1.92 (0.37) 1.79 (0.53) Answer time other 1142.24 (446.29) 1082.13 (294.93) 1118.20 (351.85) Answer time self 1135.46 (425.56) 992.64 (266.31) 1076.48 (306.47) Answer time total 1138.93 (425.45) 1037.40 (261.78) 1097.20 (303.49) Block DESD 11.5 (3.25) 11.75 (2.53) 4.86 (2.73) Table 3 \| Regions of peak activations in the Control group. Contrast Cluster level - p -FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime other > Prime self 0.001 2623 Right Occipital lobe, cuneus BA18 5.10 3, −76, 19 0.001 Right Limbic lobe, posterior cingulate BA30 5.06 9, −67, 10 0.001 Left Occipital lobe, cuneus BA17 4.72 −21, −82, 13 0.004 2272 Right Frontal lobe, superior frontal gyrus BA6 5.04 6, 32, 64 0.004 Left Frontal lobe, middle frontal gyrus BA46 4.07 −51, 29, 19 0.004 Right Frontal lobe, superior frontal gyrus BA6 3.90 21, 26, 64 3PP > 1PP 0.000 4098 Right Limbic lobe, cingulate gyrus BA23 4.90 3, −31, 28 0.000 Right Occipital lobe, cuneus BA18 4.55 6, −73, 16 0.000 Left Occipital lobe, middle occipital gyrus BA18 4.52 −21, −85, 16 0.025 1692 Left Frontal lobe, precentral gyrus BA6 3.69 −39, 2, 40 0.025 Left Frontal lobe, superior frontal gyrus BA6 3.68 −3, 17, 67 0.025 Left Frontal lobe, superior frontal gyrus BA9 3.57 −18, 41, 43 Table 2 \| Evaluation, response time, and Block DESD in each group. Table 3 \| Regions of peak activations in the Control group. Table 3 \| Regions of peak activations in the Control group. July 2013 \| Volume 7 \| Article 329 \| 5 Frontiers in Human Neuroscience Control group Prime period. The prime other > prime self contrast was associated with activations in a ﬁrst cluster (2623 vox- els, p = 0.001), including signiﬁcant activations in the right cuneus BA18, right posterior cingulate BA30 and left cuneus BA17 (see Table 3). A second cluster (2272 voxels, p = 0.004) included signiﬁcant activations in the right supe- rior frontal gyrus BA6, left middle frontal gyrus BA46 and right superior frontal gyrus BA6 (see Table 3). No signif- icant results were obtained in the prime self > prime other contrast. fMRI DATA ANALYSIS For post-hoc examination of potential associations between hallucination-proneness scores and activations resulting from group comparisons, we planned to extract local brain activity of regions of interest (ROIs) using SPM8 toolbox Marsbar (http:// marsbar.sourceforge.net/). The ROIs were delimited around the peak of signiﬁcant activations in prime self > prime other, prime other > prime self, 1PP > 3PP, 3PP > 1PP contrasts for the group comparisons. A 5 mm radius sphere was deﬁned around the cen- ter of mass for each subject to extract Beta Values. We performed Pearson correlations between Beta Values obtained for different ROIs and CAPS components for each subjects (AHs distress, intrusiveness, frequency, total scores, as well as subscale scores) (Bell et al., 2006; Debbané et al., 2011). With regard to response time (see Table 2), results yielded a non-signiﬁcant effect of diagnosis [F(2, 44) = 0.319, p = 0.729], a non-signiﬁcant effect of condition [F(1, 44) = 2.054, p = 0.159] and a non-signiﬁcant interaction between diagnosis and condi- tion [F(2, 44) = 0.593, p = 0.557]. www.frontiersin.org Comparisons between 22q11.2DS and AH Comparisons between 22q11.2DS and AH Prime period. We observed signiﬁcant results for the AH > 22q11.2DS comparison (see Table 6). The prime self > prime other contrast was associated with activations in a single clus- ter (1468 voxels, p = 0.041) with signiﬁcant activations in the left caudate body, right anterior cingulate gyrus BA32 and right superior frontal gyrus BA 10. The prime other > prime self contrast was associated with activations in a single cluster (1696 voxels, p = 0.020) with signiﬁcant acti- vations in the right postcentral gyrus BA3, left superior frontal gyrus BA10 and right superior frontal gyrus BA8. No signiﬁcant clusters were detected in the action simulation period. NEUROIMAGING RESULTS: GROUP COMPARISONS Comparisons between control and AH groups Action simulation period. We observed signiﬁcant results for the Control > AH comparison in the action simulation period (see Table 4). Speciﬁcally, the 3PP > 1PP contrast was asso- ciated with activations in a single cluster (5569 voxels, p = 0.000) including signiﬁcant activations in the left middle occip- ital gyrus BA19, left cingulate gyrus BA31 and in the right precuneus BA31. Comparisons between control and 22q11.2DS groups Prime period. We observed signiﬁcant results for the Control > 22q11.2DS comparison (see Table 5). The prime other > prime self contrast was associated with activations in a single cluster (2716 voxel, p = 0.001) with signiﬁcant activations in the left cuneus BA18, left precuneus BA31, right middle temporal gyrus BA39. ROIs analyses Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) 3PP > 1PP 0.000 5569 Right Occipital lobe, middle occipital gyrus 19 4.42 33, −76, 19 0.000 Left Limbic lobe, cingulate gyrus 31 4.17 0, −37, 31 0.000 Right Occipital lobe, precuneus 31 4.09 24, −79, 31 Table 5 \| Regions of peak activations for group comparisons Control > 22q11.2DS. Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime other > Prime self 0.001 2716 Left Occipital lobe, cuneus BA18 4.52 −6, −82, 19 0.001 Left Parietal lobe, precuneus BA31 4.13 −18, −73, 25 0.001 Right Temporal lobe, middle temporal gyrus BA39 3.98 30, −67, 22 3PP > 1PP 0.000 7020 Right Occipital lobe, superior occipital gyrus BA19 5.37 36, −76, 25 0.000 Left Sub-lobar, caudate, caudate tail 4.93 −18, −25, 19 0.000 Left Parietal lobe, precuneus BA7 4.92 −21, −73, 31 Table 6 \| Regions of peak activations for group comparisons AH > 22q11.2DS. Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime self > 0 041 1468 Left Sub lobar caudate caudate body 3 79 12 26 16 Table 5 \| Regions of peak activations for group comparisons Control > 22q11.2DS. Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime other > Prime self 0.001 2716 Left Occipital lobe, cuneus BA18 4.52 −6, −82, 19 0.001 Left Parietal lobe, precuneus BA31 4.13 −18, −73, 25 0.001 Right Temporal lobe, middle temporal gyrus BA39 3.98 30, −67, 22 3PP > 1PP 0.000 7020 Right Occipital lobe, superior occipital gyrus BA19 5.37 36, −76, 25 0.000 Left Sub-lobar, caudate, caudate tail 4.93 −18, −25, 19 0.000 Left Parietal lobe, precuneus BA7 4.92 −21, −73, 31 Table 5 \| Regions of peak activations for group comparisons Control > 22q11.2DS Table 6 \| Regions of peak activations for group comparisons AH > 22q11.2DS. July 2013 \| Volume 7 \| Article 329 \| 6 ROIs analyses No signiﬁcant results were obtained for Pearson correlations between T-values activations in Control > AH, Control > 22q11.2DS and AH > 22q11.2DS group comparisons and CAPS subscales scores for each subjects. Table 4 \| Regions of peak activations for group comparisons Control > AH. Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) 3PP > 1PP 0.000 5569 Right Occipital lobe, middle occipital gyrus 19 4.42 33, −76, 19 0.000 Left Limbic lobe, cingulate gyrus 31 4.17 0, −37, 31 0.000 Right Occipital lobe, precuneus 31 4.09 24, −79, 31 Table 5 \| Regions of peak activations for group comparisons Control > 22q11.2DS. Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime other > Prime self 0.001 2716 Left Occipital lobe, cuneus BA18 4.52 −6, −82, 19 0.001 Left Parietal lobe, precuneus BA31 4.13 −18, −73, 25 0.001 Right Temporal lobe, middle temporal gyrus BA39 3.98 30, −67, 22 3PP > 1PP 0.000 7020 Right Occipital lobe, superior occipital gyrus BA19 5.37 36, −76, 25 0.000 Left Sub-lobar, caudate, caudate tail 4.93 −18, −25, 19 0.000 Left Parietal lobe, precuneus BA7 4.92 −21, −73, 31 Table 6 \| Regions of peak activations for group comparisons AH > 22q11.2DS. Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime self > Prime other 0.041 1468 Left Sub-lobar, caudate, caudate body 3.79 −12, 26, 16 0.041 Right Limbic lobe, anterior cingulate BA32 3.67 15, 32, −8 0.041 Right Frontal lobe, superior frontal gyrus BA10 3.24 24, 59, 10 Prime other > Prime self 0.020 1696 Right Parietal lobe, postcentral gyrus BA3 3.22 66, −19, 37 0.020 Left Frontal lobe, superior frontal gyrus BA10 2.88 −12, 71, 16 0.020 Right Frontal lobe, superior frontal gyrus BA8 2.84 6, 38, 52 Frontiers in Human Neuroscience www.frontiersin.org July 2013 \| Volume 7 \| Article 329 \| 6 Table 4 \| Regions of peak activations for group comparisons Control > AH. Frontiers in Human Neuroscience BEHAVIORAL RESULTS Contrast Cluster level - p -FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime other > Prime self 0.001 2623 Right Occipital lobe, cuneus BA18 5.10 3, −76, 19 0.001 Right Limbic lobe, posterior cingulate BA30 5.06 9, −67, 10 0.001 Left Occipital lobe, cuneus BA17 4.72 −21, −82, 13 0.004 2272 Right Frontal lobe, superior frontal gyrus BA6 5.04 6, 32, 64 0.004 Left Frontal lobe, middle frontal gyrus BA46 4.07 −51, 29, 19 0.004 Right Frontal lobe, superior frontal gyrus BA6 3.90 21, 26, 64 3PP > 1PP 0.000 4098 Right Limbic lobe, cingulate gyrus BA23 4.90 3, −31, 28 0.000 Right Occipital lobe, cuneus BA18 4.55 6, −73, 16 0.000 Left Occipital lobe, middle occipital gyrus BA18 4.52 −21, −85, 16 0.025 1692 Left Frontal lobe, precentral gyrus BA6 3.69 −39, 2, 40 0.025 Left Frontal lobe, superior frontal gyrus BA6 3.68 −3, 17, 67 0.025 Left Frontal lobe, superior frontal gyrus BA9 3.57 −18, 41, 43 www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. including activations in the right cingulate gyrus BA23, right cuneus BA18 and left middle occipital gyrus BA18. A second clus- ter (1692 voxels, p = 0.025) included signiﬁcant activations in the left precentral gyrus BA6, left superior frontal gyrus BA6 and left superior frontal gyrus BA9. No signiﬁcant results were obtained in the 1PP > 3PP contrast. Action simulation period. The 3PP > 1PP contrast was associated with activations in a single cluster (7020 voxels, p = 0.000) with signiﬁcant activations in the right superior occipital gyrus BA19, left caudate tail and left precuneus BA7 (see Table 5). DISCUSSION remembering familiar objects and places (Sugiura et al., 2005) and autobiographical memory (Summerﬁeld et al., 2009; Van Der Meer et al., 2010). The mental simulation of actions may involve the retrieval of memorized visual representations (Farah, 1984; Annett, 1995) of the imaginary action. This study is the ﬁrst to compare neural correlates in self- other priming cues and action simulation using a 1PP or 3PP in typically developing adolescents (Control group), ado- lescents with transient AHs (AH group) and adolescents at genetic risk for schizophrenia (22q11.2DS group). The three objectives of this study were (1) to delineate the neural cor- relates sustaining mental simulation of actions involving 1PP and 3PP during adolescence; (2) to identify potential atypi- cal neural activations during self-other priming and/or action simulation in hallucination-prone adolescents; (3) to exam- ine whether differential risk for hallucination-proneness (clini- cal vs. genetic) is also associated with differential impairments in self-related cues and in action simulation. Our ﬁndings showed that (1) the Control group activated the key areas involved in other related cues when primed for their best friend compared to themselves, and in action simulation performed by others; (2) in the 3PP condition both hallucination-prone groups exhibited decreased activation in the parieto-occipital region, which has been related to self-other distinction of imag- ined actions (Jeannerod and Anquetil, 2008); (3) the priming period for both self and other related cues showed decreased activations in subjects with 22q11.2DS compared to those at clinical risk. Areas in the parieto-occipital region lobe are activated when processing visuo-spatial information in the context of action rep- resentation (Kilintari et al., 2011), object-distance representation (Berryhill and Olson, 2009), including position and prediction of moving objects (Maus et al., 2010), coherent moving visual motion (McKeefry et al., 1997; Braddick et al., 2001), and motor imagery of hand action (Willems et al., 2009). The 3PP contrast might engage supplementary areas devoted to motion and visuo- spatial information, as an other’s perspective implies a shift in visual-spatial perspective (Vogeley et al., 2004). Activations in the frontal lobe could reﬂect the self-relevance evaluation of the prime period and the motor cognition aspect of action simulation. For the prime other > prime self contrast, we found signiﬁcant increased activations in the right superior frontal part of the medial prefrontal cortex and the left dorso- lateral prefrontal cortex DLPFC (Mayka et al., 2006; Northoff et al., 2006; Murray et al., 2012). DISCUSSION These results are consistent with recent ﬁndings on self-other related processes in healthy adults. According to a recent meta-analysis, the dorsomedial prefrontal cortex DMPFC, DLPFC, and PCC act together in the evalua- tion and decision-making processes of self versus other relevant information (Van Der Meer et al., 2010). Control group activation patterns during prime and action simulation periods will ﬁrst be discussed. Then, the unique acti- vations in the AH group and the 22q11.2DS group will be brought into consideration, followed by a discussion concerning the differences between the two hallucination-prone groups. For the action simulation period, we obtained signiﬁcant results in the ventral part of the dorsal premotor cortex (Grezes and Decety, 2001; Mayka et al., 2006), the left pre-supplementary motor area (pre-SMA) (Mayka et al., 2006) and the DLPFC. These regions could be recruited by the task’s motor aspects. It has been shown that the ventral part of the dorsal premo- tor cortex plays a role in motor preparation (Hoshi and Tanji, 2007), the pre-SMA in maintaining an action representation (Stadler et al., 2011) and the DLPFC in the cognitive control of motor behavior (Passingham, 1993; Hoshi, 2006; Cieslik et al., 2012). ROIs analyses Contrast Cluster level - P FEW-corr Cluster level - Ke - voxels Side Brain regions activation Brodmann area T-value X, Y, Z (MNI) Prime self > Prime other 0.041 1468 Left Sub-lobar, caudate, caudate body 3.79 −12, 26, 16 0.041 Right Limbic lobe, anterior cingulate BA32 3.67 15, 32, −8 0.041 Right Frontal lobe, superior frontal gyrus BA10 3.24 24, 59, 10 Prime other > Prime self 0.020 1696 Right Parietal lobe, postcentral gyrus BA3 3.22 66, −19, 37 0.020 Left Frontal lobe, superior frontal gyrus BA10 2.88 −12, 71, 16 0.020 Right Frontal lobe, superior frontal gyrus BA8 2.84 6, 38, 52 Frontiers in Human Neuroscience www frontiersin org July 2013 \| Volume 7 \| Article 329 \| 6 Table 6 \| Regions of peak activations for group comparisons AH > 22q11.2DS. Action simulation, hallucinations, and adolescence Dahoun et al. SIMILARITIES AND DIFFERENCES FOR THE AH GROUP AND THE 22q11.2DS GROUP COMPARED TO CONTROL GROUP Both hallucination-prone groups showed signiﬁcant decreased activations for the 3PP > 1PP contrast compared to the control group. The AH group and the 22q11.2DS group presented an atypical pattern of activations in the parieto-occipital region with signiﬁcant decreased activations in the occipital gyrus BA19 and the precuneus. As reviewed above, it has been shown that the right superior occipital BA19 is speciﬁcally devoted to 3PP in an action imagery task focusing on a visuo-spatial perspective switch (Jeannerod and Anquetil, 2008). According to the authors, the mental sim- ulation of actions performed by others ﬁrst occurs through a shift in space in order to mentally represent the other’s place, and is then followed by the action simulation per se. In this framework, BA 19 would be a key area for self-other distinc- tion by evaluating the difference in spatial localization between oneself and someone else. This interpretation is supported by data demonstrating the role of BA19 in the manipulation of spa- tial relationships between objects (Haxby et al., 1991; Kosslyn et al., 1998) and further conﬁrmed by a meta-analysis (Zacks, 2008). Clinical studies have also shown that posterior parietal lesions provoke visuo-spatial dysfunction (Mendez, 2001; Harvey and Rossit, 2012) or disturbances in the capacity to represent relative location of objects with respect to the subject (Aguirre and D’Esposito, 1999). Recent evidence shows that the parieto- occipital junction responds to both gaze- and body-centered representation when reaching a target visually presented (Bernier and Grafton, 2010). This could be an argument in favor of a gaze and body reference computed by parieto-occipital junction during shift in 3PP. From a structural point of view, a signiﬁcant volume reduc- tion of the parietal lobe has been described in 22q11.2DS (Schaer et al., 2010). Results from functional connectivity also show atyp- ical connectivity involving the left precuneus and PCC regions during resting state (Debbane et al., 2012). Concerning patients with hallucinations however, no clear alterations of the precuneus have been identiﬁed as far as we know (Allen et al., 2008). In addition to the BA 19 and posterior parietal similarities, the at-risk groups showed unique differences in comparison to the controls. Compared to the control group decreased activation in the left parieto-occipital junction and the right posterior tempo- ral BA39 was found in the 22q11.2DS group, but not in the AH group. This ﬁnding might correspond to a diminished salience for other related cues in 22q11.2DS. CONTROL GROUP Typically developing adolescents showed signiﬁcant increased activations for the “other” condition compared to the “self” condition in both the prime and the action simulation periods. For the 3PP, we found increased activations in the PCC and the parieto-occipital regions (see Figure 2). These regions may underlie the inﬂuence of visuo-spatial components and episodic memory when adolescents imagine actions performed by their best friend. PCC is involved in the processing of familiar stim- uli (Qin and Northoff, 2011; Qin et al., 2012), and it has been shown that it plays an important role in memory tasks such as remembering familiar people (Maddock et al., 2001), In summary, our results showed that the mental simula- tion of actions performed by others engage increased activa- tions in the posterior midline structure including PCC and the parieto-occipital region. Our results may reﬂect the visuo-spatial FIGURE 2 \| Activations during 3PP > 1PP contrast in Control group at a statistical threshold of p < 0.05. Slice views at MNI coordinates (x = 0, y = −70, z = 25). The bar on the left shows the range of T-values. FIGURE 2 \| Activations during 3PP > 1PP contrast in Control group at a statistical threshold of p < 0.05. Slice views at MNI coordinates (x = 0, y = −70, z = 25). The bar on the left shows the range of T-values. July 2013 \| Volume 7 \| Article 329 \| 7 Frontiers in Human Neuroscience Frontiers in Human Neuroscience www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. during 3PP. The precuneus responds to a wide range of cog- nitive processes including internal self-representation, episodic memory retrieval, visuo-spatial imagery, 1PP and agency pro- cesses (Cavanna and Trimble, 2006). The anatomical and con- nectivity data reviewed by them converges toward a functional subdivision between the anterior (y closer to −60 mm) and pos- terior (y closer to −70 mm) precuneus (Cavanna and Trimble, 2006). Our results for both the AH group and the 22q11.2DS group correspond to a decreased activation in the posterior region. Importantly when considering left and right disparity between the AH and 22q11.2DS groups no evidence of inter- hemispheric specialization emerged. Whereas the anterior region responds to self-centered mental imagery strategies, the poste- rior region is involved in successful episodic memory retrieval (Cabeza and Nyberg, 2000; Cavanna and Trimble, 2006). CONTROL GROUP Episodic memory relies on the ability to remember past experiences (Tulving, 1972) with autobiographical references (Tulving, 1983) and plays a role in mental imagery (Tulving, 1983; Cabeza and Nyberg, 2000; Rubin et al., 2003; Daselaar et al., 2008). Clinical data have pointed out to a link between impaired episodic memory and auditory verbal hallucinations (Seal et al., 2004; Badcock et al., 2005; Berenbaum et al., 2008; Daselaar et al., 2008). and episodic memory components of self-other discrimination for imagined actions. and episodic memory components of self-other discrimination for imagined actions. SIMILARITIES AND DIFFERENCES FOR THE AH GROUP AND THE 22q11.2DS GROUP COMPARED TO CONTROL GROUP As mentioned in the last section, posterior parietal cortex, especially the posterior part of the pre- cuneus, is particularly involved in successful retrieval of episodic memory (Wagner et al., 2005; Cavanna and Trimble, 2006; Elman et al., 2013) and in remembering familiar people (Maddock et al., 2001). The right temporo-occipital region (BA39) has been impli- cated in face processing (Puce et al., 1995; Dichter et al., 2009) and in the increased attention to salient social information because of its interactive processing with emotional information (Norris et al., 2004). Interestingly, it has recently been shown that the right temporo-occipital region presents decreased activation in response to affective social versus affective non-social images in schizophrenia (Bjorkquist and Herbener, 2013). Clinical data indicates that the 22q11.2DS syndrome exposes to an increased risk of social withdrawal, poor social functioning and emotion recognition deﬁcits (Baker and Skuse, 2005; Debbane et al., 2006; Campbell et al., 2009). Concerning the 22q11.2DS, Bearden et al. (2009) interestingly detected an decreased cortical thickness in the right parieto- occipital cortex, while to our knowledge, no clear structural alter- ations have been identiﬁed in this region in hallucination-prone subjects (Allen et al., 2008). Moreover, it has been shown that children with 22q11.2DS tend to present signiﬁcant decreased activation in the parietal and occipital lobe during a visuo-spatial working memory task (Azuma et al., 2009). From a clinical point of view, visuo-spatial impairments have been extensively reported in the syndrome (Wang et al., 2000, 2011; Simon et al., 2005a; Jacobson et al., 2010). Together, these ﬁndings argue in favor of an atypical neuro-development of the parieto-occipital region in 22q11.2DS, which could lead to deﬁcits in visuo-spatial perspective shifting in actions with objects. The second region showing decreased activation during 3PP > 1PP contrast for both hallucination-prone groups was located in the right precuneus BA 31 (AH group) and the left pre- cuneus BA7 (22q11.2DS group). In the PET study previously mentioned, Ruby and Decety (2001) found stronger activation bilaterally in the precuneus for 3PP > 1PP, and thus considered the region as speciﬁcally involved in distinguishing self and other action imagery. Frontiers in Human Neuroscience SIMILARITIES AND DIFFERENCES FOR THE AH GROUP AND THE 22q11.2DS GROUP COMPARED TO CONTROL GROUP According to their view, the precuneus would play a role in the self’s representation with an overactivation In summary, our results show that both groups at risk (clin- ical and genetic) for hallucinations exhibited decreased activa- tion in the parieto-occipital region during 3PP compared to the Control group (see Figures 3 and 4), which has been related to July 2013 \| Volume 7 \| Article 329 \| 8 www.frontiersin.org www.frontiersin.org www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. FIGURE 3 \| Activations during 3PP > 1PP contrast in group comparisons (Control group > AH group) at a statistical threshold of p<0.05. Slice views at MNI coordinates (x = 0, y = −70, z = 25). The bar on the left shows the range of T-values. FIGURE 4 \| Activations during 3PP > 1PP contrast in group comparisons (Control group > 22q11.2DS group) at MNI coordinates (x = 0, y = −70, z = 25). Statistical threshold of p<0.05. The bar on the left shows the range of T-values. FIGURE 3 \| Activations during 3PP > 1PP contrast in group comparisons (Control group > AH group) at a statistical threshold of p<0.05. Slice views at MNI coordinates (x = 0, y = −70, z = 25). The bar on the left shows the range of T-values. FIGURE 3 \| Activations during 3PP > 1PP contrast in group comparisons (Control group > AH group) at a statistical threshold of p<0.05. Slice views at MNI coordinates (x = 0, y = −70, z = 25). The bar on the left shows the range of T-values. FIGURE 4 \| Activations during 3PP > 1PP contrast in group comparisons (Control group > 22q11.2DS group) at MNI coordinates (x = 0, y = −70, z = 25). Statistical threshold of p<0.05. The bar on the left shows the range of T-values. FIGURE 4 \| Activations during 3PP > 1PP contrast in group comparisons (Control group > 22q11.2DS group) at MNI coordinates (x = 0, y = −70, z = 25). Statistical threshold of p<0.05. The bar on the left shows the range of T-values. midline structure and especially the anterior cingulate cortex in self-speciﬁc stimuli processing (Van Der Meer et al., 2010; Murray et al., 2012; Qin et al., 2012). SIMILARITIES AND DIFFERENCES FOR THE AH GROUP AND THE 22q11.2DS GROUP COMPARED TO CONTROL GROUP It has also been shown that the caudate nucleus and the anterior cingulate cortex are engaged in reward and personal relevance, i.e., valuing external and internal stimuli with regard to their meaning for the subject (Enzi et al., 2009). self-other distinction of imagined actions. We suggest that an impaired shift perspective and/or episodic memory dysfunctions might alter self-other distinction in hallucination-prone subjects. Consequently, the lack of reliable representations of the actions performed by others could account for SM action impairments previously observed by Larøi et al. (2005) and Debbane et al. (2008). Our results also argue in favor of a decreased salience toward others in the 22q11.2DS, as illustrated by the decreased activations in regions sustaining social cognition and episodic memory. Our results might therefore reﬂect a decreased salience toward self-related cues in the 22q11.2DS compared to the AH group. The differences between the two groups could be related to neuro- structural alterations in the 22q11.2DS. Indeed reduced volume grey matter and cortical thickness have been described in the anterior cingulate cortex (Dufour et al., 2008; Bearden et al., 2009) and several studies have shown an increased volume of the caudate nucleus (Eliez et al., 2002; Kates et al., 2004; Gothelf et al., 2007). However according to several studies these regions are relatively spared in adolescents and adults with schizotypal traits (Spencer et al., 2007; Moorhead et al., 2009; Ettinger et al., 2012). Frontiers in Human Neuroscience www.frontiersin.org REFERENCES groups. Neurosci. Biobehav. Rev. 36, 431–438. doi: 10.1016/j.neubiorev. 2011.07.010 groups. Neurosci. Biobehav. Rev. 36, 431–438. doi: 10.1016/j.neubiorev. 2011.07.010 validated measure of anoma- lous perceptual experience. Schizophr. Bull. 32, 366–377. doi: 10.1093/schbul/sbj014 validated measure of anoma- lous perceptual experience. Schizophr. Bull. 32, 366–377. doi: 10.1093/schbul/sbj014 Biobehav. Rev. 32, 175–191. doi: 10.1016/j.neubiorev.2007.07.012 Biobehav. Rev. 32, 175–191. doi: 10.1016/j.neubiorev.2007.07.012 Achenbach, T. (1991). Manual for the Youth Self-Report and 1991 Proﬁle. Burlington, VT: University of Vermont, Department of Psychiatry. 431–438. doi: 10.1016/j.neubiorev. 2011.07.010 Achenbach, T. (1991). 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Visuospatial work- ing memory in children and adolescents with 22q11.2 dele- tion syndrome; an fMRI study. J. Neurodev. Disord. 1, 46–60. doi: 10.1007/s11689-009-9008-9 Bearden, C. E., Van Erp, T. G., Dutton, R. A., Lee, A. D., Simon, T. J., Cannon, T. D., et al. (2009). ACKNOWLEDGMENTS We wish to thank all the participants and their families who kindly volunteered for this study. We extend our special thanks to Sarah Menghetti, Marine Lazouret and Eleonora Rizzi for their help with data collection, processing and analysis. This research was supported by research grants from the Swiss National Science Foundation (100014-135311/1) to Martin Debban and (PP00B- 102864) to Stephan Eliez, by the National Center of Competence in Research (NCCR) “Synapsy-The Synaptic Bases of Mental Diseases” (SNF, Grant number: 51AU40_125759) to Stephan Eliez and Tarik Dahoun, as well as from the Gertrude Von Meissner Foundation (ME 7871) to Stephan Eliez and Martin Debbane. These funding bodies had no role in the analysis and interpretation of data, in the writing of the manuscript, or in the decision concering submission for publication. CONCLUSION This study constitutes the preliminary step of a neuroscientiﬁc examination targeting the neural correlates of self-other discrimi- nation in mental imagery for hallucination-proneadolescents. We suggest that impairment in the capacity to shift perspective and/or episodic memory dysfunction may alter self-other distinction in hallucination-prone subjects. In summary, in comparison to subjects at clinical risk, ado- lescents with 22q11.2DS showed atypical patterns of activations when primed for themselves and their best friend. More pre- cisely, decreased activations were found in regions involved in self-relevance, emotion processing and attribution. AT-RISK GROUPS: DIFFERENCES BETWEEN THE AH GROUP AND THE 22q11.2DS GROUP We only obtained results for the prime condition when compar- ing the AH group and the 22q11.2DS group. This comparison indicated that the salience of self-other priming cues was differ- ent between the two groups at-risk for hallucinations, whereas no signiﬁcant ﬁndings emerged for the 1PP and 3PP contrasts. Compared to the AH group, adolescents with 22q11.2DS exhibited decreased activations of the caudate body, anterior cin- gulate BA32 and right superior frontal BA 10 for the prime self > prime other contrast. In line with our results, a signiﬁ- cant lower level of activation was found in the caudate nucleus and the anterior cingulate cortex during self-reﬂective processing in adolescents with 22q11.2DS (Schneider et al., 2012). Several meta-analyses have highlighted the role of the anterior cortical Compared to the AH group, adolescents with 22q11.2DS exhibited decreased activations in the right postcentral gyrus BA3 (somatosensory cortex S1) and anterior prefrontal cortex BA10 for prime other > prime self-contrast. The decreased activation of the somatosensory cortex in the 22q11.2DS group for the prime other > prime self contrast is in contradiction with previous work indicating that this region responds speciﬁcally to 1PP July 2013 \| Volume 7 \| Article 329 \| 9 www.frontiersin.org www.frontiersin.org www.frontiersin.org Action simulation, hallucinations, and adolescence Dahoun et al. (Ruby and Decety, 2001, 2003, 2004). In our task however the role of the right somatosensory cortex and the anterior pre- frontal cortex BA10 might be related respectively to emotion processing (Adolphs et al., 2000; Pourtois et al., 2004; Hooker et al., 2008; Saxbe et al., 2012) and mental states attribution (Gilbert et al., 2006; Burgess et al., 2007; Benoit et al., 2010). The reduced activations in these regions are in line with clinical evi- dence showing impairments in cognitive theory of mind tasks in 22q11.2DS (Chow et al., 2006; Campbell et al., 2011; Ho et al., 2012). children and adults data or data with longitudinal follow-up. More research exploring shift perspective and agency processes may further contribute to a better understanding of action misat- tribution biases in hallucination-prone subjects. LIMITATIONS The present study must be considered with limitations. First, the restricted sample sizes make it difﬁcult to completely exclude the absence of signiﬁcant results for the 1PP > 3PP contrasts. Future studies with increased statistical power could address this issue. Concerning the group selection, the 22q11.2DS group had lower IQ scores compared to the Control group. However, the behav- ioral results showed that response times and difﬁculty ratings did not signiﬁcantly differ between groups. This suggests that 22q11.2DS subjects were not put in a more difﬁcult position due to the intellectual deﬁcits they might present. The functional imaging paradigm did not include a cognitive control for the prime and action simulation period other than the perspective-taking variants, which could be included in a future version of this paradigm. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://www.frontiersin.org/HumanNeuroscience/10. 3389/fnhum.2013.00329/abstract The Supplementary Material for this article can be found online at: http://www.frontiersin.org/HumanNeuroscience/10. 3389/fnhum.2013.00329/abstract Future studies should address the neurodevelopmental issues of action simulation during adolescence by also comparing REFERENCES REFERENCES Alterations in midline cortical thickness and gyriﬁcation patterns mapped in children with 22q11.2 deletions. Cereb. Cortex 19, 115–126. doi: 10.1093/cercor/bhn064 Bentall, R. P., Fernyhough, C., Morrison, A. P., Lewis, S., and Corcoran, R. (2007). Prospects for a cognitive-developmental account of psychotic experiences. Br. J. Clin. Psychol. 46, 155–173. doi: 10.1348/014466506X123011 Aguirre, G. K., and D’Esposito, M. (1999). Topographical disorienta- tion: a synthesis and taxonomy. 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Neuroimage 35, 1181–1191. doi: 10.1016/j.neuroimage.2007. 01.008 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. Ruby, P., and Decety, J. (2004). How would you feel versus how do you think she would feel. A neu- roimaging study of perspective- taking with social emotions. J. Cogn. Neurosci. 16, 988–999. doi: 10.1162/0898929041502661 Vinogradov, S., Luks, T. L., Schulman, B. J., and Simpson, G. V. (2008). Deﬁcit in a neural cor- relate of reality monitoring in schizophrenia patients. Cereb. Cortex 18, 2532–2539. doi: 10.1093/cercor/bhn028 Received: 08 March 2013; accepted: 12 June 2013; published online: 04 July 2013. Citation: Dahoun T, Eliez S, Chen F, Badoud D, Schneider M, Larøi F and Debbane M (2013) Action simulation in hallucination-prone adolescents. Front. Hum. 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Neurosci. 7:329. doi: 10.3389/ fnhum.2013.00329 Vogeley, K., May, M., Ritzl, A., Falkai, P., Zilles, K., and Fink, G. R. (2004). Neural correlates of ﬁrst-person perspective as one constituent of human self-consciousness. J. Cogn. Neurosci. 16, 817–827. doi: 10.1162/089892904970799 Stadler, W., Schubotz, R. I., Von Cramon, D. Y., Springer, A., Graf, M., and Prinz, W. (2011). Predicting and memorizing observed action: differential premotor cortex involvement. Hum. Brain Mapp. 32, 677–687. doi: 10.1002/hbm.20949 Copyright © 2013 Dahoun, Eliez, Chen, Badoud, Schneider, Larøi and Debbane. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduc- tion in other forums, provided the origi- nal authors and source are credited and subject to any copyright notices concern- ing any third-party graphics etc. Copyright © 2013 Dahoun, Eliez, Chen, Badoud, Schneider, Larøi and Debbane. 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https://openalex.org/W2922244872	http://journals.ed.ac.uk/lithicstudies/article/download/2745/3870	English	null	Middle Palaeolithic lithic tools: Techno-functional and use-wear analysis of target objects from SU 13 at the Oscurusciuto rock shelter, Southern Italy	Journal of lithic studies	2,018	cc-by	12,017	Middle Palaeolithic lithic tools: Techno-functional and use-wear analysis of target objects from SU 13 at the Oscurusciuto rock shelter, Southern Italy Giulia Marciani 1,2,3, Simona Arrighi 3,4, Daniele Aureli 3,4,5, Vincenzo Spagnolo 3, Paolo Boscato 3, Annamaria Ronchitelli 3 1. Dipartimento di Studi Umanistici, Sezione di Scienze Preistoriche e Antropologiche, Università degli Studi di Ferrara. C.so Ercole I d’Este 32, 44100 Ferrara, Italy. Email: giulia.marciani@unife.it 2. Department of History, History of Art, Universitat Rovira I Virgili Tarragona. Av. Catalunya, 35, 43002 Tarragona, Spain. Middle Palaeolithic lithic tools: Techno-functional and use-wear analysis of target objects from SU 13 at the Oscurusciuto rock shelter, Southern Italy Giulia Marciani 1,2,3, Simona Arrighi 3,4, Daniele Aureli 3,4,5, Vincenzo Spagnolo 3, Paolo Boscato 3, Annamaria Ronchitelli 3 1. Dipartimento di Studi Umanistici, Sezione di Scienze Preistoriche e Antropologiche, Università degli Studi di Ferrara. C.so Ercole I d’Este 32, 44100 Ferrara, Italy. Email: giulia.marciani@unife.it 2. Department of History, History of Art, Universitat Rovira I Virgili Tarragona. Av. Catalunya, 35, 43002 Tarragona, Spain. 3. Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente, UR Preistoria e Antropologia, Università degli Studi di Siena. Strada Laterina 8, 53100 Siena, Italy. Email: Arrighi: s.arrighi@hotmail.com; Aureli: danieleaureli1@gmail.com; Spagnolo: orpheus.85@hotmail.it; Boscato: paolo.boscato@unisi.it; Ronchitelli: annamaria.ronchitelli@unisi.it 4. Dipartimento di Beni Culturali, Università di Bologna. Via degli Ariani 1, 48121 Ravenna, Italy. 5. UMR 7041 ArScAn équipe AnTET. 21 Allée de l’Université, F 92023 Nanterre, France. g p 3. Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente, UR Preistoria e Antropologia, Università degli Studi di Siena. Strada Laterina 8, 53100 Siena, Italy. Email: Arrighi: s.arrighi@hotmail.com; Aureli: danieleaureli1@gmail.com; Spagnolo: orpheus.85@hotmail.it; Boscato: paolo.boscato@unisi.it; Ronchitelli: annamaria.ronchitelli@unisi.it 3. Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente, UR Preistoria e Antropologia, Università degli Studi di Siena. Strada Laterina 8, 53100 Siena, Italy. Email: Arrighi: s.arrighi@hotmail.com; Aureli: danieleaureli1@gmail.com; Spagnolo: orpheus.85@hotmail.it; Boscato: paolo.boscato@unisi.it; Ronchitelli: annamaria.ronchitelli@unisi.it 4. Dipartimento di Beni Culturali, Università di Bologna. Via degli Ariani 1, 48121 Ravenna, Italy. 5. UMR 7041 ArScAn équipe AnTET. 21 Allée de l’Université, F 92023 Nanterre, France. 4. Dipartimento di Beni Culturali, Università di Bologna. Via degli Ariani 1, 48121 Ravenna, Italy. 5. UMR 7041 ArScAn équipe AnTET. 21 Allée de l’Université, F 92023 Nanterre, France. Except where otherwise noted, this work is licensed under a CC BY 4.0 licence. Published by the School of History, Classics and Archaeology, University of Edinburgh ISSN: 2055-0472. URL: http://journals.ed.ac.uk/lithicstudies/ Except where otherwise noted, this work is licensed under a CC BY 4.0 licence. 1. Introduction The Oscurusciuto site is a Middle Palaeolithic rock shelter located in Southern Italy with a very rich record, essential for the definition of Neanderthals technical behaviour, as related both to the management of the raw material on the territory and the crafting and use of lithic tools. The lithic collections of Oscurusciuto show substantial uniformity even though each level displays its own peculiarity. The acquisition of raw material is local; Neanderthals used pebbles of jasper, chert, cherty limestone and quartz sandstone available in the terraces and areas near the site. The recurrent Levallois method is the most commonly noted concept of debitage especially in its unipolar modality aiming at producing elongated supports, both convergent or not. Furthermore, there is an additional volumetric exploitation aiming at producing bladelets (Boscato et al., 2011; Marciani, 2013; Marciani et al., 2016; Ranaldo, 2005; 2017; Ronchitelli et al., 2011; Spagnolo, 2013; 2017; Spagnolo et al., 2016; Villa et al., 2009). As it is widely known, the Levallois reduction sequence is an integrated concept that allows for the production of a great quantity of predetermined products with specific characteristics and dimensions (Boëda, 1991; 1993; 1994; 1995; 2013; Schlanger, 1996; Van Peer, 1992). The degree of predetermination of this concept regards both obtaining of a certain quantity of target products (resulting from the lineal\preferential or recurrent Levallois debitage) and their quality (Boëda, 1991; 1993; 1994; 1995; 2013). It also testifies to a specific economic strategy with regard to the maximization of cutting edge productivity obtained from cores (Brantingham & Kuhn, 2001; Lycett & Eren, 2013). The Levallois production at the Oscurusciuto stratigraphic unit 13 was aimed at producing a clear set of debitage goals: flakes, convergent flakes and backed flakes. In previous studies we have noted the peculiar and recurrent features of these objects and their particular role in the economy of the level (Marciani, 2013; Marciani et al., 2016). We assumed that these target objects had been imported as finished objects into the site, or had resulted from an in situ debitage process (Marciani, 2013; Marciani et al., 2016; Spagnolo et al., 2016). As the target objects are the answer to specific needs and necessities which motivated the flaking activity itself (Boëda, 2013), we examined the role played by Levallois target objects in the society where they were produced. Abstract: The Oscurusciuto rock shelter (Ginosa, Puglia, southern Italy) is a Middle Palaeolithic site characterized by a significant stratigraphy made up by several anthropic levels. The stratigraphic unit 13, consisting of a sandy compact deposit mixed with pyroclastic sediment, is a short palimpsest situated on a layer of tephra, identified as Mt. Epomeo green tuff (dated Ar/Ar ~ 55 ka BP). From a technological point of view, the aims of the production were backed flakes, convergent flakes, and other flakes obtained by means of a Levallois debitage, plus (less represented) bladelets produced by an additional volumetric reduction system. Our aim in this research was to examine a selection of the above-mentioned target objects produced by debitage in order to understand the manufacture and life-cycle of each single tool from a dynamic perspective. We integrated techno-functional and use-wear analyses: the first was implemented to globally comprehend each tool, identifying each single techno-functional unity (prehensile and transformative portions), whereas the second revealed the way in which these tools had been used, proceeding to identify the activity involved (e.g., piercing, cutting and/or scraping), and the type of material (vegetable/animal, soft/hard) on which these activities had been carried out. The combined use of these two approaches allows us to ascertain the intention of the prehistoric craftsmen, the gestures and procedures involved in making the tools, and the way they had been used. From one single object we are thus able to reconstruct a series of complex behaviours, encompassing the creation, the life-cycle and finally the ‘death’ or repurposing of the tool in question. Keywords: technical behaviour; techno-functional approach; use-wear analysis; Middle Palaeolithic; Neanderthals; western Europe Neanderthals; western Europe Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 Published by the School of History, Classics and Archaeology, University of Edinburgh ISSN: 2055-0472. URL: http://journals.ed.ac.uk/lithicstudies/ Except where otherwise noted, this work is licensed under a CC BY 4.0 licence. G. Marciani et al. 2 G. Marciani et al. 1. Introduction In particular, our main objective for this work was to focus on the Levallois target flakes produced at Oscurusciuto SU 13. We wanted to test (1) if the target objects of a reduction sequence (the production-aim), were actually used by Neanderthals to perform their activities (the functional-aim); (2) if each production-aim corresponded to a single functional-aim i.e. each tool was used solely for one activity, or, on the contrary, if it was used for a multitude of purposes; and finally (3) if tools used for a specific activity also had a specific structure. To accomplish these goals we integrated techno-functional and use-wear analyses: the first was implemented to globally comprehend each tool, identifying each single techno- functional unity (prehensile and transformative portions), whereas the second revealed the way in which these tools had been used, proceeding to identify the activity involved (e.g., piercing, cutting and/or scraping), and the type of material (vegetable/animal, soft/hard) on which these activities were carried out. From a methodological point of view, we note that both techno-functional and use-wear analyses have been applied on several lithic assemblages coming from diverse archaeological contexts and periods (e.g., Boëda, 1997; 2001; Soriano, 2000; Da Costa, 2017; Lourdeau, 2010), however few works have attempted to combine them (Aureli et al., 2015; 2016; Boëda et al., 2015; Bonilauri, 2010; Pedergnana, 2017). In the case of the site of Ficoncella (Rome - Italy; dated back to 500,000 years BP), the combined use of techno-functional and use-wear DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 3 G. Marciani et al. approaches was essential to determine the technical structure and the peculiarities of some active unities (trihedral, mini-rostrum and brute cutting edge) barely described before in lower Palaeolithic literature (Aureli et al., 2015; 2016). Another outstanding example is the case of Umm el Tlel site (central Syria - dated back to 40,000 years BP). Here the authors were able to evidence the technical role of the bladelets. These very well-known tools actually resulted from different production systems, performed several tasks, and were hafted in different ways (Boëda et al., 2015). The challenging point about applying these two approaches together is that there is an added value by their combined use. 1. Introduction In fact, the technological analysis permits us to understand the production procedure of lithic tools, and the techno-functional analysis allows us to understand the structure of the tools, as well as the functional potential of the parts constituting the tools. Finally, the use-wear analysis verifies or rejects these hypotheses. Tracceologists can also use the techno-functional study as a proxy for the selection of their samples, i.e. defining significant criteria to select the pieces to analyse, and examining traces guided by technical parameters. It is worth remembering that these approaches work in a continuous dialogue, comparing and integrating the observations from each approach. As a matter of fact our contribution with this paper is to demonstrate the complementarity and added value of combination of these approaches, which produce new and stimulating insights into “the ways of existing” of prehistoric technical objects (Du mode d’existence des objets techniques Simondon, 1958) 2. The site The Oscurusciuto rock shelter is situated in the region of Puglia, Southern Italy, namely in the ravine of Ginosa (Taranto) (Figure 1). The Oscurusciuto rock shelter is situated in the region of Puglia, Southern Italy, namely in the ravine of Ginosa (Taranto) (Figure 1). Figure 1. Panoramic view of the ravine of Ginosa, in the red circle the Oscurusciuto rock shelter (Photo A. Ronchitelli). Figure 1. Panoramic view of the ravine of Ginosa, in the red circle the Oscurusciuto rock shelter (Photo A. Ronchitelli). Panoramic view of the ravine of Ginosa, in the red circle the Oscurusciuto rock shelter (Photo A. lli) The site, opened into the Pleistocene calcarenite (Calcareniti di Gravina), stands at an elevation of 235 m above sea level, at about 15 meters from the current bottom of the ravine, and at about 20 km far from the actual Ionic coastline (Figure 2). The site, opened into the Pleistocene calcarenite (Calcareniti di Gravina), stands at an elevation of 235 m above sea level, at about 15 meters from the current bottom of the ravine, and at about 20 km far from the actual Ionic coastline (Figure 2). 2 g The archaeological deposit measures 60 m2 at its base, and has a thickness of more than 6 meters. This sequence extension downwards gradually increases because the hill erosion damaged the deposit in the shelter, especially on the upper layers (Figure 3). Since 1998 until the present day, the first 3 meters of the sequence corresponding to nine main Middle Palaeolithic occupation phases have been investigated by the U.R. “Preistoria e Antropologia” under the “Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente - University of Siena, Italy”. The chronological limits of the Neanderthal occupation of the rock shelter are obtained by two dates. The first, obtained by the C14 method (on collagen), is referred to the bottom of Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 4 4 G. Marciani et al. G. Marciani et al. SU 1 which is datable to 38,500 ± 900 BP, cal. 42,724 ± 716 BP Beta 181165 (Ramsey & Lee, 2013; Reimer et al., 2013). The former derives from the identification of the tephra layer (SU 14) as Mount Epomeo green tuff (Marciani et al., 2016; Spagnolo et al., 2016) datable to about 55,000 BP (Allen et al., 2000). Figure 2. Location of Oscurusciuto (Modified from Google Maps). Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 2. The site Figure 2. Location of Oscurusciuto (Modified from Google Maps). The faunal associations found at Oscurusciuto are characterized by the main presence of Bos primigenius, Equus ferus, Cervus elaphus and Dama dama in different variations and associations, which show both little paleo-climatic fluctuations and the coexistence of different biomes, possibly related to a micro-scale landscape variability linked to the ravine environment. Essentially, the studied samples (from SU 15 to SU 1) show that the Neanderthal hunters exploited two different environments: a forest-steppe area, probably present on the hilly relieves, and a moister territory with wooden coverage, inside the ravine (Boscato & Crezzini, 2012; Boscato, 2017). SU 13 (Figure 4) consists of the sedimentary interface between the SU 11, sandy layer above, and the SU 14, the proper tephra deposit. It represents the first stable re-colonization of the rock shelter which occurred during the final phase of volcanic ashes deposition (Marciani Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 G. Marciani et al. 5 et al., 2016; Spagnolo et al., 2016) (Figure 3). It is a short palimpsest excavated for the extension of 11 square meters. The level is characterized by the presence of quite abundant faunal remains; however, only six elements were identified, due to high fracture grade (1 = Equus ferus and 5 = Bos primigenius). Above SU 13 a series of structured hearts made in prepared dimple was exposed which seems to be arranged in a line that divides the space of the rock shelter into two portions: inside and outside the line of fires (Spagnolo et al., 2016). Figure 3. Stratigraphic sequence of the upper portion of Oscurusciuto rock shelter (sequence so far excavated) (Relief made by P. Boscato; drawing by A. Ronchitelli). Figure 3. Stratigraphic sequence of the upper portion of Oscurusciuto rock shelter (sequence so far excavated) (Relief made by P. Boscato; drawing by A. Ronchitelli). Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 3.1. Lithic production In SU 13, 7504 well preserved lithic artefacts were found. As in all the upper stratigraphic units, the dominant raw materials are jasper and cherty limestone in their fine granulometry, found in the form of pebbles, which can still be found on the sea-terraces and river deposits around the site (nowadays almost between few tens of meters and over than 20 km far from the site) (Marciani, 2013; Marciani et al., 2016). The occupation of SU 13 is a short palimpsest that was possible to disentangle in at least two (if not most) settlement events (Spagnolo et al., 2016; Spagnolo, 2017). The lithic collection corroborates this idea because there are several fragmented reduction sequences. Namely, the lithic material was entered into the site at different stages of debitage, which means in the form of rough objects (pebbles), or as semi-finished items (decortication happened outside the rock shelter), and as finished tools (target objects or retouched tools). However, there is evidence of pieces exported from the site, especially the target objects. Only 30 pieces have been retouched (Marciani et al., 2016). y p Only two concepts of debitage were utilized at the level 13: the Levallois concept and the additional volumetric reduction sequence. Namely, the latter refers to an additional production aimed at producing bladelets where the striking platform is prepared but the convexities are not prepared and only the natural convexities of the raw block are used, i.e. C2 type of cores according to Boëda classification (Boëda, 2013). DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 6 6 G. Marciani et al. In this paper, we will focus only on the Levallois products: flakes, convergent flakes and backed flakes. From a totality of 385 target pieces (Marciani, 2013; Marciani et al., 2016) we selected 312 target objects with clearly recognizable technical features (fragmented or post- depositional altered pieces were excluded from the study). In this paper, we will focus only on the Levallois products: flakes, convergent flakes and backed flakes. From a totality of 385 target pieces (Marciani, 2013; Marciani et al., 2016) we selected 312 target objects with clearly recognizable technical features (fragmented or post- depositional altered pieces were excluded from the study). Figure 4. SU 13 planimetry (Relief made by Boscato, draw by Spagnolo). Figure 4. SU 13 planimetry (Relief made by Boscato, draw by Spagnolo). 3.1. Lithic production In the text we refer to the objective of debitage\end-products with the expression “target objects” (target flakes, target blade, etc.) because we think that even if not very common in bibliography it expresses very well the strong degree of intentionality used in order to produce them. In the text we refer to the objective of debitage\end-products with the expression “target objects” (target flakes, target blade, etc.) because we think that even if not very common in bibliography it expresses very well the strong degree of intentionality used in order to produce them. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 3.2. Techno-functional analysis The techno-functional analysis is the study of the structure and potentiality of tools which are defined as objects consisting of three main parts: transformative part, transmitting part and prehensile part. These parts are defined as techno-functional unities: the UTFt - transformative techno-functional unit corresponds to the active portion of the tool plus the edge, which is the part which actually enters into contact and modifies the material; the UTFp The techno-functional analysis is the study of the structure and potentiality of tools which are defined as objects consisting of three main parts: transformative part, transmitting part and prehensile part. These parts are defined as techno-functional unities: the UTFt - transformative techno-functional unit corresponds to the active portion of the tool plus the edge, which is the part which actually enters into contact and modifies the material; the UTFp - prehensile techno-functional unit is the holding portion. Between those there is the transmitting techno-functional units (UTFtr) which is conceived as an intermediate factor t itti th f f th h dl t th t f ti ti (B ëd 1997 2001 - prehensile techno-functional unit is the holding portion. Between those there is the transmitting techno-functional units (UTFtr) which is conceived as an intermediate factor transmitting the force from the handle to the transformative portion (Boëda, 1997; 2001; 2013; Bonilauri, 2010; Da Costa, 2017; Lepot, 1993; Lourdeau, 2010; 2015; Soriano, 2000). - prehensile techno-functional unit is the holding portion. Between those there is the transmitting techno-functional units (UTFtr) which is conceived as an intermediate factor transmitting the force from the handle to the transformative portion (Boëda, 1997; 2001; 2013; Bonilauri, 2010; Da Costa, 2017; Lepot, 1993; Lourdeau, 2010; 2015; Soriano, 2000). p ) The techno-functional unities are indispensable for the tool operation, as is the synergy between them that makes the tool capable of realizing an action. Identifying these parts and understanding their synergy enables us to understand the potential capacity of each single Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 7 G. Marciani et al. tool. Furthermore, in addition to prehensile, active and transmitting parts, we should not forget the role of the gesture. 3.2.1. Defining the blank The blank is actually the support on which the UTFt and UTFp are installed. At Oscurusciuto SU 13 we defined blanks on the basis of four parameters: shape, section, elongation index and size of the pieces. The shape is rectangular, when there are 2 parallel\sub parallel edges plus a transversal edge (these edges could be sharp of backed; in this group trapezoidal and oval shapes are also considered). Alternatively, it is described as triangular when there are 2 edges converging into a point (Figure 5). The section of the piece takes into consideration the section-shape of the items but also the number of cutting edges. This means that triangular and trapezoidal pieces have 2 cutting edges, whereas rectangular-triangle and rectangular-trapeze have only one cutting edge opposed to a backed side (Figure 5). The elongation index is given by the ratio between height and width. If the result is less than 1.4 it is a flake, if the result is comprised between 1.5 and 2 it is a long flake, if the result is major then 2.1 it is a blade (Figure 5). The size of the pieces is based on the graphic of dispersion of the height of these pieces of Oscurusciuto SU 13. We define a piece as “small” if its maximum height is until 20 mm, “medium” if it is comprised between 21 and 34 mm and “big” if its height is higher than 35 mm (Figure 5). 3.2. Techno-functional analysis Namely, the specific gesture involved in moving a tool is indispensable for the proper functioning of the tool, whereby we mean the kind of action, as well as the actual movement, both of which made the tool technically worthwhile (Leroi Gourhan, 1973). The identification, location and characterization of UTFt and UTFp lay in empirical observation of the objects and collection of objective data. Whereas the identification of the transmitting part is much more difficult to perceive, for this reason in this work we consider together the prehensile and transmitting parts (Boëda, 2001; Bonilauri, 2010; Da Costa, 2017; Lourdeau, 2010; Soriano, 2000). After analysing each single piece, comprising the identification, characterization and localization of each UTF, we have identified groups of pieces that share the same structural composition, i.e. techno-type. Going more in detail, a techno-type is defined by pieces with the same number and characteristic UTFt; and the same position and number of UTFp. The sub-types are inner variations of techno-types in our case expressed by the delineation and position of cutting edges. After defining the techno-types and sub-types, i.e. the combination of UTFt and UTFp, we have identified how these combinations were arranged on each blank. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 3.2.2. Defining transformative parts (UTFt) We identified two types of UTFt: the cutting edge and the trihedron. The cutting edge is a dihedron made up by the intersection of two surfaces, which delimits a plan section and an angle able to cut (Lepot, 1993; Soriano, 2000). In order to define the cutting edges we consider its delineation (from frontal and profile view, which could be: rectilinear, convex, concave and denticulate), location (distal, mesial, proximal, mesio-distal, mesio-proximal; left or right), extension (expressed in mm) angles, and surfaces relation (Plan/Plan; Convex/Plan; Concave/Plan; Convex/Convex; Concave/Concave) (Abruzzese et al., 2016; Boëda, 1997; 2001; Bonilauri, 2010; Da Costa, 2017; Lepot, 1993; Lourdeau, 2010; Lucas, 2014). The trihedron is defined as a geometric figure composed of three planes meeting at a single vertex. As UTFt the trihedron is a punctual UTF, which means that it does not have an extension. In order to define the trihedron we consider its location, surface relations, angles, that is the angle made by the surfaces (section plan between ventral and dorsal surface of the Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 8 G. Marciani et al. G. Marciani et al. item) and the openness angles which is the angle made by the two lateral sides (left and right side of the items) (Aureli et al., 2016; Rocca, 2013) (Figure 6). item) and the openness angles which is the angle made by the two lateral sides (left and right side of the items) (Aureli et al., 2016; Rocca, 2013) (Figure 6). Figure 5. Shape, section, elongation index and size of the pieces (the size is based on the height of the pieces box plot realized with Past 3.16). Figure 5. Shape, section, elongation index and size of the pieces (the size is based on the height of the pieces box plot realized with Past 3.16). Figure 6. UTFt cutting edge and UTFt trihedron. If there is more than one UTFt on the same blank we register which kind of relation exists between them: contiguous if the two (or more UTF) are in an adjacent position and in contact between them, and opposed if the two (or more UTF) are in front of each other. All the parameters are considered with the flake oriented in the technological axis. Figure 6. UTFt cutting edge and UTFt trihedron. Figure 6. UTFt cutting edge and UTFt trihedron. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 3.2.3. Defining prehensile parts (UTFp) In the absence of evident grip-traces or glue residues, so far, we consider as potential UTFp all the backed sides, the cortical edges and more in general a discontinuous and irregular edges (Soriano, 2000). The butt is also considered as potential grip, in fact especially for Levallois chapeau de gendarme, the ergonomic features of its shape are already attested in experimental studies (Baena Preysler et al., 2016). We consider the thickness, location, angles and extension of UTFp. A notable parameter when defining the structure of a tool is the relation between backed sides i.e. a single backed side, two continuous backed sides, two opposed backed sides, or three framed backed sides (Da Costa, 2017). 3.3. Use-wear analysis The use-wear analysis was performed on 34 items identified as Type-blank A (see paragraph 4.1). The study was carried out by means of both the low power approach (LPA) (Odell, 1981; Odell & Odell-Vereecken, 1980; Tringham et al., 1974, and for more recent discussions or applications see Lemorini, 2000; Plisson, 2007; Rots, 2010) and the high power approach (HPA) (Keeley, 1980; Plisson, 1985; Van Gijn, 2010). LPA is focused on analysis and interpretation of macro use-wear (edge-removals, edge- rounding) while HPA is based on the observation of micro use-wear (micro edge-rounding, polishes, striations). Macro use-wear was observed at low magnification (20x - 80x) by means of a Hirox KH 7700 3D digital microscope, using a MX-G 5040Z body equipped with an AD-5040Lows and an AD-5040HS lens. Micro-wear analysis was performed using the mentioned Hirox microscope fitted out with a MXG-10C body and an OL-140II lens (140x- 480x). The microscope enables us to obtain in-focused pictures through the overlapping of planes taken at different focus levels (Arrighi et al., 2016; Moretti et al., 2015). The traces on the archaeological lithic tools were interpreted by means of the comparison with the experimental reference collection of the U.R. Preistoria e Antropologia - University of Siena, Italy. 3.2.2. Defining transformative parts (UTFt) If there is more than one UTFt on the same blank we register which kind of relation exists between them: contiguous if the two (or more UTF) are in an adjacent position and in contact between them, and opposed if the two (or more UTF) are in front of each other. All the parameters are considered with the flake oriented in the technological axis. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 G. Marciani et al. 9 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 4.1. Blank The majority of blanks (almost 50% of the complex) are of medium size (Table 1). Based on the elongation index, the main objects pursued were flakes (Table 2) and most of them could be considered as rectangular (with at least two cutting edges) (Table 3); 34 pieces have a backed side (Table 4). Table 1. Size of target objects. Size N. Big 77 Medium 148 Small 87 Total 312 Table 1. Size of target objects. Size N. Big 77 Medium 148 Small 87 Total 312 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 G. Marciani et al. 10 Table 2. Elongation index of target objects. Elongation Index N. Blade 52 Long Flake 71 Flake 189 Total 312 Table 3. Shape of target objects. Shape N. Rectangular 270 Triangular 42 Total 312 Table 4. Section of target objects. Section N. Trapezoidal\triangular 278 Rectangular trapeze or triangle 34 Total 312 Table 2. Elongation index of target objects. Elongation Index N. Blade 52 Long Flake 71 Flake 189 Total 312 Table 3. Shape of target objects. Shape N. Rectangular 270 Triangular 42 Total 312 Table 4. Section of target objects. Section N. Trapezoidal\triangular 278 Rectangular trapeze or triangle 34 Total 312 Table 2. Elongation index of target objects. Table 3. Shape of target objects. ble 4. Section of target objects. ction N. apezoidal\triangular 278 ctangular trapeze or triangle 34 tal 312 Table 4. Section of target objects. Section N. Trapezoidal\triangular 278 Rectangular trapeze or triangle 34 Total 312 Combining these technical traits, we obtained the blanks on which the active and prehensile parts of the instrument are imposed (Figure 7). The majority of Oscurusciuto SU 13 target objectives of Levallois debitage are rectangular flakes of medium and small size, followed by rectangular big and medium long flakes and big blades. We note that elongated products are mostly big and medium sized, whereas flakes are mostly small. Convergent flakes are scarcely represented, no matter what the size. Except for convergent medium flakes (19 pieces), other sizes do not account for more than ten unities (Figure 7). According to these characteristics, we can sub-divide the macro-class of blanks into: type-blank A, B or C. Type-blank A is made by rectangular blanks with one cutting edge opposed to a backed side. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 4.2. Type-blank A Type-blank A encompassed 34 pieces, most of them are elongated supports (blades or long flakes) (Table 5). Table 5. Type-blank A rectangular blanks with one cutting edge opposed to a backed side. Table 5. Type-blank A rectangular blanks with one cutting edge opposed to a backed side. Table 5. Type-blank A rectangular blanks with one cutting edge opposed to a backed side. Blank Quantity Big blade 8 Medium blade 5 Big long flake 6 Medium long flake 2 Small long flake 1 Medium flake 9 Small flake 3 Total 34 On these supports several UTFt are imposed: 21 UTFt trihedrons (from now UTFt T or just T) and 39 UTFt cutting edges (from now UTFt C or just C) (Table 6). On these supports several UTFt are imposed: 21 UTFt trihedrons (from now UTFt T or just T) and 39 UTFt cutting edges (from now UTFt C or just C) (Table 6). Table 6. presence of UTFt and UTFp. UTF types Quantity UTFt Rectilinear cutting edge 30 Denticulate cutting edge 5 Convex cutting edge 3 Concave cutting edge 1 Trihedron 21 UTFp 1 backed side 2 2 backed sides 19 3 backed sides 13 The most represented UTFt is the cutting edge. It could be found alone, in association with a trihedron, or with other cutting edges (Table 7). The majority of cutting edges show a rectilinear delineation, less represented are denticulate, convex and concave forms (Table 6). Usually cutting edges are in a lateral position (32) and occupy 3\4 or the totality of the edge of the tool. The 21 UTFt trihedron are made up by 3 planes encompassing the dorsal and ventral faces of the flakes plus the butt, or plus a third face constituted by a rib made up of two negatives on the dorsal surface or by a broken portion of the flakes, which lets us suppose an intentional use of the fracture. Consequently, these UTFt are slightly difficult to identify, as their features, (when the rib or butt is the third face), mostly occur during the production of the pieces and not through intentional action (like in the case of intentional fracture). This issue could have caused an overestimation of this UTFt. 4.1. Blank This group contains small, medium and big rectangular flakes, long flakes and blades with rectangular-trapeze and triangular-trapeze sections, which means one cutting edge opposed to a back (34 pieces). Type-blank B is made by the rectangular blanks with at least two cutting edges. This group comprehends small, medium and big rectangular flakes, long flakes and blades with trapezoidal and triangular sections, which means pieces with at least 2 cutting edges (233 pieces). Type-blank C is made by convergent blanks with at least two cutting edges. This group comprehends small, medium and big convergent flakes, long flakes and blades with trapezoidal and triangular sections, which means pieces with at least 2 cutting edges (42 pieces). Having defined these three classes of blanks, in this paper, we decided to focus only of the type-blank A in order to give extensive attention to each category (further work will focus on the other two blank type, B and C) (Figure 7). Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 11 G. Marciani et al. Figure 7. a. Type-blank A rectangular blanks with one cutting edge opposed to a backed side. b. Type-blank B: rectangular blanks with at least two cutting edge. c. Type- blank C: convergent blanks with at least two cutting edges (the scale of colour indicates the number of items: darker = more frequent and lighter = less frequent). Figure 7 a Type-blank A rectangular blanks with one cutting edge opposed to a backed side b Type-blank B: rectangular blanks with at least two cutting edge c Type- Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 12 G. Marciani et al. G. Marciani et al. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 4.2. Type-blank A of Pieces Plus UTFt trihedron A: 1 Cutting edge + 2 backed sides (CDD) 13 3 A.1 Cutting edge with denticulate delineation 3 1 A.2 Cutting edge with rectilinear delineation 6 2 A.3 Cutting edge with transversal position (rectilinear) 1 0 A.4 Cutting edge with convex delineation 2 0 A.5 Cutting edge with concave delineation 1 0 B: 1 Cutting edge + 1 backed side (CD) 2 0 C: 2 Cutting edge + 2 backed sides (CCDD) 6 3 C.1 Contiguous cutting edges 4 1 C.2 Separated cutting edges 2 2 D: 1 Cutting edge + 3 backed sides (CDDD) 12 12 D.1 Cutting edge with denticulate delineation 2 1 D.2 Cutting edge with rectilinear delineation 9 9 D.3 Cutting edge with convex delineation 1 2 E: 1 Trihedron + 3 backed sides (TDDD) 1 1 E: 1 Trihedron + 3 backed sides (TDDD) We recognized 5 techno-types, presenting a variety of sub-types (Figure 8, Table 7). To sum up the most common techno-type was A: 1 cutting edge + 2 backed sides (CDD), present in sub-types 1, 2, 3, 4 and 5. Sub-types 1, 2, 4, and 5 have the cutting edge opposed to the lateral backed side, whereas sub-type 3 is the only one that has just one cutting edge in a transversal position (Figure 8, Table 7). Highly represented is also techno-type D: 1 cutting edge + 3 backed sides (CDDD), whose peculiarity the 3 backed sides constituting a frame for the cutting edge. Not surprisingly this is where we find the major number of trihedrons as the backed side is actually the third surface that enhanced the creation of the trihedron itself. The pieces of techno-type B: 1 cutting edge + 1 backed side (CD), are peculiar because in both cases the UTFp is made through retouch. Finally, we have only one case of techno-type E: 1 trihedron + 3 backed sides (TDDD). This piece is very interesting as it is the smallest tool in the collection; here the intention of creating 3 backed sides as prehensile portion leaving free only the distal trihedron become clear. This is also the only case were the trihedron is found alone and not connected with other UTFt (Figure 8, Table 7). These above-mentioned techno-types and sub-types could be found imposed on various blanks. 4.2. Type-blank A The UTFp are found in several different combinations: the most relevant one is the presence of 2 backed sides (most of them proximal plus lateral) and the 3 backed sides that actually forms a frame around the piece (from now on we refer to backed side with D) (Table 6, Table 7). Going into more detail, we focused on the combination among the active parts (UTFt C and UTFt T), plus their association with the prehensile parts (UTFp). In this way we were Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 G. Marciani et al. 13 able to individuate classes of tools that have the same structure (techno-type). For the nomenclature, we describe each tool based on the combination of UTFt and UTFp (UTFt cutting edge = C, UTFt Trihedron = T; UTFp backed side = D), the repetition of each letter represents the frequency of the unity. The techno-type is indicated with a letter and the sub- type is indicated by a number. It this way each piece is described by a code i.e. CDD-A1 means a piece with one UTFt cutting edge: C + two backed sides: DD, belonging to techno- type A, subtype 1. Table 7. Techno-type and subtype defined by the combination of UTFt and UTFp. The column “plu trihedron” indicates how many trihedrons are present on the pieces (please note that this column re number of UTFt Trihedrons and not to the number of pieces). Techno - Type and Sub-type N. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 4.2. Type-blank A Starting from the blades, these pieces are found in big and medium sizes; generally, we noted a recurrence of 2 baked sides and an opposite cutting edge. Incidentally, each tool has its own peculiarities; they are supports for different sub-types (Table 8, Figures 8, 9, and 10). The case of the pieces ID 919 and ID 989 (techno-type B) is interesting because the retouch is implemented to improve the adherence potential of the grip, and it is adjacent to the cutting edge opposed to the backed side. This configuration of the tool introduces the hypothesis of a hafting (Figure 9). DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx G. Marciani et al. 14 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 Figure 8. Schematic composition of techno-type and sub-type, plus examples of tools for each category. Figure 8 Schematic composition of techno type and sub type plus examples of tools for each category Figure 8. Schematic composition of techno-type and sub-type, plus examples of tools for each category. gure 8. Schematic composition of techno-type and sub-type, plus examples of tools for each category. DOI: https://doi.org/10.2218/jls.2745 15 G. Marciani et al. Table 8. Synthetic table of techno-types and sub-types (C: cutting edge, T: trihedron: D: backed side; n. refers to the total number of pieces of each type), how they are disposed on the blank, plus information regarding the use: action, worked material and angles of the active edge (for the blank: BB: big blade, MB: medium blade, BL: big long flake, ML: medium long flake, SL: small long flake, MF: medium flake, SF: small flake). long flake, ML: medium long flake, SL: small long flake, MF: medium flake, SF: small flake). Techno-type Sub- type N. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 4.2. Type-blank A Blank Action Material Angles ID pezzi BB MB BL ML SL MF SF A: CDD n13 A.1 3 1 1 1 cut soft, hard 20°, 20°, -30° 28, 894, 214 A.2 6 1 1 1 1 2 cut semi-hard, hard 20°, 30°, 20°, -30°, 40° 983, 47, 318, 1512, 531, 906 A.3 1 1 \ \ 20° 959 A.4 2 1 1 cut soft 20° 400, 320 A.5 1 1 cut semi-hard, 20° 82 B: CD n.2 B 2 2 scrape, cut soft 20° 919, 989 C: CCDD n.6 C.1 4 2 1 1 cut, scrape semi-hard, hard 20°, 30°, 40° 633, 1384, 1632, 846 C.2 2 2 scrape soft 20°, 30°, 50° 852, 853 D: CDDD n.12 D.1 2 1 1 cut, carve soft 20°, 30°, -40° 213, 991 D.2 9 2 2 4 1 cut, scrape semi-hard, hard 20°, -40°, 20°, -40°, 30°, 40°, 50° 495, 102, 165, 636, 371, 712, 632, 1012, 414 D.3 1 1 \ \ 30° 1628 E: TDDD n.1 E 1 1 carve hard 882 Total 34 8 5 6 2 1 9 3 \ \ DOI: https://doi.org/10.2218/jls.2745 DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 16 G. Marciani et al. G. Marciani et al. Figure 9. Techno-functional analysis and description of use wear traces of the big blades. Figure 9. Techno-functional analysis and description of use wear traces of the big blades. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 17 G. Marciani et al. Figure 10. techno-functional analysis and description of use wear traces of the medium blades. Figure 10. techno-functional analysis and description of use wear traces of the medium blades. Considering the medium blades, the pieces ID 852 and ID 853 refits, in this case it is interesting that the same configuration of the tool is created in the same core Namely pieces Figure 10. techno-functional analysis and description of use wear traces of the medium blades. Considering the medium blades, the pieces ID 852 and ID 853 refits, in this case it is interesting that the same configuration of the tool is created in the same core. 4.2. Type-blank A Namely, pieces that technologically play the role of predetermining-predetermined pieces from a techno- functional point of view present identical the same prehensile and active portions (sub-type C2) (Table 8, Figures 8 and 10). As for the long flakes, we encountered specimens of every sizes: big, medium, and small in varying numbers (Figures 11 and 12). This is the blank with the most variations in techno- types, in fact of 9 pieces we noted 8 different sub-types (Table 8, Figures 8, 11, and 12). Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 18 G. Marciani et al. Figure 11. Techno-functional analysis and description of use wear traces of the big long flakes. Flakes are represented only in small and medium sizes (Figures 13 and 14). The main defining feature in these pieces is the presence of 3 framed backed sides (techno-type D) (Table 8, Figures 8 and 13). Probably because of their small dimensions, these small pieces, show a particular prehensile need that could be met by the 3 backed sides, artifice that permit the creation of trihedrons. Figure 11. Techno-functional analysis and description of use wear traces of the big long flakes. Flakes are represented only in small and medium sizes (Figures 13 and 14). The main defining feature in these pieces is the presence of 3 framed backed sides (techno-type D) (Table 8, Figures 8 and 13). Probably because of their small dimensions, these small pieces, show a particular prehensile need that could be met by the 3 backed sides, artifice that permit the creation of trihedrons. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 19 G. Marciani et al. Figure 12. Techno-functional analysis and description of use wear traces of the medium long flakes and small long flakes. no-functional analysis and description of use wear traces of the medium long flakes and small Figure 12. Techno-functional analysis and description of use wear traces of the medium long flakes and small long flakes. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 4.3. Use-wear analysis Of the examined sample (34), 21 tools show evidence of use, 9 pieces display unclear or uncertain traces because of post-depositional modifications, 4 artefacts do not reveal use-wear at all. We noted that in the majority of the cases use-wear traces were found on the portion of the pieces recognized as active by the techno-functional analysis. Only in one case traces were found on another edge which was not recognized by techno-functional analysis (ID 991, Figure 13). In other cases, there was not enough microscopic evidence to prove the techno- functional reading. Going more into detail, 22 UTFt were used, in particular we noted 20 cutting edges (UTFt C) and 2 trihedrons (UTFt T). In addition, traces were visible on 2 prehensile portions (UTFp). Each artefact showed evident traces on only one UTFt, with the exception of the piece ID 1384 where its 3 UTFt showed traces (Figure 15). Due to the fact that micro-wear traces (polishes) are by nature not very evident, for several pieces it was inferred only the action carried out and general information about the hardness of the worked material. Nevertheless, as a general functional scene, we may deduce that techno-type analysed was regularly used for various tasks (Tables 8 and 9). The tools were used for processing hard (9), semi-hard (8) and soft materials (5). Such a variety is confirmed also when worked materials have been detected, as both vegetal (4) and animal tissues (4) were processed (Table 9, Figure 16). Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 20 G. Marciani et al. G. Marciani et al. Figure 13. Techno-functional analysis and description of use wear traces of the medium flakes. Figure 13. Techno-functional analysis and description of use wear traces of the medium flakes. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 21 G. Marciani et al. Figure 14. Techno-functional analysis and description of use wear traces of the small flakes. Figure 14. Techno-functional analysis and description of use wear traces of the small flakes. Figure 14. Techno-functional analysis and description of use wear traces of the small flakes. Figure 15. Use-wear polish on the big blade ID 1384 (the scale bar is 500 µm.). Figure 15. Use-wear polish on the big blade ID 1384 (the scale bar is 500 µm.). DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 4.3. Use-wear analysis xx-xx 22 G. Marciani et al. Table 9. Correlation among active parts (UTFt C: cutting edge and UTFt T: trihedron), movement and worked material. Type of material and UTF Longitudinal motion Transversal motion Rotational motion Long+trans motion Total UTFt C UTFt T UTFt C UTFt T UTFt C UTFt T UTFt C UTFt T Soft 2 1 3 Semi-hard 5 5 Hard 3 1 2 6 Wood 1 1 1 3 Herbaceous plants 1 1 Soft animal tissues 1 1 Semi-hard animal tissues 2 1 3 Total 15 1 4 0 1 1 0 22 22 UTFt C are used mainly for longitudinal actions and to a lesser extent in transversal movements. The transversal actions are carried out by cutting edges with wider angles, in particular when hard materials were processed. Comparing worked materials and motions, we may conclude that the transversal actions were mostly performed on hard material, while the longitudinal ones on all types of material (Tables 8 and 9, Figures 9 to 14 and 16). Few items are employed in mixed actions. A single UTFt C, of the flake ID 1012, was used with both longitudinal and transversal movements for processing vegetal material (Figure 13, Tables 8 and 9). Whereas two different UTFt C of the big blade ID 1384 were both used for longitudinal actions and UTFt T was employed in a rotational motion. In both cases animal tissue was processed (Figure 15, Tables 8 and 9). By means of a techno-functional analysis, 21 trihedrons were identified, but traces were found on only two UTFt T of which one was involved in a rotational motion (pieces ID 1384 - Figure 15), and the other in a longitudinal action (pieces ID 882 - Figure 17) (Tables 8 and 9). These tools were used for processing hard and semi-hard material. Evidence of probable hafting traces on UTFp is detected on two pieces (ID 1020 and ID 919), confirming the hypothesis of the techno-functional analysis. In both cases, the traces are located on the backed sides of the tools (Figures 9 and 10). In both cases, the traces are few bright spots located on the backed sides of the tools. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 5. Combined use of techno-functional and use-wear analysis The result obtained by the combined use of the techno-functional and use-wear analysis gives a consistent result. In fact, in the sampled pieces, we note that in the majority of the pieces the use-wear analysis confirmed the interpretation given in the techno-functional study. The result obtained by the combined use of the techno-functional and use-wear analysis gives a consistent result. In fact, in the sampled pieces, we note that in the majority of the pieces the use-wear analysis confirmed the interpretation given in the techno-functional study. We note that sometimes the same techno-type are installed on different blanks, as in the case of sub-type A2 and D2, a recurrent combination in almost all blanks. Then again, other techno-types seem to be specific to some blanks, such as techno-type B. Moreover, some blanks are the support for several techno-types, e.g., the big blades present quite a number of different combinations, and they seem to be made to purpose unique tools. In contrast, other p y p g y We note that sometimes the same techno-type are installed on different blanks, as in the case of sub-type A2 and D2, a recurrent combination in almost all blanks. Then again, other techno-types seem to be specific to some blanks, such as techno-type B. Moreover, some blanks are the support for several techno-types, e.g., the big blades present quite a number of different combinations, and they seem to be made to purpose unique tools. In contrast, other blanks support few techno-types, as is the case with medium flakes where most of them support the techno-type D (Table 8). DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx G. Marciani et al. 23 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.274 Figure 16. A. ID 852 - use-wear polish interpreted as due to processing soft wood (the scale bar is 500 µm) B. ID 906 - edge rounding associated with use-wear polish from cutting dry skin (the scale bar is 200 µm); C. ID 531 - edge scarring related to cutting hard material (the scale bar is 0.5 cm ). Figure 16. A. ID 852 - use-wear polish interpreted as due to processing soft wood (the scale bar is 500 µm); B. ID 906 - edge rounding associated with use-wear polish from cutting dry skin (the scale bar is 200 µm); C. 5. Combined use of techno-functional and use-wear analysis ID 531 - edge scarring related to cutting hard material (the scale bar is 0.5 cm ). DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 24 G. Marciani et al. Figure 17. Use-wear polish associated with edge rounding of the small flake ID 882 (the scale bar is 200 µm). gure 17. Use-wear polish associated with edge rounding of the small flake ID 882 (the scale bar is 200 µm A very remarkable fact is that the techno-types are consistent with their actual use, in fact we noted that some techno-types were used for specific activities, as is the case with techno- type A, which is mostly used for cutting (longitudinal movement). On the other hand, there are techno-types, such as C and D, which are employed in a variety of activities, such as cutting and scraping (respectively longitudinal and transversal movements). In addition, some techno-types met a particular need, as is the case with techno-type E, which can be considered unicum in the collection both from a structural and a functional point of view, in fact this piece (a UTFt T framed by 3 backed sides) was used for carving (Figure 17). Finally, in techno-type B a very specific construction of the piece (also produced by retouch) does not correspond the same activity, one is used for scraping and the other for cutting, however both pieces are related to butchering activities (Table 8). G. Marciani et al. G. Marciani et al. 25 The encouraging results of this study motivated us to continue in this direction, analysing the other two categories (type-blanks B and C) in order to obtain a larger set of statistically significant data. The encouraging results of this study motivated us to continue in this direction, analysing the other two categories (type-blanks B and C) in order to obtain a larger set of statistically significant data. Lastly, one question remains: the trihedron was identified by techno-functional analysis but presented traces in only 2 cases. For this reason, we want to know why there is such little evidence of usage on these pieces. Is it because the action did not leave strong visible traces, or because this part was not used? We could also hypothesise an artificial increase due to possibly misleading criteria of identification. To be able to answer these questions we are planning to set up an experimental protocol to verify the functional potential or the trihedron, possibly applying more restrictive criteria for its identification, based on the technical criteria present on the two items which showed clear use-wear traces. Acknowledgements The authors are indebted to the Soprintendenza Archeologia, Belle Arti e Paesaggio per le province di Brindisi, Lecce e Taranto, Municipality of Ginosa, Onlus CESQ, to Piero di Canio and to all the students and colleagues who participated, together with the authors, in the excavation of 2011. This paper was presented at 11th International Symposium on Knappable Materials held in University of Buenos Aires between 7 and 12 November 2017. We would like to warmly acknowledge Roxane Rocca, Marcos César Pereira Santos, Birgitte Hoiberg Nielsen and the two reviewers Antonella Pedergnana and Amelie Da Costa for their precious and helpful advice. We thank also the session organizers Leslye Valenzuela, Otis Crandell, and Patrick Julig. Author contributions Giulia Marciani performed the technological and techno-functional analysis supervised by Daniele Aureli. Simona Arrighi performed the use-wear analysis. Vincenzo Spagnolo is responsible for the planimetries and spatial data. Paolo Boscato analyzed the faunal remains, and is the director of the excavation together with Annamaria Ronchitelli. 6. Conclusions A first methodological observation was that the integrated use of techno-functional analysis and use-wear analysis yields consistently matching results to the benefit of both technologists and tracceologists. Thanks to this combined method we get much closer to the human realities where there were several needs to solve, and several tools to be manufactured. This work answered the three main queries posed at the beginning of this rese 1. We proved that the target objects (production-aims) were also actually used as functional objects (functional-aims), i.e. a large number of items shows use-wear. 1. We proved that the target objects (production-aims) were also actually used as functional objects (functional-aims), i.e. a large number of items shows use-wear. 2. We noted that each production-aim actually comprised several techno-types employed for different purposes. Namely, through the technological approach we learned that the production-aims was flakes, convergent flakes and backed flakes. Thanks to this study, we perceive great diversity in both structure and functionality of these tools, noting that to each class of target objects corresponds a set of different functional objects, as each category actually comprises several blanks, and each blank could be the support for several techno-types. yp 3. Finally, we also proved that tools with a specific structure aimed to solve a specific task. yp 3. Finally, we also proved that tools with a specific structure aimed to solve a specif Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745 G. Marciani et al. 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Originality of the first lithic industries in central Europe, in the framework of the first settlements in Europe”) URL: http://www.theses.fr/2013PA100060 DOI: https://doi.org/10.2218/jls.2745 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx 30 G. Marciani et al. Ronchitelli, A., Ferguglia, M., Longo, L., Moroni, A. & Ranaldo, F. 2011, Studio tecno- funzionale dei supporti a morfologia triangolare dell’US 8 del Riparo L’Oscurusciuto (Ginosa - Taranto). Rivista di Scienze Preistoriche, 61(5-20): 22-22. (in Italian) (“Techno-functional analysis of triangular tools from layer 8 of the Oscurusciuto Rock Shelter (Ginosa, Taranto - southern Italy)”) Rots, V. 2010, Prehension and Hafting Traces on Flint Tools. A Methodology. Leuven University Press, Leuven, 296 p. URL: http://www.jstor.org/stable/j.ctt9qf05s Schlanger, N. Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx References 1996, Understanding Levallois: Lithic Technology and Cognitive Archaeology. Cambridge Archaeological Journal, 6(02): 231-254. doi:10.1017/s0959774300001724 Simondon, G. 1958, Du mode d’existence des objets techniques. Philosophie. Aubier, Paris, 340 p. (in French) (“On the mode of existence of technical objects”) Soriano, S. 2000, Outillage bifacial et outillage sur éclat au Paléolithique ancien et moyen : coexistence et interaction. Doctorat en Histoire thesis at Université Paris X- Nanterre, Nanterre, 459 p. (in French) (“Bifacial tools and flake tools of the Lower and Middle Palaeolithic: Coexistence and interaction”) URL: http://www.theses.fr/2000PA100137 Spagnolo, V. 2012, Analisi spaziale di un contesto musteriano: riparo l’Oscurusciuto (Ginosa - Ta). Tesi di laurea in Protostoria Europea thesis no. 10074504 at the Facoltà di Beni Culturali, Corso di Laurea Magistrale in Archeologia, Università del Salento, Lecce, 210 p. (in Italian) (“Spatial analysis of a Mousterian context: The Oscurusciuto shelter (Ginosa - Ta)”) URL: https://www.researchgate.net/publication/284723658 Spagnolo, V. 2017, Studio delle strategie insediative del Paleolitico Medio in Italia centro- meridionale. Doctoral thesis at the Scienze della Terra, Ambientali e Polari, Università di Siena, Siena, 443 p. (in Italian) (“Study of settlement strategies of the Middle Palaeolithic in central-southern Italy”) URL: http://hdl.handle.net/11365/1011000 Spagnolo, V., Marciani, G., Aureli, D., Berna, F., Boscato, P., Ranaldo, F. & Ronchitelli, A. 2016, Between hearths and volcanic ash: The SU 13 palimpsest of the Oscurusciuto rock shelter (Ginosa - Southern Italy): Analytical and interpretative questions. Quaternary International, 417: 105-121. doi:10.1016/j.quaint.2015.11.046 Tringham, R., Cooper, G., Odell, G., Voytek, B. & Whitman, A. 1974, Experimentation in the Formation of Edge Damage: A New Approach to Lithic Analysis. Journal of Field Archaeology, 1(1-2): 171-196. doi:10.1179/jfa.1974.1.1-2.171 Villa, P., Boscato, P., Ranaldo, F. & Ronchitelli, A. 2009, Stone tools for the hunt: points with impact scars from a Middle Paleolithic site in southern Italy. Journal of Archaeological Science, 36(3): 850-859. doi:10.1016/j.jas.2008.11.012 Journal of Lithic Studies (2018) vol. 5, nr. 2, p. xx-xx DOI: https://doi.org/10.2218/jls.2745
https://openalex.org/W4391364515	https://pubs.aip.org/aip/adv/article-pdf/doi/10.1063/5.0188122/19223851/015065_1_5.0188122.pdf	English	null	Two-relaxation-time lattice Boltzmann model of the velocity profiles and volumetric flow rate of generalized Newtonian fluids in a single-screw extruder	AIP advances	2,024	cc-by	11,034	RESEARCH ARTICLE \| JANUARY 30 2024 Two-relaxation-time lattice Boltzmann model of the velocity profiles and volumetric flow rate of generalized Newtonian fluids in a single-screw extruder Zhuo Meng  ; Liguo Liu ; Yujing Zhang; Yize Sun Zhuo Meng  ; Liguo Liu ; Yujing Zhang; Yize Sun AIP Advances 14, 015065 (2024) https://doi.org/10.1063/5.0188122 AIP Advances 14, 015065 (2024) https://doi.org/10.1063/5.0188122 RESEARCH ARTICLE \| JANUARY 30 2024 Two-relaxation-time lattice Boltzmann model of the velocity profiles and volumetric flow rate of generalized Newtonian fluids in a single-screw extruder Zhuo Meng  ; Liguo Liu ; Yujing Zhang; Yize Sun RESEARCH ARTICLE \| JANUARY 30 2024 AFFILIATIONS College of Mechanical Engineering, Donghua University, Shanghai 201620, China a)Author to whom correspondence should be addressed: mz@dhu.edu.cn ABSTRACT Single-screw extruders and injection molding machines are essential equipment in polymer processing. It is of great importance for the optimization of operating parameters and the design of extrusion screw to predict the throughput of an extruder and the metering time of an injection molding machine according to the geometric parameters of the screw, operating parameters, and the rheological behavior of materials. Most polymer melts exhibit non-Newtonian behavior. The lattice Boltzmann method has many advantages in simulating the flow of non-Newtonian fluids. Herein, the dimensionless velocity profiles and dimensionless volumetric flow rate of generalized Newtonian fluids in a screw channel have been studied using the two-relaxation-time lattice Boltzmann method (TRT–LBM). The numerical results of power- law fluids are in good agreement with the analytical solutions, which verifies the validity of TRT–LBM. Through research, the change rule of the dimensionless volumetric flow rate of Bingham fluids with dimensionless pressure gradient has been obtained. It was found that the rheological properties of polymer melts and the dimensionless pressure gradient significantly affect the dimensionless velocity profiles and dimensionless volumetric flow rate. The dimensionless volume flow rate has some unexpected changes with the increase of the dimensionless pressure gradient. This study can provide theoretical guidance for the optimization of operating parameters and the design of extrusion screws. 24 October 2024 04:01:23 © 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0188122 Two-relaxation-time lattice Boltzmann model of the velocity profiles and volumetric flow rate of generalized Newtonian fluids in a single-screw extruder Cite as: AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 Submitted: 19 November 2023 • Accepted: 4 January 2024 • Published Online: 30 January 2024 Cite as: AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 Submitted: 19 November 2023 • Accepted: 4 January 2024 • Published Online: 30 January 2024 Zhuo Meng,a) Liguo Liu, Yujing Zhang, and Yize Sun Articles You May Be Interested In Predicting the pumping capability of single-screw extruders: A comparison of two- and three-dimensional modeling approaches AIP Conf. Proc. (May 2023) Time‐dependent compressible extrudate‐swell problem with slip at the wall J. Rheol. (November 1994) Flow analysis of screw extrusion in three-dimensional concrete printing Physics of Fluids (February 2024) Predicting the pumping capability of single-screw extruders: A comparison of two- and three-dimensional modeling approaches 24 October 2024 04:01:23 AIP Advances ARTICLE pubs.aip.org/aip/adv I. INTRODUCTION In early studies, polymer melts were considered Newtonian fluids, and the analytical solutions of polymer melts flow in the down-channel and cross-channel were obtained. However, most polymer melts exhibit non-Newtonian behavior. The governing equations of non- Newtonian fluids are nonlinear. The mathematical analysis becomes very complicated. Therefore, some researchers began to use the numerical simulation method to study the flow of polymer melt in an extruder.1,2 McKelvey,6 Tadmor, and Gogos7 summarized and expanded these basic studies. p y gll The two-relaxation-time lattice Boltzmann method (TRT–LBM) was proposed by Ginzburg et al.32 in 2005. The TRT–LBM absorbs the simple and efficient advantages of the SRT–LBM and the stable and accurate advantages of the MRT–LBM.33–35 There are two relaxation times τ+ and τ−in the TRT–LBM. The relaxation time τ+ is determined by the apparent viscosity v(˙γ) of the fluid. The relaxation time τ−is a free parameter. The magic parameter Λ is a function of the relaxation time τ+and τ−. If the apparent viscosity in the fluid domain changes, τ+ also changes, and by adjusting τ−to keep Λ constant, the accuracy error of TRT–LBM can be independent of viscosity.8,33–35 For non-Newtonian fluids, the v(˙γ) of each grid point in the flow field is very different. Therefore, TRT–LBM is very suitable for simulating the flow of non-Newtonian fluids. However, there are few studies on the non-Newtonian fluids flow using TRT–LBM.li The lattice Boltzmann method (LBM) has been widely used in porous media flow, multiphase flow, and blood flow.8,9 LBM can calculate shear rate tensor locally without calculating velocity gra- dient and is easy to parallel.10–12 The single-relaxation-time lattice Boltzmann method (SRT–LBM) has been widely used. Boyd and Buick13,14 studied three-dimensional non-Newtonian blood flow in the human carotid artery using the SRT–LBM and analyzed the blood velocity and shear rate distribution. Chen et al.15 studied the convective heat transfer of generalized Newtonian fluids in an impeller-stirred tank using the SRT–LBM. Deng et al.16 proposed an injection molding simulation method based on the SRT–LBM to simulate the filling process of polymer melt in the mold. Young17 simulated the low Reynolds number flow of polymer melts using SRT–LBM. Buick18 studied the flow of Newtonian and power-law fluids in a screw channel using SRT–LBM and discussed the effects of different power-law indices on the flow field. 24 October 2024 04:01:23 24 October 2024 04:01:23 For non-Newtonian fluids, the relaxation time τ varies with the apparent viscosity v(˙γ) of the fluid. According to the equation τ = 3v(˙γ) + 0.5, if the apparent viscosity v(˙γ) ≫0.17, the relax- ation time τ ≫1. When the apparent viscosity v(˙γ) approaches 0, the relaxation time τ approaches 0.5. The truncation error of SRT–LBM is proportional to (τ/Δt −1/2)2.20 If τ ≫1, the accu- racy of SRT–LBM will decrease. The necessary condition for the stability of SRT–LBM is that the relaxation time τ > 1/2.8 If τ approaches 0.5, the stability of SRT-LBM will decrease. In the equa- tion τ = 3v(˙γ) + 0.5, the units of relaxation time τ and apparent viscosity v(˙γ) are lattice units. The physical units of the actual fluid can be easily converted into lattice units. To overcome the short- comings of SRT–LBM, some researchers have made improvements to SRT–LBM.12,21–25 The dimensionless volumetric flow rate determines the throughput of a single-screw extruder and the metering time of an injection molding machine. The dimensionless pressure gradient is determined by the geometric parameters of the screw, operat- ing conditions, and polymer materials. Therefore, the study of the relationship between the dimensionless volumetric flow rate of gen- eralized Newtonian fluids and the dimensionless pressure gradient can provide theoretical guidance for the optimization of operating parameters and the design of extrusion screw. McKelvey6 studied the dimensionless volumetric flow rate of a Newtonian fluid in a screw channel and obtained an analytical solution. Hirshberger7,36 studied the change rule of the dimensionless volumetric flow rate of power-law fluids with the dimensionless pressure gradient and obtained the analytical solution. However, the unknown parameters in the analytical solution still need to be obtained by numerical cal- culation. Béreaux et al.37 developed a model that can be used to The multiple-relaxation-time lattice Boltzmann method (MRT–LBM) was proposed by Humières26 in 1992. MRT-LBM has multiple free parameters. Compared with SRT–LBM, MRT–LBM has great advantages in calculation accuracy and stability. Some researchers have studied the flow of non-Newtonian fluids using MRT–LBM.27–31 In their study, they chose the same set of relax- ation parameters and did not give an optimization method for the relaxation parameters. This set of relaxation parameters is S = diag(0, 1.1, 1.0, 0, 1.2, 0, 1.2, 1/τ, 1/τ). I. INTRODUCTION extruder and the metering time of an injection molding machine according to the geometric parameters of the screw, operating para- meters, and the rheological behavior of materials. The plasticizing and mixing process of polymer is carried out in a closed barrel, so it is difficult to observe the plasticizing and mixing process of polymer. Therefore, it is of great importance to study the effects of screw geometric parameters, operating parameters, and the rheo- logical behavior of materials on the extrusion process by using the mathematical modeling method.l Single-screw extruder is the essential equipment in the produc- tion process of pipe, plate, profile, film, and so on.1,2 Single-screw extruders are widely used in mixing, granulation, and chemical reac- tion of polymer materials. Single-screw extruders are also commonly used in food processing, pharmaceutical processing, feed process- ing, and 3D printing. An injection molding machine is the essential equipment of plastic product molding. Some researchers have studied the complex flow of polymer melts in the screw channel of an extruder. Rowell and Finlayson3 first analyzed the flow of viscous oil in the screw channel of an extruder. They simulated the drag flow and pressure flow of isother- mal Newtonian fluids. Carley et al.4 applied the analytical method The production capacity of an extruder is determined by the dimensionless volumetric flow rate. The working cycle of an injec- tion molding machine is determined by the metering time. It is of great importance for the optimization of operating parameters and the design of extrusion screw to predict the throughput of an AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-1 14, 015065-1 ARTICLE AIP Advances pubs.aip.org/aip/adv only through the study of relaxation parameters and linear stability analysis, it can give full play to its advantages.8 However, for non- Newtonian fluids, it is difficult to improve the accuracy and stability through the relaxation parameter optimization and linear stability analysis because MRT–LBM has many relaxation parameters.8 Therefore, the MRT–LBM faces difficulty in reaching its highest potential when studying the flow of non-Newtonian fluids. of Rowell and Finlayson to study the flow of polymer melt in the screw channel. Yao et al.5 studied the flow pattern of Newtonian fluids in the screw channel using the finite difference method. I. INTRODUCTION Mohebbi et al.19 used the SRT–LBM to study the flow and convective heat transfer of power-law fluids between parallel plates containing partially porous media. ll When using LBM to simulate actual flow fields, there are two methods to choose from. The first method is to use the LBM to simulate the actual flow field directly. The second method is to use the similarity theory to dimensionless the parameters of the actual flow field, and then simulate. When the first method is adopted, the physical units of the actual flow field parameters, such as velocity, density, viscosity, and geometric parameters, need to be converted into lattice units first and then simulated. The τ has great effects on the accuracy and stability of SRT–LBM. The τ of SRT–LBM can- not approach 0.5 and cannot be much greater than 1. According to the equation τ = 3v(˙γ) + 0.5, the v(˙γ) of the actual fluid can- not be much greater than 0.17 (lattice unit) and cannot approach 0. TRT–LBM can effectively overcome these shortcomings of SRT-LBM. When the second method is adopted, the parameters of the actual flow field need to be dimensionless according to the sim- ilarity theory first and then simulated. The simulation parameters can be flexibly adjusted to make the apparent viscosity within a reasonable range under the condition that the dimensionless para- meters, such as Reynolds number, dimensionless pressure gradient, and geometric parameters, remain unchanged.l III. NUMERICAL METHOD AND BOUNDARY CONDITIONS For the power-law model, the apparent viscosity can be expressed as40 24 October 2024 04:01:23 It was selected according to the research of Lallemand and Luo.26 It was not the optimal parameters obtained through mathematical analysis. According to previous studies, the relaxation parameters considerably affect the accuracy and stability of MRT–LBM.8,31 For the MRT–LBM, 14, 015065-2 14, 015065-2 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 AIP Advances pubs.aip.org/aip/adv FIG. 1. Mixing section of extruders and injection molding machines. FIG. 1. Mixing section of extruders and injection molding machines. n > 1 represent Newtonian fluids, shear-thinning fluids, and shear- thickening fluids, respectively. predict the throughput of an extruder and the metering time of an injection molding machine. However, this model is only applica- ble to power-law fluids. It is difficult to obtain analytical solutions for non-Newtonian fluids. Therefore, numerical simulation is very important for studying non-Newtonian fluids. Herein, the dimen- sionless velocity profiles and dimensionless volumetric flow rate of generalized Newtonian fluids in a screw channel have been studied by using TRT–LBM for the first time. II. GENERALIZED NEWTONIAN FLUIDS FLOW A Th l fl id A. The power-law fluids B. The Bingham fluids For the Bingham model, the constitutive equations can be expressed as41 ⎧⎪⎪⎪⎪⎨⎪⎪⎪⎪⎩ τ = ( τ0 ∣˙γ∣+ μp)˙γ, ∣τ∣≥τ0, ˙γ = 0, ∣τ∣< τ0, (2) i The extrusion screw of the extruder and injection molding machine is composed of solid conveying section, melting section, and mixing section. The mixing section is shown in Fig. 1(a). It mainly consists of a screw and a barrel. In general, the effects of the curvature of the screw and barrel on melt flow can be ignored, and the screw can be unwound along the down-channel direction.6,7 The screw can be considered fixed, and the barrel can be considered as rotating according to the principle of reverse motion.38 Therefore, as shown in Fig. 1(b), the model of the mixing section can be simplified as a moving flat plate and a rectangular channel.6,39 In general, the ratio of height H to width W of the screw channel is given as H/W < 0.1. As shown in Fig. 1(c), when the effects of screw flight on melt flow are ignored, the model can be simplified into one-dimensional form.6,7 Figure 1(c) shows that the polymer melts are driven by two forces: one is the drag force exerted on the surface of the barrel and the other is the flow resistance exerted by the end of the extruder and injection molding machine.6,7 (2) where μp is the plastic viscosity, τ is the shear stress tensor, and τ0 is the Bingham yield stress. To overcome the discontinuous character in Eq. (2), a modified equation has been proposed by Papanastasiou,42 which can be expressed as τ = [μp + (1 −e−m∣˙γ ∣) τ0 ∣˙γ∣]˙γ, (3) (3) where m is the stress growth exponent. The apparent viscosity can be determined as follows: where m is the stress growth exponent. The apparent viscosity can be determined as follows: v(˙γ) = τ ˙γ = μp + (1 −e−m∣˙γ ∣) τ0 ∣˙γ∣. (4) (4) A. The TRT–LBM v(˙γ) = m∣˙γ∣n−1, (1) The TRT–LBM was developed by Ginzburg et al.32 The TRT–LBM absorbs the simple and efficient advantages of the SRT–LBM and the stable and accurate advantages of the MRT–LBM.33–35 (1) where m is the flow consistency coefficient of fluids, ˙γ is the shear rate tensor, n is the power-law index of fluids, and n = 1, n < 1, and 14, 015065-3 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-3 © Author(s) 2024 AIP Advances For the D2Q9 TRT–LBM, the evolution equation can be expressed as For the D2Q9 TRT–LBM, the evolution equation can be expressed as fi(x + eiΔt, t + Δt) = fi(x, t) + Ωi(x, t), (i = 0 −8), (5) (5) where fi is the distribution function, Ωi is the collision operator, and ei are the discrete velocity vectors of the D2Q9 model (see Fig. 2), which can be determined by ei = ⎧⎪⎪⎪⎪⎪⎨⎪⎪⎪⎪⎪⎩ (0, 0), i = 0, (cos ((i −1)π/2), sin ((i −1)π/2)), i = 1, 2, 3, 4, √ 2(cos ((i −1)π/2 + π/4), sin ((i −1)π/2 + π/4)), i = 5, 6, 7, 8. (6) (6) where f eq i and f eq i are the equilibrium distribution functions of ei and ei (ei = −ei), respectively, and the symmetric part f eq+ i and antisymmetric part f eq− i can be expressed as For TRT–LBM, the distribution function can be decomposed into the symmetric part f + i and antisymmetric part f − i , fi = f + i + f − i , fi = f + i −f − i , (7) (7) (7) f eq+ i = 1 2(f eq i + f eq i ), f eq− i = 1 2(f eq i −f eq i ). A. The TRT–LBM (11) (11) where fi and fi are the distribution functions of ei and ei (ei = −ei), respectively, and the symmetric part f + i and antisymmetric part f − i can be expressed as where fi and fi are the distribution functions of ei and ei (ei = −ei), respectively, and the symmetric part f + i and antisymmetric part f − i can be expressed as The magic parameter Λ is a function of the relaxation time τ+ and τ−, and τ−, f + i = 1 2(fi + fi), f − i = 1 2(fi −fi), (8) (8) Λ = (τ+ −1 2)(τ−−1 2). (12) (12) where f eq i is the equilibrium distribution function of the D2Q9 model, which is given by43 The relaxation time τ+ can be determined by The relaxation time τ+ can be determined by τ+ = 3v(˙γ) + 1 2. (13) f eq i (x, t) = ωiρ[1 + ei ⋅u c2 s + (ei ⋅u)2 2c4 s −u2 2c2 s ], (9) (13) (9) The relaxation time τ−can be calculated using Eq. (12), which can be determined by where ω0 = 4/9, ωi = 1/9 for i = 1–4, and ωi = 1/36 for i = 5–8, cs = 1/ √ 3 is the sound speed. τ−= 2Λ 2τ+ −1 + 1 2. (14) / The equilibrium distribution function of TRT–LBM also can be decomposed into the symmetric part f eq+ i and antisymmetric part f eq− i , (14) f eq− i , For TRT–LBM, the collision operator Ωi was given by Ginzburg et al 32 as For TRT–LBM, the collision operator Ωi was given by Ginzburg et al.32 as et al.32 as f eq i = f eq+ i + f eq− i , f eq i = f eq+ i −f eq− i , (10) f eq i = f eq+ i + f eq− i , f eq i = f eq+ i −f eq− i , ( FIG. 2. Discrete velocity vectors of the D2Q9 model at a grid point. (10) (10) Ωi(x, t) = −ω+(f + i (x, t) −f eq+ i (x, t)) −ω−(f − i (x, t) −f eq− i (x, t)), (15) where ω+ and ω−are the relaxation rates; ω+ = 1/τ+ and ω−= 1/τ−. A. Dimensionless parameters The actual flow field can be dimensionless according to the sim- ilarity theory. The dimensionless parameters of the flow field can be defined as Sαβ = − 1 2ρc2 s τ+Δt 8 ∑ i=0 eiαeiβ[fi(x, t) −f eq i (x, t)]. (20) (20) x∗= x H , y∗= y H , v∗ z = vz Vbz , (26) (26) The density ρ(x, t) and velocity u(x, t) of the fluid can be calculated directly by where H is the height of the screw flight and Vbz is the velocity component of the barrel in the down-channel direction of the screw. For power-law fluids, the Reynolds number can be defined as where H is the height of the screw flight and Vbz is the velocity component of the barrel in the down-channel direction of the screw. F l fl id th R ld b b d fi d ρ(x, t) = 8 ∑ i=0 fi(x, t), u(x, t) = 1 ρ(x, t) 8 ∑ i=0 ei fi(x, t). (21) p For power-law fluids, the Reynolds number can be defined as (21) Rep = Vbz 2−nHn m , (27) (27) A. The TRT–LBM The evolutionary steps of TRT–LBM: where ω+ and ω−are the relaxation rates; ω+ = 1/τ+ and ω−= 1/τ−. The evolutionary steps of TRT–LBM: (1) The collision step, (1) The collision step, (1) The collision step, f ∗ i (x, t) = fi(x, t) −ω+(f + i (x, t) −f eq+ i (x, t)) −ω−(f − i (x, t) −f eq− i (x, t)), (16) (16) where f ∗ i (x, t) is the distribution function after collisions. where f ∗ i (x, t) is the distribution function after collisions. 2) The propagation step, f i ( ) (2) The propagation step, fi(x + eiΔt, t + Δt) = f ∗ i (x, t). (17) (17) For power-law fluids and Bingham fluids, the apparent viscosity v(˙γ) can be calculated using Eqs. (1) and (4), respectively. The shear rate ˙γ can be defined as FIG. 2. Discrete velocity vectors of the D2Q9 model at a grid point. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-4 14, 015065-4 © Author(s) 2024 pubs.aip.org/aip/adv ˙γ = √ 2DII, (18) (18) where u1 represents the velocity at nodes x1, uN represents the velocity at nodes xN, x0 = x1 −Δx is an additional layer of virtual nodes at the inlet, xN+1 = xN + Δx is an additional layer of virtual nodes at the outlet, Pin and Pout are the pressure values at the inlet and outlet, respectively, and the pressure drop Δp = pout −pin. where DII is the second invariant of the strain-rate tensor, which can be determined by where DII is the second invariant of the strain-rate tensor, which can be determined by DII = 2 ∑ α,β=1 SαβSαβ, (19) (19) B. Boundary conditions All the unknown f ∗ i at the virtual nodes can be derived from the following equations: The dimensionless volumetric flow rate can be defined as The dimensionless volumetric flow rate can be defined as q∗= q qd , (31) (31) where q∗represents the dimensionless volumetric flow rate, q is the flow rate of pressure flow, and qd is the flow rate of drag flow. where q∗represents the dimensionless volumetric flow rate, q is the flow rate of pressure flow, and qd is the flow rate of drag flow. g Inlet boundary condition: IV. NUMERICAL SIMULATION where Sαβ is the strain-rate tensor at each node, which can be deter- mined locally by the following equation that has been proposed by Artoli:44 B. Boundary conditions At the moving and stationary walls, the non-equilibrium extrapolation non-slip boundary conditions are applied following the work of Guo et al.45 According to the non-equilibrium extrap- olation scheme, the fi at the boundary nodes can be decomposed into the equilibrium part f (eq) i and non-equilibrium part f (neq) i . The f (eq) i is computed by using the velocity of the boundary node and the density of the nearest fluid node. The f (neq) i at the boundary nodes is replaced by the f (neq) i at the nearest fluid nodes. Therefore, all the unknown fi at the boundary can be determined by the following equations: where m is the flow consistency coefficient and n is the power-law index. For Bingham fluids, the Reynolds number and Bingham number can be defined as ReB = VbzH μp , Bn = τ0H μpVbz , (28) (28) where τ0 is the Bingham yield stress and μp is the plastic viscosity.l p The dimensionless pressure gradient of power-law fluids can be defined as GP = Hn+1 6mVn bz (∂P ∂z ), (29) fi(xb, t) = f (eq) i (xb, t) + f (neq) i (xb, t), (22) (29) (22) fi(xb, t) = f (eq) i (ρ(x f , t), uw) + [fi(x f , t) −f (eq) i (x f , t)], (23) where ∂P/∂z is the pressure gradient in the down-channel direction. For Bingham fluids, the dimensionless pressure gradient can be defined as where ∂P/∂z is the pressure gradient in the down-channel direction. For Bingham fluids, the dimensionless pressure gradient can be defined as defined as where xb represents the boundary node, x f represents the nearest fluid node, and uw are the velocities of moving walls and stationary walls, i = 4, 7, 8 for moving walls, i = 2, 5, 6 for stationary walls. GB = H2 6μpVbz (∂P ∂z ). (30) (30) At the inlet and outlet, we impose the generalized periodic boundary conditions that have recently been proposed by Kim and Pitsch.46 We add an additional layer of virtual nodes at the inlet and outlet, respectively. The fi at the virtual nodes can be decomposed into the equilibrium part f (eq) i and non-equilibrium part f (neq) i . A. Validation of the TRT–LBM for non-Newtonian fluids f q f i (2) The f + i and f − i can be calculated using Eq. (8), respec- tively. The f eq+ i and f eq− i can be calculated using Eq. (11), respectively. At present, there are few studies on the non-Newtonian flu- ids flow using TRT–LBM. To verify the validity of TRT–LBM, the Poiseuille flow of power-law fluids and Bingham fluids between par- allel plates was simulated. At the inlet and outlet, we impose the generalized periodic boundary conditions. The constant pressure gradient is ∇P = −8 × 10−6. The non-equilibrium extrapolated no- slip boundaries are applied to the upper and lower walls. The grid number 51 × 19 is fine enough for the two-dimensional Poiseuille flow.ll p y (3) Implement the collision step using Eq. (16). (4) The generalized periodic boundary conditions are applied at the inlet and outlet. All the unknown f ∗ i on these virtual nodes can be determined by Eqs. (24) and (25). 24 October 2024 04:01:23 (5) Implement the propagation step using Eq. (17). (6) The non-equilibrium extrapolation non-slip boundary con- ditions are applied at the moving and stationary walls. All the unknown fi at the boundary can be calculated using Eq. (23). (6) The non-equilibrium extrapolation non-slip boundary con- ditions are applied at the moving and stationary walls. All the unknown fi at the boundary can be calculated using Eq. (23). (7) Calculate the ˙γ, DII, and Sαβ at each node using Eqs. (18)–(20), respectively. l First, we took the Poiseuille flow of power-law fluids as an example to verify the TRT–LBM. The analytical solution can be expressed as f y g q (7) Calculate the ˙γ, DII, and Sαβ at each node using Eqs. (18)–(20), respectively.l q p y (8) Calculate the v(˙γ) of the power-law fluids and the Bingham fluids using Eqs. (1) and (4), respectively. The τ+ can be derived from Eq. (13). The τ−can be calculated by Eq. (14). (9) Calculate the ρ and u using Eq. (21). q p y (8) Calculate the v(˙γ) of the power-law fluids and the Bingham fluids using Eqs. (1) and (4), respectively. The τ+ can be derived from Eq. (13). The τ−can be calculated by Eq. (14). u(y) = n n + 1(−1 μp ∂P ∂x ) 1/n [(H 2 ) n+1/n −∣y∣ n+1 n ], (33) (33) FIG. 3. Dimensionless velocity profiles of the Poiseuille flow. B. Simulation parameters f ∗ i (x0, y, t) = f (eq) i (Pin, uN) + (f ∗ i (xN, y, t) −f (eq) i (xN, y, t)), i = 1 , 5 , 8. (24) As shown in Fig. 1(c), the computational domain is divided into 51 × 19 grid points in the y- and z-directions, respectively. In addition, to impose generalized periodic boundary conditions, it is needed to add a layer of virtual nodes at the inlet and outlet, respectively. After verification, the grid point number 51 × 19 is fine enough to derive grid-independent results. The Reynolds number of polymer melt flow is generally very small.7 The Reynolds number is (24) Outlet boundary condition: Outlet boundary condition: f ∗ i (xN+1, y, t) = f (eq) i (Pout, u1) + (f ∗ i (x1, y, t) −f (eq) i (x1, y, t)), i = 3 , 6 , 7 , (25) (25) AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-5 © Author(s) 2024 AIP Advances (10) Go back to step (2), proceed to the next time step, until the following convergence condition is satisfied: set to 1 (Rep = ReB = 1). For power-law fluids, the n varies from 0.2 to 1.7, and the dimensionless pressure gradient Gp varies from −1 to 1. For Bingham fluids, the Bingham number Bn varies from 0 to 3, and the dimensionless pressure gradient GB varies from −1.5 to 1.5. The magic parameter Λ in Eq. (12) is set to 1/6. If the v(˙γ) in the fluid domain changes, τ+ also changes, and by adjusting τ−to keep Λ constant, the accuracy error of TRT–LBM can be independent of viscosity.8,33–35 ∑∣u(x, t + 40) −u(x, t)∣ ∑∣u(x, t)∣ ≤10−7, (32) (32) where u(x, t) represents the velocity at the grid point x when the TRT–LBM program iterates “t” steps. u(x, t + 40) represents the velocity at the grid point x when the TRT–LBM program iterates “t + 40” steps. To save computational time and memory, we make the TRT–LBM program judge whether the convergence condition is met every 40 steps, while saving the data and updating the flow field image. V. NUMERICAL RESULTS AND DISCUSSION A. Validation of the TRT–LBM for non-Newtonian fluids (1) Initialize the flow field, setting the density ρ(x, t) = 1 and the velocity u(x, t) = 0. The f eq i can be calculated using Eq. (9). Make the fi equal to the f eq i . C. Implementation steps of TRT–LBM for generalized Newtonian fluids The TRT–LBM of generalized Newtonian fluids can be imple- mented using the following procedure. Herein, we used MATLAB software 2018a for calculation. the maximum velocity can be derived as Second, we took the Poiseuille flow of Bingham fluids as an example to verify the TRT–LBM. The analytical solution can be expressed as the maximum velocity can be derived as um velocity can be derived as umax = n n + 1(−1 μp ∂P ∂x ) 1/n (H 2 ) n+1/n , (34) Second, we took the Poiseuille flow of Bingham fluids as an example to verify the TRT–LBM. The analytical solution can be expressed as umax = n n + 1(−1 μp ∂P ∂x ) 1/n (H 2 ) n+1/n , (34) example to verify the TRT–LBM. The analytical solution can be expressed as umax = n n + 1(−1 μp ∂P ∂x ) 1/n (H 2 ) n+1/n , (34) ex ex u(y) = ⎧⎪⎪⎪⎪⎪⎪⎨⎪⎪⎪⎪⎪⎪⎩ 1 2up (−∂P ∂x )[(H 2 ) 2 −y2 τ] −τ0 up (H 2 −yτ), 0 ≤∣y∣≤yτ, 1 2up (−∂P ∂x )[(H 2 ) 2 −y2] −τ0 up (H 2 −∣y∣), yτ < ∣y∣≤H 2 , (36) and the dimensionless velocity can be expressed as u∗= u(y) umax . (35) (35) We simulated the velocity profiles of Poiseuille flow under dif- ferent n (n = 0.3, 0.6, 1, 1.3, and 1.7) using the TRT–LBM. As shown in Fig. 3(a), the TRT–LBM results agree well with the analytical solutions. where yτ = −τ0/(∂P/∂x) indicates the yield point of the material. The maximum velocity can be derived from Eq. (36) as where yτ = −τ0/(∂P/∂x) indicates the yield point of the material. The maximum velocity can be derived from Eq. (36) as FIG. 4. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 5. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 0.5. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 4. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. 24 October 2024 04:01:23 less velocity profiles of power-law fluids at different Gp values when n = 1. The dots and solid lines represent the TRT–LBM results an FIG. 4. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 1. A. Validation of the TRT–LBM for non-Newtonian fluids The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. ss velocity profiles of the Poiseuille flow. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 3. Dimensionless velocity profiles of the Poiseuille flow. The dots and solid lines represent the TRT–LBM results and analytical so FIG. 3. Dimensionless velocity profiles of the Poiseuille flow. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-6 14, 015065-6 © Author(s) 2024 AIP Advances the maximum velocity can be derived as The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 5. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 0.5. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 5. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 0.5. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 14, 015065-7 © Author(s) 2024 AIP Advances ARTICLE pubs.aip.org/aip/adv AIP Advances umax = 1 2up (−∂P ∂x )[(H 2 ) 2 −y2 τ] −τ0 up (H 2 −yτ), (37) McKelvey’s analytical solution can be expressed as McKelvey’s analytical solution can be expressed as (37) v∗ z = y∗(1 −3Gp + 3y∗Gp). (39) (39) and the dimensionless velocity can be expressed as Hirshberger’s analytical solution can be expressed as u∗= u(y) umax . (38) (38) v∗ z = vz Vbz = ∣6Gp∣s sign Gp (1 + s) (∣y∗−λ∣ 1+s −∣λ∣1+s) = ∣y∗−λ∣ 1+s −∣λ∣1+s ∣1 −λ∣1+s −∣λ∣1+s (40) We simulated the velocity profiles of Poiseuille flow under dif- ferent yield stresses τ0 (τ0 = 0, 0.000 04, 0.000 08, 0.000 12). As shown in Fig. 3(b), the TRT–LBM results agree well with the analytical solutions. (40) and and B. Simulation and verification of the dimensionless velocity profiles and volumetric flow rate of power-law fluids in a screw channel ∣λ∣1+s −∣1 −λ∣1+s + 1 + s ∣6Gp∣s sign Gp = 0, (41) (41) where s = 1/n and sign Gp = Gp ∣Gp∣. where s = 1/n and sign Gp = Gp ∣Gp∣. where s = 1/n and sign Gp = Gp ∣Gp∣. McKelvey6 studied the dimensionless velocity profiles and vol- umetric flow rate of a Newtonian fluid in a screw channel and obtained an analytical solution. Hirshberger7,36 studied the change rule of the dimensionless volumetric flow rate of power-law fluids with the dimensionless pressure gradient and obtained the analytical solution. Herein, we studied the dimensionless velocity profiles and volumetric flow rate of power-law fluids in a screw channel using TRT–LBM and compared them with the analytical solutions. ∣ p∣ λ can be solved by Eq. (41) combined with the following conditions: ∣ p∣ λ can be solved by Eq. (41) combined with the following conditions: if ∣Gp∣≥1 6(1 + s)n then 0 ≤λ ≤1, if ∣Gp∣≤1 6(1 + s)n and if Gp > 0 then λ ≤0, if ∣Gp∣≤1 6(1 + s)n and if Gp < 0 then λ ≥1. (42) (42) 1. Effects of power-law index and dimensionless pressure gradient on the dimensionless velocity profiles As shown in Figs. 4–6, n and Gp have considerable effects on the velocity profiles. As shown in Figs. 4(a)–6(a), when the dimensionless pressure gradient Gp ≤0, the dimensionless velocity v∗ z ≥0. There is no circulation flow in the fluid domain. For a fixed n, with the increase in the absolute value of Gp, the dimension- less velocity v∗ z increases corresponding to the same y∗-position. As shown in Figs. 4(b)–6(b), when the dimensionless pressure gradient Gp = 0 or 1/3, the dimensionless velocity v∗ z gradually decreases from 1 to 0 along the y∗-position. Therefore, there is no circulation flow 24 October 2024 04:01:23 The down-channel dimensionless velocity profiles of power- law fluids at different Gp values when n = 1, 0.5, and 1.5 are shown in Figs. 4–6, respectively. The dimensionless pressure gradients Gp are −1, −2/3, −1/3, 0, 1/3, 2/3, and 1, respectively. When the n = 1, the TRT–LBM results of the velocity profiles are in good agreement with the analytical solutions of Mckelvey.6,7 When the n = 0.5 and 1.5, the TRT–LBM results of velocity profiles are in good agreement with the analytical solutions of Hirshberger.7,36 FIG. 6. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 1.5. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 6. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 1.5. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 6. Down-channel dimensionless velocity profiles of power-law fluids at different Gp values when n = 1.5. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-8 © Author(s) 2024 AIP Advances q = VbzWH 2 −WH3 12μ (∂P ∂z ), (43) qd = VbzWH 2 , (44) q∗= q qd = 1 − H2 6μVbz (∂P ∂z ) = 1 −GP. (45) q = VbzWH 2 −WH3 12μ (∂P ∂z ), (43) in the fluid domain. When the dimensionless pressure gradient Gp = 2/3 or 1, the dimensionless velocity v∗ z gradually decreases from 1 to 0 along the y∗-position and then becomes negative. There is a circulation flow in the fluid domain. (43) qd = VbzWH 2 , (44) (44) 2. Effects of power-law index and dimensionless pressure gradient on the dimensionless volumetric flow rate When n = 1.5 and Gp = 0, the v(˙γ) of the fluid domain increases uniformly. When Gp > 0, the velocities corresponding to n = 0.5, 1, and 1.5 are distributed along a nonlinear curve, and there are significant differences in shear rates in the fluid domain. Therefore, when n = 0.5 and Gp > 0, the v(˙γ) of the fluid domain decreases unevenly. When n = 1.5 and Gp > 0, the v(˙γ) of the fluid domain increases unevenly. Therefore, the negative velocity region at n = 0.5 is significantly larger than that at n = 1, as shown in Figs. 4(b) and 5(b). The negative velocity region at n = 1.5 is sig- nificantly smaller than that at n = 1, as shown in Figs. 4(b) and 6(b). As shown in Figs. 4(b)–6(b), when Gp > 2/3, the reflux area of shear-thinning fluids in the screw channel is significantly larger than that of shear-thickening fluids. Therefore, for a fixed Gp (Gp > 0), the dimensionless volumetric flow rate of shear-thickening fluids is significantly higher than that of shear-thinning fluids. (46) qd = VbzH 2 , (47) (47) q∗= q qd = 2∣6Gp∣s sign Gp (1 + s)(2 + s) × [(1 −λ)∣1 −λ∣1+s + λ∣λ∣1+s −(2 + s)∣λ∣1+s]. (48) (48) As shown in Figs. 7 and 8, n and Gp have considerable effects on q∗. The q∗determines the throughput of an extruder and the backward speed of the screw of an injection molding machine. For n < 1, the smaller n, the more pronounced the nonlinearity. For n > 1, the larger n, the more pronounced the nonlinearity. 2. Effects of power-law index and dimensionless pressure gradient on the dimensionless volumetric flow rate q∗= q qd = 1 − H2 6μVbz (∂P ∂z ) = 1 −GP. (45) (45) Figure 7 shows the effects of n and Gp of the shear-thinning flu- ids on q∗. The effects of n and Gp of the shear-thickening fluids on q∗ are shown in Fig. 8. As depicted in these figures, for Newtonian flu- ids (n = 1), there is a linear relationship between q∗and Gp, and the TRT–LBM results are in good agreement with the analytical solution of McKelvey.6,7 McKelvey’s analytical solution can be expressed as When n ≠1, the relationship between q∗and Gp is nonlinear, and the TRT–LBM results are in good agreement with the analytical solution of Hirshberger.7,36 Hirshberger’s analytical solution can be expressed as FIG. 7. Dimensionless volumetric flow rate of power-law fluids at different Gp values when n ≤1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 7. Dimensionless volumetric flow rate of power-law fluids at different Gp values when n ≤1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 8. Dimensionless volumetric flow rate of power-law fluids at different Gp values when n ≥1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. 24 October 2024 04:01:23 FIG. 7. Dimensionless volumetric flow rate of power-law fluids at different Gp values when n ≤1. The dots and solid lines represent the TRT–LBM results and analytical solutions respectively FIG. 8. Dimensionless volumetric flow rate of power-law fluids at different Gp values when n ≥1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 8. Dimensionless volumetric flow rate of power-law fluids at different Gp values when n ≥1. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-9 © Author(s) 2024 AIP Advances ARTICLE ARTICLE AIP Advances pubs.aip.org/aip/adv pubs.aip.org/aip/adv q = VbzH∣6Gp∣s sign Gp (1 + s)(2 + s) × [(1 −λ)∣1 −λ∣1+s + λ∣λ∣1+s −(2 + s)∣λ∣1+s], (46) qd = VbzH 2 , (47) q = VbzH∣6Gp∣s sign Gp (1 + s)(2 + s) × [(1 −λ)∣1 −λ∣1+s + λ∣λ∣1+s −(2 + s)∣λ∣1+s], (46) gradient (shear rate) is the same. Therefore, when n = 0.5 and Gp = 0, the apparent viscosity v(˙γ) of the fluid domain decreases uniformly. C. Simulation of the dimensionless velocity profiles and volumetric flow rate of Bingham fluids in a screw channel As shown in Figs. 7(a) and 8(a), when Gp ≤0, the q∗increases with the increase of the absolute value of Gp for a fixed n. For a fixed Gp (Gp < 0), the larger n, the smaller q∗. As shown in Figs. 7(b) and 8(b), when Gp > 0, the q∗decreases with the increase of Gp for a fixed n. For a fixed Gp (Gp > 0), the larger n, the larger q∗.l At present, there are no reports on the study of the dimension- less velocity profiles and volumetric flow rate of Bingham fluids in a screw channel. We studied the dimensionless velocity profiles and volumetric flow rates of Bingham fluids using TRT–LBM. Through research, the change rule of the dimensionless volumetric flow rate of Bingham fluids with the dimensionless pressure gradient has been obtained. i For Newtonian fluids (n = 1), when Gp = 1, q∗= 0. At this time, the extruder has no throughput, the screw of the injection molding machine does not move backward, and the polymer melts circu- late in the screw channel. For shear-thickening fluids, when Gp = 1, the q∗> 0. At this time, the extruder still has throughput, and the screw of the injection molding machine is still moving backward. For shear-thinning fluids, when Gp = 1, the q∗< 0. In actual production, this phenomenon does not exist. 24 October 2024 04:01:23 3. Effects of rheological properties of power-law fluids on flow field As shown in Figs. 4–6, when Gp = 0, the velocity profiles cor- responding to n = 0.5, 1, and 1.5 are the same, and the velocity Figure 9 shows the down-channel dimensionless velocity pro- files of Bingham fluids at different GB values when Bn = 0. The FIG. 9. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 0. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 9. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 0. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. FIG. 9. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 0. The dots and solid lines represent the TRT–LBM results and analytical solutions, respectively. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-10 © Author(s) 2024 AIP Advances ARTICLE pubs.aip.org/aip/adv FIG. 10. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 0.5. The dots represent the TRT–LBM results. The solid lines represent the line chart of the TRT–LBM results. AIP Advances pubs.aip.org/aip/adv FIG. 10. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 0.5. The dots represent the TRT–LBM results. The solid lines represent the line chart of the TRT–LBM results. As shown in Figs. 9–11, the dimensionless pressure gradient GB has great effects on the dimensionless velocity profiles. As shown in Figs. 9(a)–11(a), when the dimensionless pressure gradient GB ≤0, the dimensionless velocity v∗ z ≥0. There is no circulation flow in the fluid domain. For a fixed Bn, with the increase of the absolute value of GB, the dimensionless velocity v∗ z increases corresponding to the same y∗-position. As shown in Figs. 9(b)–11(b), when the dimensionless pressure gradient GB = 0 or 1/3, the dimensionless velocity v∗ z ≥0. There is no circulation flow in the fluid domain. When the dimensionless pressure gradient GB = 1, the dimension- less velocity gradually decreases along the y∗-position from 1 to 0 and then becomes negative, generating a circulation flow in the fluid domain. dimensionless pressure gradients GB are −1, −2/3, −1/3, 0, 1/3, 2/3, and 1, respectively. As can be seen from these figures, when Bn = 0, the Bingham fluids become Newtonian fluids. 3. Effects of rheological properties of Bingham fluids on flow field As shown in Figs. 9–12, when ∣GB∣< 0.4, the shear stress τ in the fluid domain is greater than the yield stress τ0 of the Bingham fluids, and the Bingham fluids exhibit the properties of Newtonian fluid. As shown in Figs. 9–11, when GB = 0 and 1/3, the velocity profiles for Bn = 0.5 and 1 are exactly the same as that of Newto- nian fluid (Bn = 0). As shown in Fig. 12, when ∣GB∣< 0.4, the q∗at Bn = 0.5 and 1 are exactly the same as those of Newtonian fluid (Bn = 0). When ∣GB∣≥0.4, the shear stress τ in some areas of the fluid domain is smaller than the τ0 of the Bingham fluids. At this time, the velocity in this part of the fluid domain remains constant and does not change with y∗. The shear rate in this area is 0, exhibiting elastic solid properties, as shown in Figs. 9–11. As shown in Fig. 12, when ∣GB∣≥0.4, the curve of q∗with GB is nonlinear, and the fluid exhibits non-Newtonian properties. 2. Effects of Bingham number and dimensionless pressure gradient on the dimensionless volumetric flow rate Figure 12 shows the effects of Bn and the dimensionless pres- sure gradient GB on the dimensionless volumetric flow rate q∗. As depicted in these figures, when Bingham number Bn = 0, the flu- ids are Newtonian fluids. As shown in Fig. 12(a), when the absolute value of the dimensionless pressure gradient ∣GB∣< 0.4, Bn has no effect on the q∗, and there is a linear relationship between q∗and GB. With the increase of ∣GB∣, the dimensionless volumetric flow rate q∗first increases greatly along the straight line with a slope of −1, then increases slowly along the nonlinear curve, and finally increases greatly along the straight line with a slope of approximately −1. It can be seen that the convex parts of curves corresponding to differ- ent Bingham numbers Bn are completely coincident, and the larger the Bn, the longer the convex part of curves. For a fixed GB, with the increase of Bn, the dimensionless volumetric flow rate q∗decreases or remains constant. 3. Effects of rheological properties of power-law fluids on flow field The TRT–LBM results of the velocity profiles are in good agreement with the analytical solution of Mckelvey.6,7 Figure 10 shows the down-channel dimensionless velocity pro- files of Bingham fluids at different GB values when Bn = 0.5. The dimensionless pressure gradients GB are −1, −2/3, −1/3, 0, 1/3, 2/3, and 1, respectively. Figure 11 shows the down-channel dimension- less velocity profiles of Bingham fluids at different GB values when Bn = 1. The dimensionless pressure gradients GB are −1, −2/3, −1/3, 0, 1/3, 2/3, and 1, respectively. FIG. 11. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 1. The dots represent the TRT–LBM results. The solid lines represent the line chart of the TRT–LBM results. FIG. 11. Down-channel dimensionless velocity profiles of Bingham fluids at different GB values when Bn = 1. The dots represent the TRT–LBM results. The solid lines represent the line chart of the TRT–LBM results. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 14, 015065-11 © Author(s) 2024 ARTICLE AIP Advances greatly along the straight line with a slope of approximately −1. It can be seen that the concave parts of the curves corresponding to different Bingham numbers Bn are completely coincident, and the larger the Bn, the longer the concave part of the curve. For a fixed GB, q∗increases or remains constant with the increase of Bn. By comparing Figs. 9(a)–11(a), it can be found that when GB = −1/3 or 0, the dimensionless velocity profiles are the same for the different Bingham numbers. By comparing Figs. 9(b)–11(b), it can be found that when GB = 1/3, the dimensionless velocity profiles are the same for the different Bingham numbers. Therefore, when GB = −1/3, 0, and 1/3, the Bn has no effect on the dimensionless velocity profiles. We also found that when GB = 2/3 or −2/3, the dimensionless velocity profiles of Bn = 0.5 and 1 are the same, but it is different from that of Bn = 0. Therefore, for the Bingham flu- ids, when GB is small, Bn has no effect on the dimensionless velocity profiles. D. Effects of the rheological properties of fluids and dimensionless pressure gradient on the throughput and the metering time We will further study the flow of viscoelastic fluids in a screw channel using TRT–LBM. of the q∗with dimensionless pressure gradient can provide theoret- ical guidance for the optimization of operating parameters and the design of extrusion screw.l As shown in Figs. 7, 8, and 12, there are some unexpected inflec- tion points in the curves. Therefore, the dimensionless volume flow rate may have some unexpected changes with the increase in the dimensionless pressure gradient. For extruders, the throughput may have some unexpected changes with the increase in die resistance. For injection molding machines, the metering time and the back- ward speed of the screw may have some unexpected changes with the increase in the back pressure. The same model can be used to simulate other generalized Newtonian fluids in the screw channel, such as Casson fluids and Herschel–Bulkley fluids. The constitutive equation of Casson fluids can be derived as47 v(˙γ) = { √τ0 ∣˙γ∣[1 −exp (− √ m∣˙γ∣)] + √μp} 2 . (49) (49) It can be seen from Eq. (49) that when τ0 = 0, the fluids become Newtonian fluids.l l For Herschel–Bulkley fluids, the constitutive equation can be derived as48,49 AUTHOR DECLARATIONS l In addition, many polymer melts exhibit viscoelasticity. How- ever, there are few studies on the viscoelastic fluids in the screw channel. LBM has some advantages when simulating viscoelastic fluids.50–54 The LBM can calculate the shear rate tensor locally without calculating the velocity gradient and is easy to parallel. D. Effects of the rheological properties of fluids and dimensionless pressure gradient on the throughput and the metering time 24 October 2024 04:01:23 In the actual production process, the pressure gradient in the mixing section of an extruder is determined by the die. The pressure gradient is generally not adjustable. However, the pressure gradient in the mixing section of an injection molding machine is determined by the back pressure. The back pressure is an important operat- ing parameter that can be easily adjusted by the relief valve. The throughput of an extruder and the metering time of an injection molding machine are determined by the q∗. Studying the change rule As shown in Fig. 12(b), when GB < 0.4, Bn has no effect on q∗, and there is a linear relationship between q∗and GB. With the increase of GB, the dimensionless volumetric flow rate q∗first decreases greatly along the straight line with a slope of −1, then decreases slowly along the nonlinear curve, and finally decreases FIG. 12. Dimensionless volumetric flow rate of Bingham fluids at different GB values. The dots represent the TRT–LBM results. The solid lines represent the line chart of the TRT–LBM results. FIG. 12. Dimensionless volumetric flow rate of Bingham fluids at different GB values. The dots represent the TRT–LBM results. The solid lines represent the line chart of the TRT–LBM results. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-12 © Author(s) 2024 ARTICLE AIP Advances pubs.aip.org/aip/adv 4. For power-law fluids, when Gp > 0 and n > 1, the shear thickening properties of the fluid make the apparent viscos- ity and internal friction in the fluid domain increase unevenly. When Gp > 0 and n < 1, the shear thinning properties of the fluid make the apparent viscosity and internal friction in the fluid domain decrease unevenly. For Bingham fluids, when ∣GB∣< 0.4, the shear stress τ in the fluid domain is greater than the yield stress τ0 of the Bingham fluids, and the Bingham flu- ids exhibit the properties of Newtonian fluid. When ∣GB∣≥0.4, the shear stress τ in some areas of the fluid domain is smaller than the τ0 of the Bingham fluids, and the fluid exhibits non- Newtonian properties. The rheological properties of polymer melts and the dimensionless pressure gradient significantly affect the dimensionless velocity profiles and volumetric flow rate. Therefore, the rheological properties of polymer melts and the dimensionless pressure gradient significantly affect the throughput of an extruder and the metering time of an injection molding machine. ACKNOWLEDGMENTS This work was supported by the International Coopera- tion Fund of Science and Technology Commission of Shanghai Municipality (Grant No. 21130750100). v(˙γ) = K∣˙γ∣n−1 + τ0 ∣˙γ∣[1 −exp (−m∣˙γ∣)]. (50) (50) 24 October 2024 04:01:23 It can be seen from Eq. (50) that when τ0 = 0, the fluids become power-law fluids. REFERENCES Phys. A: Math. Gen. 39, 14241–14247 (2006). 34L. S. Luo, W. Liao, X. Chen, Y. Peng, and W. Zhang, “Numerics of the lat- tice Boltzmann method: Effects of collision models on the lattice Boltzmann simulations,” Phys. Rev. E 83(5), 056710 (2011). ewtonian flow model,” J. Phys. A: Math. Gen. 39, 14241–14247 (2 24 October 2024 04:01:23 11O. Malaspinas, G. Courbebaisse, and M. Deville, “Simulation of generalized Newtonian fluids with the lattice Boltzmann method,” Int. J. Mod. Phys. C 18(12), 1939–1949 (2007). 35D. D’Humières and I. 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Author Contributions 1. The velocity distributions of the Poiseuille flow of power- law fluids and Bingham fluids have been simulated using TRT–LBM. The TRT–LBM results agree well with the analyt- ical solutions, which verifies the validity of TRT–LBM.i Zhuo Meng: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). Liguo Liu: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & edit- ing (equal). Yujing Zhang: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal). Yize Sun: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal). i 2. The dimensionless velocity profiles and dimensionless vol- umetric flow rate of power-law fluids under the different dimensionless pressure gradients have been studied using TRT–LBM. The numerical results of power-law fluids agree well with the analytical solutions, which verifies the validity of TRT–LBM. The effects of the different power-law indices and dimensionless pressure gradients on the dimensionless veloc- ity profiles and dimensionless volumetric flow rate have been compared.i p 3. The dimensionless velocity profiles and dimensionless vol- umetric flow rate of Bingham fluids under the different dimensionless pressure gradients have been studied using the TRT–LBM. Through research, the change rule of the dimen- sionless volumetric flow rate of Bingham fluids with dimen- sionless pressure gradient has been obtained. The effects of the different Bingham numbers and dimensionless pressure gradients on the dimensionless velocity profiles and dimen- sionless volumetric flow rate have been compared. AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 © Author(s) 2024 AIP Advances 14, 015065 (2024); doi: 10.1063/5.0188122 14, 015065-13 ARTICLE AIP Advances pubs.aip.org/aip/adv DATA AVAILABILITY 24W. Wu, X. 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https://openalex.org/W2151666380	https://www.scielo.br/j/jbchs/a/QtZwfWdX5DxzMMQ6hjPcGGb/?lang=en&format=pdf	English	null	Synthesis of N-Methyl-N-formyltyramine, a new beta-phenethylamide derivative isolated from Cyathobasis fruticulosa (Bunge) Aellen	Journal of the Brazilian Chemical Society	2,006	cc-by	3,476	Enrique L. Larghi and Teodoro S. Kaufman* Instituto de Química Orgánica de Síntesis (IQUIOS, CONICET-UNR) and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (S2002LRK) Rosario, República Argentina Este trabalho descreve a síntese da N-metil-N-formiltiramina, um novo derivado β-fenetilamídico isolado de Cyathobasis fructiculosa (Bunge) Aellen. O produto natural foi preparado em seis etapas com bom rendimento, partindo-se do 4-hidroxibenzaldeído. The synthesis of N-methyl-N-formyltyramine, a novel β-phenethylamide derivative isolated from Cyathobasis fructiculosa (Bunge) Aellen, is reported. The natural product was prepared in six steps and good overall yield from 4-hydroxybenzaldehyde. Keywords: synthesis, β-phenethylamide derivative, Cyathobasis fructiculosa, natural product * e-mail: tkaufman@fbioyf.unr.edu.ar Synthesis of N-Methyl-N-formyltyramine, a New βββββ-Phenethylamide Derivative Isolated from Cyathobasis fruticulosa (Bunge) Aellen Synthesis of N-Methyl-N-formyltyramine, a New βββββ-Phenethylamide Derivative Isolated from Cyathobasis fruticulosa (Bunge) Aellen J. Braz. Chem. Soc., Vol. 17, No. 3, 599-602, 2006. Printed in Brazil - ©2006 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 J. Braz. Chem. Soc., Vol. 17, No. 3, 599-602, 2006. Printed in Brazil - ©2006 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 J. Braz. Chem. Soc., Vol. 17, No. 3, 599-602, 2006. Printed in Brazil - ©2006 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 Short Report E-1-Benzyloxy-4-(2’-nitrovinyl)-benzene (15) Benzyl chloride (0.52 mL, 4.54 mmol) was added to a stirred suspension of 4-hydroxybenzaldehyde (13, 482 mg, 3.95 mmol) and K2CO3 (818 mg, 5.92 mmol) in absolute EtOH (4 mL). The slurry was refluxed until complete consumption of the starting material (by TLC), and the solvent was then evaporated under reduced pressure. After addition of H2O (5 mL), the aqueous phase was extracted with EtOAc (3 x 25 mL) and the combined organic extracts were successively washed with 10% Na2CO3 (2 x 10 mL) and brine (10 mL). The organic phase was dried (Na2SO4), concentrated in vacuo and chromatographed, to give 14 (830 mg, 99%), as a solid; mp 66-68 ºC (lit. 72 ºC);15 IR (KBr) νmax/cm-1: 3034, 2830, 2740, 1690, 1600, 1577, 1508, 1454, 1312, 1258, 1160, 1022, 1005, 830, 738, 697; 1H NMR δ 5.15 (s, 2H, C6H5CH2O-), 7.07 (d, J 8.6, 2H, H-3 and H-5), 7.33-7.45 (m, 5H, C6H5CH2O-), 7.83 (d, J 8.6, 2H, H-2 and H-6), 9.88 (s, 1H, CHO); 13C NMR δ 70.2 (C6H5CH2O-), 115.1 (C-3 and C-5), 127.4 (m-C6H5CH2O-), 128.2 (p-C6H5CH2O-), 128.6 (o-C6H5CH2O-), 130.1 (C-1), 131.9 (C-2, and C-6), 135.9 (ipso-C6H5CH2O-), 163.6 (C- 4), 190.6 (ArCHO). Without further purification, a solution of aldehyde 14 (258 mg, 1.21 mmol), MeNO2 (0.2 mL, 3.64 mmol) and anhydrous [NH3CH2CH2NH3]2+. 2AcO– (22 mg, 0.12 mmol) in dry tBuOH (3.7 mL) was heated at 65 ºC. After 18 h, the mixture was diluted with EtOAc (40 mL) and successively washed with water (3 x 15 mL) and brine Scheme. a: BnCl, K2CO3, EtOH, reflux (99%); b: MeNO2, (H3NCH2CH2NH3)++ . 2AcO-, tBuOH, 60 ºC, 18 h (90%); c: LiAlH4, THF, reflux, 3 h; d: HCO2Et, reflux, 8 h (44%, 2 steps); e: 1. NaH, DMF, 65 ºC, 10 min; 2. MeI, 45 ºC (79%); f: H2, 1 atm, 10% Pd/C, 4 h (92%). Next, 17 was N-methylated with methyl iodide, and the resulting amide 18 obtained in 79% yield was subjected to a final 10% Pd/C mediated catalytic hydrogenation, furnishing 92% of synthetic 12, the spectral data of which fully agreed with those previously reported for the natural product.13 Clear signals of the two conformers of 12 and 18 were also observed in their 1H and 13C NMR spectra. Interestingly, despite that the use of benzyl ether intermediates required additional protection and deprotection steps, increasing the length of the sequence, it allowed convenient manipulation of amine and amide reaction intermediates. Introduction β-Phenethylamides, exemplified by compounds 1 and 2,1 constitute a relatively widespread family of natural products, the members of which have been found mainly as soluble constituents of cell-wall fractions of higher plants.2 Several amides, structurally related to them, have been found in different plant species,3 and N-trans- feruloyltyramine (3) has demonstrated to have an ubiquitous occurrence.4,5 There is no definite conclusion yet about the functions of the β-phenethylamides; however, it has been discussed their possible role in plant growth processes, as well as their participation in self defense, due to their antimicrobial and antiviral effects.6 In addition, β-phenethylamides such as 4-7 have been informed to display interesting biological activities, ranging from DNA strand scission7 to anti- mutagenic and anticarcinogenic,8 and including inhibition of the lipopolysaccharide-induced nitric oxide production in macrophages,9 as well as inhibition of acetyl- cholinesterase.10 Noteworthy, N-acetyl tyramine (8) is an inducible phytoalexin found in soybean seeds,11 while Paik and coworkers reported the isolation of the unusual compounds 9-11 from Xenorhabdus nematophilus, a bacterial strain which grows symbiotic with a nematode. These β-phenethylamides were shown to be cytotoxic against five human cancer cell lines.12 Very recently, Topçu and coworkers13 isolated N- methyl-N-formyltyramine (12) from the aerial parts and roots of Cyathobasis fruticulosa (Bunge) Aellen (Chenopodiaceae), the only species of the genus Cyathobasis of the Turkish flora, which grows most commonly in Central Anatolia. In view of the potential interest of this type of compounds, we decided to prepare 12, and herein we disclose a short synthesis of the natural product from commercial 4-hydroxybenzal- dehyde (13). Larghi and Kaufman 600 J. Braz. Chem. Soc. General procedures Williamson etherification of 13 with benzyl chloride provided 99% of aldehyde 14 (Scheme), which was subjected to a Henry condensation with nitromethane in the presence of ethylenediammonium diacetate as base,14 furnishing 90% of nitrostyrene 15. In turn, this was submitted to reduction with lithium aluminum hydride, giving β-phenethylamine 16, as an oil which easily darkened upon contact with air; therefore, this was immediately amidated in refluxing ethyl formate, providing formamide 17 in 44% overall yield. Noteworthy, the 1H and 13C NMR spectra of 17 exhibited signals of two conformers, even at 70 ºC in DMSO-d6. Melting points (uncorrected) were taken on an Ernst Leitz Wetzlar model 350 hot-stage microscope. FT-IR spectra were determined with a Shimadzu IR Prestige 21 spectrophotometer. The 1H and 13C NMR spectra were acquired in CDCl3 employing TMS as internal standard, with a Bruker AC200-E spectrometer operating at 200.13 and 50.33 MHz, respectively; coupling constants (J) are expressed in Hertz. HRMS data were obtained from Kent Electronics (UK). The reactions were carried out under dry argon atmospheres, employing oven-dried glassware. All new compounds gave single spots on TLC plates run in different hexane-EtOAc solvent systems. Spots were visualized by exposure to UV light (254 and 365 nm), followed by spraying with ethanolic p-anisaldehyde/ sulfuric acid reagent and careful heating. Flash column chromatographies were carried out with silica gel 60 H, eluting with hexane-EtOAc mixtures under positive pressure and employing gradient techniques. Scheme. a: BnCl, K2CO3, EtOH, reflux (99%); b: MeNO2, (H3NCH2CH2NH3)++ . 2AcO-, tBuOH, 60 ºC, 18 h (90%); c: LiAlH4, THF, reflux, 3 h; d: HCO2Et, reflux, 8 h (44%, 2 steps); e: 1. NaH, DMF, 65 ºC, 10 min; 2. MeI, 45 ºC (79%); f: H2, 1 atm, 10% Pd/C, 4 h (92%). E-1-Benzyloxy-4-(2’-nitrovinyl)-benzene (15) Drying (Na2SO4) and concentration of the organic phase, followed by flash chromatography furnished 18 (37 mg, 79%), as a yellowish waxy solid; mp 56-58 ºC; IR (KBr) νmax/cm-1: 2929, 2855, 1675, 1515, 1455, 1397, 1242, 1179, 1016, E-1-Benzyloxy-4-(2’-nitrovinyl)-benzene (15) In conclusion, a simple synthesis of 12 was achieved in six steps from commercial 4-hydroxybenzaldehyde. Protection of the starting phenol as a benzyl ether allowed convenient manipulation of the reaction intermediates without sacrificing efficiency of the synthetic sequence. Synthesis of N-Methyl-N-formyltyramine, a New β-Phenethylamide Derivative 601 Vol. 17, No. 3, 2006 (2 x 15 mL). The organic phase was dried (Na2SO4), most of the solvent was removed under reduced pressure, and the resulting yellow solution was crystallized on settling, affording nitrostyrene derivative 15 (249 mg, 90%), as a solid; mp 116-118 ºC (lit. 120 ºC, EtOH);15 IR (KBr) νmax/ cm-1: 3109, 2941, 2875, 2217, 1600, 1509, 1492, 1341, 1252, 1175, 1003, 969, 828, 752, 699; 1H NMR δ 5.13 (s, 2H, C6H5CH2O-), 7.03 (d, J 8.8, 2H, H-3 and H-5), 7.30- 7.43 (m, 5H, C6H5CH2O-), 7.51 (d, J 8.6, 2H, H-2 and H-6), 7.52 (d, J 13.4, 1H, ArCH=CHNO2), 7.98 (d, J 13.4, 1H, ArCH=CHNO2); 13C NMR δ 70.1 (C6H5CH2O-), 115.7 (C- 3 and C-5), 122.7 (C-1), 127.3 (m-C6H5CH2O-), 128.2 (p- C6H5CH2O-), 128.6 (o-C6H5CH2O-), 131.1 (C-2 and C-6), 135.0 (ArCH=CHNO2), 135.9 (ipso-C6H5CH2O-), 138.8 (ArCH=CHNO2), 161.9 (C-4); HRMS (CI) Found: 256.09755 (M+ + 1). Calc. for C15H14NO3: 256.09736. NMR δ 34.4 (ArCH2CH2N), 39.1 (ArCH2CH2N), 69.9 (C6H5CH2O-), 114.9 (C-3 and C-5), 127.3 (m-C6H5CH2O-), 127.8 (p-C6H5CH2O-), 128.4 (o-C6H5CH2O-), 129.5 (C-2 and C-6), 131.7 (C-1), 136.9 (ipso-C6H5CH2O-), 157.4 (C- 4), 169.9 (NHCHO); HRMS (CI) Found: 256.13390 (M+ + 1). Calc. for C16H18NO2: 256.13375. N-[2’-(4-Hydroxy-phenyl)-ethyl]-N-methyl-formamide (N- methyl-N-formyltyramine, 12) Anhydrous DMF (1.0 mL) was added to a 50% NaH dispersion in mineral oil (10 mg, 0.207 mmol), and the system was heated 10 min at 65 ºC, and cooled to room temperature. Then, a solution of formamide 17 (44 mg, 0.172 mmol) in DMF (1.0 mL) was introduced, the system was warmed to 65-70 ºC for 10 min, cooled to -10 ºC, and treated with MeI (38 μL, 0.602 mmol). The reaction was warmed to 45 ºC until complete consumption of the starting material; brine (5 mL) was added and the reaction product was extracted with Et2O (5 x 10 mL). E-1-Benzyloxy-4-(2’-nitrovinyl)-benzene (15) Drying (Na2SO4) and concentration of the organic phase, followed by flash chromatography furnished 18 (37 mg, 79%), as a yellowish waxy solid; mp 56-58 ºC; IR (KBr) νmax/cm-1: 2929, 2855, 1675, 1515, 1455, 1397, 1242, 1179, 1016, 819, 746, 698; 1H NMR δ Z-form 2.74-2.81 (m, 2H, ArCH2CH2N), 2.84 (s, 3H, NCH3), 3.53 (t, J 6.9, 1H, ArCH2CH2N), 5.03 (s, 2H, C6H5CH2O-), 6.91 (m, 2H, H- 3 and H-5), 7.14 (d, J 8.6, 2H, H-2 and H-6), 7.31-7.45 (m, 5H, C6H5CH2O-), 8.01 (s, 1H, -NCHO); E-form 2.74- 2.81 (m, 2H, ArCH2CH2N), 2.88 (s, 3H, NCH3), 3.43 (t, J 6.8, 2H, ArCH2CH2N), 5.03 (s, 2H, C6H5CH2O-), 6.91 (m, 2H, H-3 and H-5), 7.04 (d, J 8.6, 2H, H-2 and H-6), 7.31- 7.45 (m, 5H, C6H5CH2O-), 7.80 (s, 1H, NCHO); 13C NMR δ Z-form 32.3 (C-2’), 35.0 (NCH3), 46.1 (C-1’), 70.1 (C6H5CH2O-), 114.9 (C-3 and C-5), 127.3 (m-C6H5CH2O-), 127.8 (p-C6H5CH2O-), 128.4 (o-C6H5CH2O-), 129.6 (C-2 and C-6), 130.8 (C-1), 136.9 (ipso-C6H5CH2O-), 157.4 (C-4), 162.3 (NCHO); E-form 29.7 (C-2’), 33.9 (NCH3), 51.4 (C-1’), 70.1 (C6H5CH2O-), 115.1 (C-3 and C-5), 127.3 (m-C6H5CH2O-), 127.8 (p-C6H5CH2O-), 128.4 (o- C6H5CH2O-), 129.6 (C-2 and C-6), 129.9 (C-1), 137.0 (ipso-C6H5CH2O-), 157.6 (C-4), 162.5 (NCHO). Without further purification, 18 (45 mg, 0.168 mmol) was dissolved in EtOH (5.7 mL), 10% Pd/C (9 mg) was added, and the black suspension was vigorously stirred 4 h under a H2 atmosphere. The catalyst was removed by filtration trough a short pad of Celite, the filtrate was concentrated under vacuum and chromatographed affording 12 (28 mg, 92%), as a solid; mp 111-113 ºC (CHCl3) [lit. 112-114 ºC (MeOH)];13 IR (KBr) νmax/cm-1: 3141, 3135, 2945, 2924, 2882 1657 1652 1614 1595 1509 1446 1394 1239 Anhydrous DMF (1.0 mL) was added to a 50% NaH dispersion in mineral oil (10 mg, 0.207 mmol), and the system was heated 10 min at 65 ºC, and cooled to room temperature. Then, a solution of formamide 17 (44 mg, 0.172 mmol) in DMF (1.0 mL) was introduced, the system was warmed to 65-70 ºC for 10 min, cooled to -10 ºC, and treated with MeI (38 μL, 0.602 mmol). The reaction was warmed to 45 ºC until complete consumption of the starting material; brine (5 mL) was added and the reaction product was extracted with Et2O (5 x 10 mL). N-[2’-(4-Benzyloxyphenyl)-ethyl]-formamide (17) A solution of nitrostyrene 15 (128 mg, 0.497 mmol) in THF (0.5 mL) was introduced via cannula during 5 min into an ice-cooled suspension of LiAlH4 (113 mg, 2.98 mmol) in THF (1.5 mL). The system was submitted to reflux for 3 h, then cooled to room temperature and treated with Na2SO4.10H2O. The slurry was diluted with Et2O (40 mL) and the resulting ethereal suspension was filtered trough Celite. Solvent removal under reduced pressure furnished crude amine 16, as a yellowish oil. IR (film) νmax/cm-1: 3361, 3033, 2927, 2865, 1610, 1511, 1454, 1381, 1241, 1177, 1026, 829, 739, 697; 1H NMR δ 1.93 (bs, 2H, NH2 J 6.8, 2H, ArCH2CH2N), 2.92 (t, J 6.8, 2H, ArCH2CH2N), 5.04 (s, 2H, C6H5CH2O-), 6.92 (d, J 8.5, 2H, H-2 and H-6), 7.11 (d, J 8.5, 2H, H-3 and H-5), 7.31-7.41 (m, 5H, C6H5CH2O-); 13C NMR δ 38.6 (ArCH2CH2N), 43.2 (ArCH2CH2N), 69.8 (C6H5CH2O-), 114.7 (C-3 and C-5), 127.3 (m-C6H5CH2O-), 127.7 (p-C6H5CH2O-), 128.4 (o-C6H5CH2O-), 129.6 (C-2 and C- 6), 131.7 (C-1), 136.9 (ipso-C6H5CH2O-), 157.1 (C-4). Without further purification, the crude amine 16 (112 mg, 0.443 mmol) was dissolved in freshly distilled HCO2Et (3 mL), and was refluxed until complete consumption of the starting amine. The solvent was removed under vacuum, and the oily residue was chromatographed to give formamide derivative 17 (51 mg, 44%), as a white solid; mp 99-101 ºC; IR (KBr) νmax/cm-1: 3231, 3035, 2928, 2886, 1653, 1510, 1379, 1232, 1111, 1010, 959, 821, 753, 699, 563; 1H NMR δ major conformer 2.78 (t, J 6.7, 2H, ArCH2CH2N), 3.54 (t, J 6.7, 2H, ArCH2CH2NH), 5.05 (s, 2H, C6H5CH2O-), 5.46 (bs, 1H, NHCHO), 6.93 (d, J 8.6, 2H, H-2 and H-6), 7.11 (d, J 8.6, 2H, H-3 and H-5), 7.31- 7.44 (m, 5H, C6H5CH2O-), 8.13 (s, 1H, -NHCHO); 13C A solution of nitrostyrene 15 (128 mg, 0.497 mmol) in THF (0.5 mL) was introduced via cannula during 5 min into an ice-cooled suspension of LiAlH4 (113 mg, 2.98 mmol) in THF (1.5 mL). The system was submitted to reflux for 3 h, then cooled to room temperature and treated with Na2SO4.10H2O. The slurry was diluted with Et2O (40 mL) and the resulting ethereal suspension was filtered trough Celite. Solvent removal under reduced pressure furnished crude amine 16, as a yellowish oil. N-[2’-(4-Benzyloxyphenyl)-ethyl]-formamide (17) IR (film) νmax/cm-1: 3361, 3033, 2927, 2865, 1610, 1511, 1454, 1381, 1241, 1177, 1026, 829, 739, 697; 1H NMR δ 1.93 (bs, 2H, NH2 J 6.8, 2H, ArCH2CH2N), 2.92 (t, J 6.8, 2H, ArCH2CH2N), 5.04 (s, 2H, C6H5CH2O-), 6.92 (d, J 8.5, 2H, H-2 and H-6), 7.11 (d, J 8.5, 2H, H-3 and H-5), 8.5, 2H, H-2 and H-6), 7.11 (d, J 8.5, 2H, H-3 and H-5), 7.31-7.41 (m, 5H, C6H5CH2O-); 13C NMR δ 38.6 (ArCH2CH2N), 43.2 (ArCH2CH2N), 69.8 (C6H5CH2O-), 114.7 (C-3 and C-5), 127.3 (m-C6H5CH2O-), 127.7 (p-C6H5CH2O-), 128.4 (o-C6H5CH2O-), 129.6 (C-2 and C- 6), 131.7 (C-1), 136.9 (ipso-C6H5CH2O-), 157.1 (C-4). Without further purification, the crude amine 16 (112 mg, 0.443 mmol) was dissolved in freshly distilled HCO2Et (3 mL), and was refluxed until complete consumption of the starting amine. The solvent was removed under vacuum, and the oily residue was chromatographed to give formamide derivative 17 (51 mg, 44%), as a white solid; mp 99-101 ºC; IR (KBr) νmax/cm-1: 3231, 3035, 2928, 2886, 1653, 1510, 1379, 1232, 1111, 1010, 959, 821, 753, 699, 563; 1H NMR δ major conformer 2.78 (t, J 6.7, 2H, ArCH2CH2N), 3.54 (t, J 6.7, 2H, ArCH2CH2NH), 5.05 (s, 2H, C6H5CH2O-), 5.46 (bs, 1H, NHCHO), 6.93 (d, J 8.6, 2H, H-2 and H-6), 7.11 (d, J 8.6, 2H, H-3 and H-5), 7.31- 7.44 (m, 5H, C6H5CH2O-), 8.13 (s, 1H, -NHCHO); 13C Larghi and Kaufman J. Braz. Chem. Soc. 602 1172, 1072, 816, 768, 655; 1H NMR δ Z-form 2.71-2.80 (m, 2H, ArCH2CH2N), 2.86 (s, 3H, NCH3), 3.55 (t, J 7.1, 2H, ArCH2CH2N), 6.75 (d, J 8.2, 2H, H-3 and H-5), 6.92 (t, J 8.2, 2H, H-2 and H-6), 7.97 (s, 2H, ArOH and NCHO); E-form 2.71-2.80 (m, 2H, ArCH2CH2N), 2.90 (s, 3H, NCH3), 3.42 (t, J 6.5, 2H, ArCH2CH2N), 6.72 (d, J 8.0, 2H, H-3 and H-5), 7.03 (t, J 8.0Hz, 2H, H-2 and H-6), 7.66 (s, 1H, NCHO), 7.97 (s, 2H, ArOH and NCHO); 13C NMR δ Z-form 32.0 (C-2’), 34.9 (NCH3), 45.9 (C-1’), 115.4 (C-3 and C-5), 129.0 (C-1), 129.5 (C-2 and C-6), 155.2 (C-4), 162.8 (NCHO); E-form 29.9 (C-2’), 33.4 (NCH3), 51.7 (C-1’), 115.7 (C-3 and 5), 128.2 (C-1), 129.6 (C-2 and C-6), 155.3 (C-4), 163.2 (-NCHO); HRMS (CI) Found: 180.10254 (M+ + 1). Calc. for C10H14NO2: 180.10245. 4. Rahman, A. U.; Bhatti, M. K.; Akhtar, F.; Choudhary, M. I.; Phytochemistry 1992, 31, 2869; Gozler, B.; Anur, M. N-[2’-(4-Benzyloxyphenyl)-ethyl]-formamide (17) A.; Bitar, S.; Hesse, M.; Helv. Chim. Acta 1992, 75, 260. 5. Achenbach, H.; Frey, D.; Waibel, R.; J. Nat. Prod. 1991, 54, 1331; Fukuda, N.; Yonemisu, M.; Kimura, T.; Chem. Pharm. Bull. 1983, 31, 156; Hussain, S. F.; Gözler, B.; Shamma, M.; Gözler, T.; Phytochemistry 1982, 21, 2979; Lajide, L.; Escoubas, P.; Mizutani, J.; Phytochemistry 1995, 40, 1105; 1331; Fukuda, N.; Yonemisu, M.; Kimura, T.; Chem. Pharm. Bull. 1983, 31, 156; Hussain, S. F.; Gözler, B.; Shamma, M.; Bull. 1983, 31, 156; Hussain, S. F.; Gözler, B.; Shamma, M.; Gözler, T.; Phytochemistry 1982, 21, 2979; Lajide, L.; Escoubas, P.; Mizutani, J.; Phytochemistry 1995, 40, 1105; Yoshihara, T.; Takamatsu, S.; Sakamura, S.; Agric. Biol. Chem. 1978, 42, 623; Claros, B. M. G.; da Silva, A. J. R.; Vasconcellos, M. L. A. A.; de Brito, A. P. P.; Leitão, G. G.; Phytochemistry 2000, 55, 859; Seca, A. M. L.; Silva, A. M. S.; Silvestre, A. J. D.; Cavaleiro, J. A. S.; Domingues, F. M. J.; Pascoal-Neto, C.; Phytochemistry 2001, 58, 1219. 6. Martin-Tanguy, J.; Negrel, J. In Plant Molecular Biology; von Wettstein, D.; Chua, N. H., eds.; Plenum: New York, 1987, pp. 253-263; Martin-Tanguy, J.; Plant Growth Regul. 1985, 3, 381. Acknowledgments 7. Ma, J.; Jones, S. H.; Hecht, S. M.; Bioorg. Med. Chem. 2004, 12, 3885. The authors gratefully acknowledge CONICET (PIP 5439) and ANPCyT (Project 06-12532) for financial support and Dr. Viviana Nicotra (UNC). E.L.L. is also thankful to CONICET for his fellowship. 8. Milic, B. L.; Milic, N. B.; Phytother. Res. 1998, 12, S3. 9. Tanaka, H.; Nakamura, T.; Ichino, K.; Ito, K.; Phytochemistry 9. Tanaka, H.; Nakamura, T.; Ichino, K.; Ito, K.; Phytochemistry 1989, 28, 2516.; Kim, Y.; Han, M. S.; Lee, J. S.; Kim, J.; Kim, Y. C.; Phytother. Res. 2003, 17, 983. 3. Adesina, S. K.; Olatunji, O. A.; Bergenthal, D.; Reisch, J.; Pharmazie 1988, 43, 517; Adesina, S. K.; Reisch, J.; Phytochemistry 1989, 28, 839; Horio, T.; Yoshida, K.; Kikuki, H.; Kawabata, J.; Mizutani, J.; Phytochemistry 1993, 33, 807; Cutillo, F.; D’Abrosca, B.; DellaGreca, M.; Di Marino, C.; Golino, A.; Previtera, L.; Zarrelli, A.; Phytochemistry 2003, 64, 1381; Kim, D. K.; Lim, J. P.; Kim, J. W.; Park, H. W.; Eun, J. S.; Arch. Pharmacal Res. 2005, 28, 39; Suzuki, T.; Holden, I.; Casida, J.; J. Agric. Food Chem. 1981, 29, 992. References 10. Kim, D. K.; Kee, K.; Arch. Pharmacal Res. 2003, 26, 735. 11. Garcez, W. S.; Martins, D.; Garcez, F. R.; Marques, M. R.; Pereira, A. A.; Oliveira, L. A.; Rondon, J. N.; Peruca, A. D.; J. Agric. Food Chem. 2000, 48, 3662. 1. Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H.; Planta Med. 1984, 50, 187. 2. Martin-Tanguy, J.; Cabanne, F.; Perdrizet, E.; Martin, C.; Phytochemistry 1978, 17, 1927. 12. Paik, S.; Park, Y. H.; Suh, S. I.; Kim, H. S.; Lee, I. S.; Park, M. K.; Lee, C. S.; Park, S. H.; Bull. Korean Chem. Soc. 2001, 22, 372. 3. Adesina, S. K.; Olatunji, O. A.; Bergenthal, D.; Reisch, J.; Pharmazie 1988, 43, 517; Adesina, S. K.; Reisch, J.; Phytochemistry 1989, 28, 839; Horio, T.; Yoshida, K.; Kikuki, H.; Kawabata, J.; Mizutani, J.; Phytochemistry 1993, 33, 807; Cutillo, F.; D’Abrosca, B.; DellaGreca, M.; Di Marino, C.; Golino, A.; Previtera, L.; Zarrelli, A.; Phytochemistry 2003, 64, 1381; Kim, D. K.; Lim, J. P.; Kim, J. W.; Park, H. W.; Eun, J. S.; Arch. Pharmacal Res. 2005, 28, 39; Suzuki, T.; Holden, I.; Casida, J.; J. Agric. Food Chem. 1981, 29, 992. 3. Adesina, S. K.; Olatunji, O. A.; Bergenthal, D.; Reisch, J.; Pharmazie 1988, 43, 517; Adesina, S. K.; Reisch, J.; Phytochemistry 1989, 28, 839; Horio, T.; Yoshida, K.; Kikuki, H.; Kawabata, J.; Mizutani, J.; Phytochemistry 1993, 33, 807; Cutillo, F.; D’Abrosca, B.; DellaGreca, M.; Di Marino, C.; Golino, A.; Previtera, L.; Zarrelli, A.; Phytochemistry 2003, 64, 1381; Kim, D. K.; Lim, J. P.; Kim, J. W.; Park, H. W.; Eun, J. S.; Arch. Pharmacal Res. 2005, 28, 39; Suzuki, T.; Holden, I.; Casida, J.; J. Agric. Food Chem. 1981, 29, 992. 13. Bahceevli, A. K.; Kurucu, S.; Kolak, U.; Topçu, G.; Adou, E.; Kingston, D. G. I.; J. Nat. Prod. 2005, 68, 956. 14. Barton, D. H. R.; Motherwell, W. B.; Zard, S. Z.; J. Chem. Soc., Chem. Commun. 1982, 551. 15. Rosenmund, K. W.; Chem. Ber. 1913, 1034. 15. Rosenmund, K. W.; Chem. Ber. 1913, 1034. Received: December 27, 2005 Published on the web: April 12, 2006 Received: December 27, 2005 Published on the web: April 12, 2006 Published on the web: April 12, 2006 I.; Casida, J.; J. Agric. Food Chem. 1981, 29, 992.
W3135848036.txt	https://www.researchsquare.com/article/rs-242151/latest.pdf	en		Energy/Exergy Conversion Factors of Low Enthalpy Geothermal Plants	Research Square (Research Square)	2,021	cc-by	10,327	Energy/Exergy Conversion Factors of Low-Enthalpy Geothermal Heat Plants Henning Francke (  francke@gfz-potsdam.de ) Helmholtz-Zentrum Potsdam GFZ: Deutsches Geoforschungszentrum Potsdam https://orcid.org/0000-0002-8086-2262 Method Article Keywords: COP, Power-to-heat, geothermal, energy, exergy, conversion, second law e ciency, system, auxiliary energy Posted Date: December 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-242151/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version: 12.12.2021 1 2 Energy/exergy conversion factors of lowenthalpy geothermal heat plants 3 Henning Francke, GFZ German Research Centre for Geosciences 4 Keywords: Geothermal, energy, exergy, efficiency, COP, heat pump 5 Abstract 6 7 8 9 10 11 12 From the system perspective, a geothermal heat plant is not only a source of heat, but, in case of wells producing liquid brine (low-enthalpy), also a sink for relevant amounts of electricity, consumed mainly by the pump(s). This electricity demand is usually not given much attention, although being decisive for operation costs and offering chances for demand side management as a variable consumer. From the perspective of an integrated energy system, geothermal installations basically move energy from the electricity sector into the heat sector. So do electrical compression heat pumps, whose performance is rated by the COP, the ratio between useful heat and invested energy. 13 14 15 16 17 18 19 20 This study transfers the COP concept to geothermal sites, by defining and determining the energy conversion factor 𝜀𝜀 (i.e. relative auxiliary energy or operating cost of heat provision expressed in electricity) for a selection of mostly German geothermal sites. Based on heterogenous data consisting of operational values for some sites and theoretical estimations for others, the calculated 𝜀𝜀 range from 12 to 116. In analogy, the concept is extended to the exergy conversion factor 𝜁𝜁, which is calculated to range from 1 to 36. A comparison with alternative heat provision technologies, such as heat pumps (COP ≤ 6) or simple electric heating (𝜀𝜀 ≈ 1), quantifies the potential service geothermal plants can render to the grid by converting electrical energy into useful heat. 21 22 Keywords: COP, Power-to-heat, geothermal, energy, exergy, conversion, second law efficiency, system, auxiliary energy Introduction 23 24 25 26 27 28 29 30 31 32 33 The integration of renewable energies into our energy system poses various challenges, among others matching the fluctuating demand and weather dependent production. Possible solutions are storage, adaptive production and curtailment, demand-side load management and energy transport over long distances. Another approach is to go beyond the electric sector and make use of other energies. Power-to-X is the fashionable name for the transformation of surplus electric energy a into another energy form which can be stored better or consumed directly. Power-to-Heat is the most promising option (Drünert et al. 2019), which in its simplest form can be implemented by basically dissipating the electric energy in an electric heater. This is a very simple technology that scales well and converts nearly 100 % of the input at any voltage, DC or AC, to useful heat at virtually any temperature, right where it is needed without residuals, byproducts or exhaust fumes. 34 35 36 37 38 39 40 41 The valuable electric energy can, however, be used much more efficiently for heat provision by deploying more advanced technologies, such as compression heat pumps (CHP) extracting heat from ambient or exhaust air, sewage water, soil and/or ground water using closed borehole heat exchangers or open groundwater circuits. Heat pumps are designed to provide multiples of the input power as heat output by moving heat from a low temperature heat source to a higher temperature level. Their key performance indicator is the coefficient of performance (COP), defined as heat output 𝑄𝑄̇out per electrical input 𝑊𝑊̇in . It is limited by the theoretical maximum, which is defined by the reversed Carnot’s law: a In Germany 2019 6.4 TWh (2.8 % of the electricity from renewables or 1.2 % of total) were throttled(Burger 2020; Bundesnetzagentur 2020) 1 Version: 12.12.2021 𝐶𝐶𝐶𝐶𝐶𝐶 = 𝑇𝑇c −1 𝑄𝑄̇out ≤ �1 − � 𝑇𝑇h 𝑊𝑊̇in (1) 1 2 3 4 5 6 7 8 9 10 11 As eq. (1) expresses, for a given heat pump technology, the COP increases with source temperature 𝑇𝑇h and decreases with falling thermal output 𝑄𝑄̇out . Ambient air as a heat source (ASHP) has low requirements and an unlimited heat reservoir, but also the inherent disadvantage of a low COP especially when air temperature is low and heat demand consequentially high. Eligibility for government requires COP of at least 3.1 (ASHP) and 4.3 (GSHP)(BAFA 2018), but, often installed in suboptimal conditions with excessive use of direct heating by the heating cartridge, not all ASHP can keep that promise, causing COPs to sink to 2 and below (Russ et al. 2010). Ground source heat pumps (GSHP) perform considerably better due to a more stable heat source, but require the installation of heat exchangers in the underground. However, they, too, often fail to reach their theoretical COPs in practice. One study found average values below 3.5 (Russ et al. 2010). Heat pumps for residential heating are considered to be potential actors in demand-side management (Bechtel et al. 2020). 12 13 14 15 16 17 18 19 Ground temperature and hence the realized COP generally increase with depth, but so do the technical effort and requirements. At a given depth the ground is warm enough to use the harvested heat directly without enhancing it by a heat pump. This reduces the electrical input more or less to the power consumption of circulation pumps. A closed-circuit heat exchanger although proposed on a MW scale (Winsloe et al. 2021; Toews et al. 2021) relies on heat transport by conduction (Winsloe et al. 2021) and therefore does not allow as much heat extraction as an open circuit, which exploits the convective heat transport as well. Such a closed circuit does, however, avoid the problems caused by reservoir hydraulics and precipitation of solutes from the brine. 20 21 22 Generally speaking, increasing the technical effort for a technology, such as increasing the depth of a geothermal well can increase the heat output, absolute and relative to the electrical input, but obviously also the financial cost (see Fig. 1). Seasonal Performance Factor, Coefficient of Performance 100 deep GT 85 °C electric boiler 55 °C flow heater 110 °C flow heater 98 °C electrode boiler HP air-water P fix HP air/water P var. 10 GSHP HP brine-water P fix large HP 35 °C HP brine-water P var HP direct evaporator HP water-water P fix HP water-water P var ASHP 35 °C electric heat 1 1 10 100 1000 10000 Pth/[kW] large HP real 35…90 °C large HP acc. to manufacturer 30…80 °C ATES 100 % permeability 38…170 °C deep geothermal 23 24 25 26 27 Fig. 1 Common thermal output and energetic conversion efficiencies of different heat provision technologies powered by electric energy. Values are based on test data of available devices in Germany (BAFA 2018) and installed devices in Switzerland (Hubacher et al. 2008) and on the energy conversion efficiencies of low-enthalpy geothermal plants from this publication (HP – heat pump, GHSP – ground source HP, ASHP – air source HP, GT – geothermal) 28 29 30 31 Fig. 1 also shows that while electric heaters can easily be scaled up or down without limits imposed by the working principle itself, CHPs are commonly available only up to a few dozen kW (BAFA 2018) as usually installed in single-family houses. A few large-scale heat pumps are in operation with thermal outputs up to a few MW (Hubacher et al. 2008). Beyond that range lies the domain of geothermal 2 Version: 12.12.2021 1 2 3 plants. Some produce from several wells at combined mass flow rates that correspond to a thermal power of hundreds of MW b. They do so at a relatively smaller electrical expense, as will be shown herein. 4 5 6 7 8 9 A geothermal plant, sketched in Fig. 2 with open loop and hydrothermal reservoir, comprises one or more production wells and usually one or more injection wells. Hot (geo)fluid is produced from the underground. Unless the reservoir is hot enough to produce steam (i.e. high-enthalpy site), a production pump is required, usually a centrifugal pump installed down-hole. At the surface, heat is extracted from the geofluid, which is then reinjected via the injection well, driven by an injection pump, if required. The recharging heat flow 𝑄𝑄̇in has a convective and a conductive component, which depend on reservoir hydraulics and the temperature gradient in the underground. Both are affected by well operation. 𝑄𝑄̇in is not necessarily in balance with the extracted heat 𝑄𝑄̇out , i.e. the geothermal exploitation is not always sustainable in the strict sense, but rather designed for a finite duration until reservoir depletion, ideally matching the lifetime of the subsurface components. Note that 𝑄𝑄̇in is not considered nor mentioned further in here. It is only depicted in Fig. 2 for completeness. No equilibrium of 𝑄𝑄̇in and 𝑄𝑄̇out is assumed or required as no energy balance is formulated. 10 11 12 13 14 15 16 𝑄𝑄̇out (useful heat) 𝑊𝑊̇in (electricity) injection pump production well injection well production pump geothermal reservoir 𝑄𝑄̇in (heat) 17 18 Fig. 2: Principle of a geothermal plant 19 20 21 22 23 24 25 26 Heat extraction rate obviously depends on production rate and reinjection temperature. The latter is thermodynamically limited by the temperature of the heat sink, usually being the return temperature of the secondary loop. The lower the temperature required by the heat sink, the more heat can be extracted from the geofluid. Fluid chemistry, however, adds another potential limitation. Temperature reduction may trigger precipitation, which, at the high mass flow rates realized in geothermal sites, can produce a considerable mass of solids, that at best ends up in filters and at worst clogs pipes, heat exchangers or the pores of the reservoir rock (Mouchot et al. 2018; DiPippo 2016; GTN 2010; Brehme et al. 2019). 27 28 Production rate is subject to friction, in the porous rock matrix of the reservoir as well as in the pipes of the wells and the surface installations. The complex hydraulic rock properties with respect to flow b Several geothermal power plants at the Geysers Geothermal Field have an installed gross electrical capacity of >100 MWel. This implies a thermal input of several hundreds of MWth, which is produced from several wells per plant(Brophy et al. 2010). 3 Version: 12.12.2021 1 2 3 into/from a well determine the productivity/injectivity. It is quantified by the productivity/injectivity index (PI/II), defined as the ratio between flow rate and the pressure drop/increase in the well during production/injection. 4 5 6 7 8 The pumps have to overcome not only said friction, but also the level difference between static water table and surface plus the production well-head-pressure. Consequently, unless the production well is artesian c and the injection well is absorbing (creates no relevant back pressure), considerable energy is required to circulate the fluid in the geothermal circuit. This is independent of the installed pump technology, be it an ESP d, a LSP e or a piston pump. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Hence, the electrical consumption of the pumps is considerable, albeit controllable by ramping up and down the production rate and with it the heat production. Technically, this is feasible at a given maximum ramp-up speed within the boundaries of minimal partial load and maximum flow-rate. Economically it may make sense to do so given the necessary capacities in storage and/or backup heat production as well as the right economic boundary conditions which gratify operation strategies stabilizing the electric grid (Aubele et al. 2020). This upgrades geothermal heat from being a renewable energy source not causing fluctuations to one instead even compensating them, thus increasing its value as a component of an energy system. The same holds true for geothermal power plants, where the electricity production offers an additional control reserve besides their pump’s consumption. Accordingly, Schlagermann predicts the shift in the operation of geothermal power plants from baseload to market oriented or even operating reserve optimized (Schlagermann 2014). Whether it is beneficial to operate an individual geothermal plant this way, is a complex question as it not only depends on the plant itself but also on the energy system characteristics. Hence, it is out of the scope of this work, but has been extensively studied by (Aubele et al. 2020). 23 24 25 26 The presented approach is applicable irrespective of useful heat application, pump technology, whether there are one or several production/injection wells, whether the reservoir is hydro/petrothermal, or whether the brine is partly/fully reinjected. The geothermal plant is simply considered as a system receiving electric energy and returning thermal energy. This paper gives an overview about the ratio of these two quantities, i.e. the harvested heat 𝑄𝑄̇out relative to the auxiliary power demand 𝑊𝑊̇in for a selection of existing geothermal sites: 27 28 29 30 31 32 33 34 35 36 37 38 39 40 𝜀𝜀 = 𝑄𝑄̇out 𝑊𝑊̇in (2) This quantity is herein referred to as the “energy conversion factor”. This 𝜀𝜀 of a geothermal plant is not limited to 𝜀𝜀 ≤ 1 by energy conservation, but rather nominally exceeds 1. If it were below 1, i.e. if more energy was invested than harvested, there would be no benefit over the much simpler direct transformation to heat by an electric heater having 𝜀𝜀 = 1. The analogy to the COP of heat pumps is obvious. Using a given amount of electrical/mechanical energy to provide a larger amount of energy as heat is the purpose of compression heat pumps (CHP). While their efficiency has the aforementioned theoretical maximum (eq. (1)), the 𝜀𝜀 of a geothermal plant as defined by eq. (2) however, is not subject to this limitation derived from the fundamental laws of thermodynamics. That is due to the fact that eq. (2) is not based on a complete energy balance, as it does not consider 𝑄𝑄̇in , but rather on the ratio output to input relevant from the energy system perspective. Hence, given an artesian or high-enthalpy steam-producing production well and an absorbing injection well, i.e. 𝑊𝑊̇in = 0, 𝜀𝜀 approaches infinity and the factor cannot be calculated. c Flowing artesian well: the reservoir fluid pressure is high enough to make the fluid rise to the well-head, resulting in flow without pumping. d Electrical submersible pump –centrifugal pump installed together with the motor in the production well. e Line shaft pump - centrifugal pump installed in the production well driven by a motor at the surface via a shaft. 4 Version: 12.12.2021 1 2 3 4 5 6 7 8 9 This key value 𝜀𝜀 combines the thermal and the hydraulic reservoir properties, but also site-specific boundary conditions, such as pump capacity or heat exchanger performance which determine design and operating parameters, primarily reinjection temperature and production rate. In combination with a maximum thermal output 𝜀𝜀 is a very simple characteristic representation of a geothermal heat plant in an energy system model. In other words, 𝜀𝜀 represents the operational cost of heat provision in terms of invested electric energy, which can be converted to monetary cost or GHG emission using the specific parameters of the electricity provider. It can be used to assess the systemic potential of geothermal plants in general or to compare the energetic performance of single sites, but also, with limitations, for comparison with other heat provision technologies in a multimodal energy system. 10 11 12 13 14 15 A fair comparison of heat provision technologies, however, requires taking into account the temperature of the delivered useful heat. Heat sources with different temperature levels are not always interchangeable, because a heat source obviously needs to be warmer than the sink for the heat to flow in the right direction (2nd law of thermodynamics). Hence, a warmer heat source can always replace a colder one, but not always vice versa, which makes warmer heat more valuable. This inherent “value of heat” is quantified by the exergy of the heat. f 16 17 18 19 Looking at the exergies driving and leaving the plant, the exergetic conversion factor 𝜁𝜁 is defined in analogy to 𝜀𝜀 as the ratio of thermal exergy 𝐸𝐸̇ ∗ out output to driving electric energy 𝑊𝑊̇in , which is pure exergy. The ratio is also called “exergetic efficiency” or “second law efficiency” (Baehr & Kabelac 2009; Backhaus & Schlichting 1984): 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 𝜁𝜁 = (3) While the energy conversion factor 𝜀𝜀 extends the concept of the COP and can be compared to it, the reference for this exergy ratio is a reversible process with 𝜁𝜁 = 1, e.g. an ideal heat pump. 𝜁𝜁 < 1 consequentially indicates destruction of exergy, while 𝜁𝜁 > 1 marks a gain of exergy. Exergy gain is possible for the system as sketched in Fig. 2, because, again, definition eq. (3) is not based on a full exergy balance, but rather on the energy system perspective. Hence, it intentionally omits 𝑄𝑄̇in , the inflow of exergy by heat in to the system considered here, as it is not invested in the sense that electric energy is. Furthermore, unlike a heat pump, the system considered here has no inherent exergy outflow by waste/excess heat. As for 𝜀𝜀, there is no theoretical upper limit to 𝜁𝜁. Another obvious reference is the heat provision by an electric heater. There, the electric energy is converted completely to heat, i.e. 𝑊𝑊̇in = 𝑄𝑄̇out ., yet only a fraction of 𝑄𝑄̇out being exergy. This fraction, depends on temperature and is directly given by the Carnot factor g. If the heater were operated at a higher temperature it would destroy less exergy, but it would still be lost unless the temperature level of the final heat use also increases. The sink temperature 𝑇𝑇out eventually determines the system exergy loss, no matter if the loss happens by dissipation in the resistor or during transfer to the heat sink. Even though eventually the economic profitability of a site usually is pivotal, the conversion factors give a first evaluation from the energetic/exergetic perspective how reasonable the operation of a geothermal plant can be with a systemic perspective. State of the Art 37 38 39 40 41 42 𝐸𝐸̇ ∗ out 𝑊𝑊̇in Thoroughly characterizing geothermal sites is complex as several key parameters must be considered (DiPippo 2016), primarily the obvious parameters thermal output power and production temperature. They are determined by technical installations (well setup, pump configuration, etc.), design and operating decisions (production rate, reinjection temperature), the hydrogeological conditions (rock permeability and porosity) and, last not least, the geochemistry. f Two heat flows A and B at different temperatures having equal exergy content implies that heat flow A could theoretically be converted to temperature B by an ideal machine, then equaling heat flow B. g For example, electric heating to 𝑇𝑇out = 100 °C at 𝑇𝑇amb = 0 °C, has an exergetic efficiency of 0.27. 5 Version: 12.12.2021 1 2 3 4 5 The aquifer geometry is often simplified to a homogenous horizontal layer of rock with a given thickness. The hydraulic behavior of the aquifer (including the connection to the well) is usually linearized and described with the coefficients productivity and injectivity relating drawdown and production rate. The brine composition is thermodynamically relevant as a high salinity affects density, heat capacity and viscosity. 6 7 8 9 10 11 12 One way to reduce the multitude of parameters to a single value is to determine the heat generation cost in terms of money (Straubel et al. 1998; Kölbel et al. 2009; Pratiwi et al. 2018) or CO2 emissions (Pratiwi et al. 2018). This requires a usually rather extensive profitability calculation considering geological, technical and economical boundary conditions. This value can be compared against alternative heat provision technologies. Schlagermann conducted a comprehensive exergo-economic analysis for the geothermal power plant in Bruchsal, Germany, focusing on the electricity production costs (Schlagermann 2014). 13 14 15 16 17 18 19 While an economic calculation is certainly indispensable for the decision about the realization of a project, it is often too complex and requires too much economic input for purely energetic considerations. For this purpose, the ratio proposed here is more suitable, as it represents a relatively inexpensive and universal evaluation method with very few input parameters: Thermal output and electrical input. It is however not common to find both values. Even the reference publication for geothermal technology by (DiPippo 2016) does not give both values for any of the sites presented in detail. 20 21 22 23 While several publications investigate the energy conversion efficiency of the power cycle or the total power plant efficiencies (Moon & Zarrouk 2015; DiPippo 2016; Eyerer et al. 2020), thereby considering the pump energy demand among others as parasitic load, only three articles are known to the author which consider it in relation to the extracted heat. 24 25 26 Bugai assessed a geothermal heat supply system and defined an “annual exergy efficiency factor” as the exergy of the useful heat divided by the exergy input by geothermal fluid, peak reheater and pump power supply. Yet no values are given. (Bugai 2014) 27 28 29 30 31 32 33 34 Kastner et al. took this method a step further and combined predictions of productivity and temperature to determine the energetic efficiencies (COP therein) of idealized virtual geothermal doublets in two aquifers below Berlin, Germany. They assume no heat loss in the well (𝑇𝑇prod = 𝑇𝑇res ), an ideal pump, disregarding pressure loss, thermal and limitations on the consumer side. Their COP is the maximum theoretically possible energetic efficiency. It depends on both production and injection temperature (𝑇𝑇prod and 𝑇𝑇inj ) and on the flow rate 𝑉𝑉̇. Kastner et al. generally assume 𝑇𝑇inj = 45 °C (based on the return temperature of a connected heating network) and determine the flow rate assuming an absorbing well without injection pump as: 35 36 37 38 39 40 41 42 43 44 45 46 47 ̇ = II𝜚𝜚𝜚𝜚(−𝑧𝑧wt ) . 𝑉𝑉max (4) This in turn requires the knowledge/assumption of the hydraulic properties of the assumed homogenous horizontal aquifer, namely the natural water table depth 𝑧𝑧wt and the injectivity which they estimate via a porosity-permeability correlation. Kastner’s approach yields rather low production rates and consequentially high conversion factors. With large uncertainties mostly due to the porosity data, they estimate the average COP, the ratio of electricity input and thermal output, to be 16.2 for the more productive one of the aquifers (Middle Buntsandstein). Their method does consider the reservoir temperature, albeit only its for the calculation of 𝑄𝑄̇out , thus effectively equating 1 MW at 63 °C from the shallower aquifer with 1 MW at 109 °C from the deeper aquifer. (Kastner et al. 2015) Banks et al. compiled hydraulic borehole data from a gas field. For 35 suitable wells with sufficient data and bottom hole temperatures between 65 and 152 °C they estimated the pumping power required if a virtual power plant were to produce an arbitrarily assumed 1 MWel. Pump efficiency was assumed constant, conversion efficiency of the power cycle was estimated from the well temperature data using an empiric correlation. They found parasitic loads between 2 and 60 %. (Banks et al. 2020) 6 Version: 12.12.2021 1 2 3 They also list parasitic loads for a few existing plants, but not thermal power nor reinjection temperature. Vice versa, Kamila et al. list reinjection temperatures for a few other sites, but without parasitic or pump load as this would be out of scope. (Kamila et al. 2021) Method 4 5 6 7 8 9 Most of the considered sites produce (also) electric energy. In order to include them, they are considered herein as heat plants with an attached separate power cycle (as indicated by the system boundary in Fig. 2), so that the energy conversion factor can be determined in the same way as for heat plants. The additional consumers which are part of a geothermal power plant such as cooling facility and feed pumps are not of interest here. 10 11 The energy input into a low-enthalpy geothermal heat plant is mainly consumed by the electrical consumption of the pumps 𝐶𝐶el , with the production pump usually having the biggest share. inj prod + 𝐶𝐶el 𝑊𝑊̇in = 𝐶𝐶el (5) 12 13 14 15 Data of pump power consumption in geothermal sites is scarce and often considered company secret. For some sites information about net and gross electricity generation is available. The difference between the two values is a hint to the pump power consumption, but probably also includes cooling effort for the power cycle and other auxiliary consumers. 16 17 𝐶𝐶el comprises the pumps’ actual hydraulic work as well as mechanical and electrical losses in the pump, motor, cable and power electronics (VSD h): 18 19 20 21 𝐶𝐶el = 25 26 If unknown, Δ𝑝𝑝pump can be estimated from the hydraulic work, using the productivity/injectivity index PI / II of the well, production rate 𝑉𝑉̇, the static water table 𝑧𝑧𝑤𝑤𝑤𝑤 < 0 and brine density 𝜚𝜚: prod Δ𝑝𝑝pump = �𝑝𝑝wh − 𝑝𝑝res � + 𝑉𝑉̇ − 𝜚𝜚g𝑧𝑧res PI (7) The productivity/injectivity describes the ability of a wellbore to produce/absorb fluid. It is quantified by productivity/injectivity index PI / II, defined as the ratio between flowrate per pressure drop induced by pumping at the well-bottom with respect to initial reservoir pressure 𝑝𝑝res : PI, −II = 𝑉𝑉̇ . 𝑝𝑝res − 𝑝𝑝wb (8) For the injection well, equation (7) is limited to Δ𝑝𝑝pump > 0 in order to avoid falsely calculating electricity gain in absorbing wells: inj 27 28 29 (6) If unavailable, the electrical consumption of the pumps can be estimated with eq. (6) from production rate and differential pump pressure Δ𝑝𝑝pump and assumed efficiencies. prod 22 23 24 Δ𝑝𝑝pump ∙ 𝑉𝑉̇ 𝜂𝜂pump 𝜂𝜂motor 𝜂𝜂cable 𝜂𝜂VSD inj Δ𝑝𝑝pump ≈ max �0, �𝑝𝑝res − 𝑝𝑝wh � + prod inj 𝑉𝑉̇ + 𝜚𝜚g𝑧𝑧res � . II (9) If both differential pressures Δ𝑝𝑝pump and Δ𝑝𝑝pump are considered in a sum and both wells are connected to the same reservoir, 𝑝𝑝res and 𝑧𝑧res cancel out leaving only the contributions by productivity/injectivity. h Variable speed drive 7 Version: 12.12.2021 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 If data about the electrical consumption of the injection pump is not available, it is assumed to be insignificant in relation to the production pump consumption. If the static water table is unknown, it is assumed to be 0. The harvested heat 𝑄𝑄̇out can be calculated as the difference of the fluid’s enthalpy at both wellheads. Disregarding pressure changes, any heat losses possibly occurring in the well or between heat extraction and delivery as well as assuming one-phase flow (no gas phase), 𝑄𝑄̇out can be approximated by the product of production rate 𝑚𝑚̇, a constant specific heat capacity 𝑐𝑐𝑝𝑝 and the temperature difference between the well heads. If the well head temperature is not available, it is assumed to equal the reservoir temperature: 𝑄𝑄̇out = 𝑚𝑚̇�ℎprod − ℎinj � ≈ 𝑚𝑚̇𝑐𝑐𝑝𝑝 �𝑇𝑇prod − 𝑇𝑇inj � . The production rate 𝑚𝑚̇ is assumed to equal the injection rate, i.e. there is no relevant fluid loss between production and injection, i.e. none of the produced geofluid is diverted without being reinjected and, in the case of HDR i reservoirs, all of the produced fluid volume is injected again. The flow rate, given as a volume flow rate 𝑉𝑉̇, is converted to mass flow rate 𝑚𝑚̇ with the fluid density 𝜚𝜚 at production temperature: 𝑚𝑚̇ = 25 26 27 28 29 30 𝑉𝑉̇ 𝜚𝜚(𝑇𝑇prod , 𝑋𝑋) (11) . Both 𝜚𝜚 and 𝑐𝑐𝑝𝑝 of the geofluid depend on temperature and salinity 𝑋𝑋. In this study, their values were estimated using the brine property model BrineProp (Francke et al. 2013) considering the respective salinity 𝑋𝑋. 𝑐𝑐𝑝𝑝 is listed here only for reproducibility. The calculations were conducted with the specific enthalpies. Unless indicated otherwise, the mean specific heat capacity 𝑐𝑐𝑝𝑝 for each site was calculated from the specific enthalpies at wellhead conditions as follows: ℎ�𝑝𝑝prod , 𝑇𝑇prod , 𝑋𝑋� − ℎ�𝑝𝑝inj , 𝑇𝑇inj , 𝑋𝑋� . 𝑇𝑇prod − 𝑇𝑇inj (12) 𝑄𝑄̇out 𝑚𝑚̇�ℎprod − ℎinj � 𝑚𝑚̇𝑐𝑐𝑝𝑝 �𝑇𝑇prod − 𝑇𝑇inj � = = . inj inj prod prod 𝑊𝑊̇in 𝐶𝐶 𝐶𝐶 + 𝐶𝐶 + 𝐶𝐶 (13) 𝑇𝑇amb 𝐸𝐸̇ ∗ out 𝑄𝑄̇out �. = ∙ �1 − ̇ ̇ 𝑇𝑇m 𝑊𝑊in 𝑊𝑊in (14) 𝑐𝑐𝑝𝑝 = 20 21 22 23 24 (10) The energetic conversion factor of a geothermal plant is hence calculated by eq. (2) as 𝜀𝜀 = el el el el In order to consider the absolute temperature or quality of the useful heat 𝑄𝑄̇out , the exergetic conversion factor is calculated by eq. (3) as the ratio of exergy output to exergy input. The exergy 𝐸𝐸̇ ∗ out contained in 𝑄𝑄̇out is calculated by applying the Carnot or quality factor j, (Baehr & Kabelac 2009). It depends on the temperature of heat provision 𝑇𝑇out and of the environment 𝑇𝑇amb . 𝜁𝜁 = Assuming a perfect seasonal storage, exergy depends on the minimum of the periodically varying ambient temperature (Pons 2009). The average minimum air temperature in Germany is about -0.4 °C (DWD 2018). This matches closely the conventional choice of 𝑇𝑇amb = 0 °C which is also assumed herein. 𝑇𝑇m is used as the upper temperature, as the brine flow is a sensible heat source. 𝑇𝑇m is the logarithmic mean temperature of the heat transfer from the brine: i Hot Dry Rock method, applied to petrothermal reservoirs Here, the Carnot factor describes the amount of work achievable by a Carnot cycle operating between the brine and the ambient temperature 𝑇𝑇amb . j 8 Version: 12.12.2021 𝑇𝑇m = 1 2 3 𝑇𝑇inj − 𝑇𝑇prod . 𝑇𝑇inj � ln � 𝑇𝑇prod (15) An alternative way of obtaining eq. (14) is by relating 𝜀𝜀 to the COP of an ideal heat pump working between 𝑇𝑇m and 𝑇𝑇amb . This is how heat pump performance is assessed independently of temperatures, albeit assuming a non-sensitive heat source (𝑇𝑇prod instead of 𝑇𝑇m ). 5 The net exergy output is defined as the difference of exergy output 𝐸𝐸̇ ∗ out and the electric input 𝑊𝑊̇in 6 7 8 9 10 Motivated by a project dealing with the German energy system, this study focuses on German deep geothermal sites and European sites with comparable low enthalpy conditions. All German sites were included where enough data could be acquired to calculate the efficiencies. All sites have in common that their wells are deeper than 1000 m and that they have at least one production and one injection well. The map Fig. 3 shows their locations. 4 11 12 13 14 15 16 17 18 19 20 21 22 23 Sites Fig. 3: Locations of the geothermal sites covered in this study (Francke n.d.) Typical operational values were used, if available. However, the source data are very heterogeneous in quality and in what quantities were disclosed. They were acquired from publications, the GeotIS database (Agemar et al. 2014), personal communication with operators, but also from project and news websites. While values for the thermal power of geothermal sites are easily found, pump consumption data are rarely disclosed by commercial operators and if so, then often design values are given instead of actually measured ones. On the other hand, values measured as a snapshot or averaged over a limited time period, may be not representative. As listed in Table 1 and visualized in Fig. 4 (Δ𝑇𝑇 over 𝑇𝑇prod ) and Fig. 5 (𝑄𝑄̇out over 𝑇𝑇prod ), the geothermal sites considered here provide between 1 and 50 MW of heat at production temperatures of 80…170 °C and reinjection temperatures of 40…80 °C, except for the outlier Klaipeda site, which is operating at lower temperatures. 9 Version: 12.12.2021 1 Baltic Basin 120 Pannonian Basin South-German Basin North-German Basin Rhinegraben ΔT =Tprod-Tinj/ [K] 100 80 60 40 20 0 20 2 3 40 60 80 100 120 140 160 180 production temperature Tprod / [°C] Fig. 4: Brine temperature differences between production and injection vs. production temperatures Baltic Basin Pannonian Basin South-German Basin North-German Basin Rhinegraben trend 50 Pth / [MW] 40 30 20 10 0 20 4 5 40 60 80 100 120 140 160 180 production temperature Tprod / [°C] Fig. 5: Thermal output vs. production temperature. Separate linear trends plotted for sites in the SGB and NGB. 6 10 Version: 12.12.2021 Results 1 2 3 4 Table 1: Geothermal sites (p – power plant, h – heat plant, () – formerly /planned) and operational parameters used in calculations, (ϱ/cp –geofluid density/specific heat capacity calculated with BrineProp (Francke et al. 2013), V� - production rate, Pth – thermal power extracted from the geofluid, PESP,el – electric consumption of the production pump, Pinj,el – electric consumption of the injection pump), NGB – North-German Basin, SGB – South-German Basin, RG – Rhinegraben, BB - Baltic Basin, PB - Pannonian Basin) Site Type Geology 𝑻𝑻𝐩𝐩𝐩𝐩𝐩𝐩𝐩𝐩 𝑻𝑻𝐢𝐢𝐢𝐢𝐢𝐢 𝒄𝒄𝒑𝒑 (p)+h p+h h p+(h) h p+h h+p h+p h+(p) p+h p+h p+h h p h h (p)+h 71.3 40…50 60 (60)[4]k 40…50 55 38…48 57.2 50 59 m 66.8 70 50 80 70 11 50 3361 4211…4214 4190 1122 928 966 4205…4209 4191 4211…4215 935 962 926 4209 4206 935 941 °C Neustadt-Glewe[1] Dürrnhaar[2] Freiham[2] Grünwald/Laufzorn Kirchstockach[2] Riem[2] Sauerlach[2] Traunreut [6] Oberhaching [8] Unterhaching Bruchsal [10] Insheim [8] Landau [8] Rittershoffen[11] Soultz-sous-Forêts[11] Klaipeda[14] Mezőberény[16] NGB SGB SGB SGB SGB SGB SGB SGB SGB SGB RG RG RG RG RG BB PB 96 138 89 127[3] 130 95 140 113.7 128 123.3[7] 126 165 160 170 150 36 80 °C J/kg/K 3837 3837 3915 3907 3844[15] 4177 𝝔𝝔 kg/m³ 982 982 968 983 1054[15] 975 𝑽𝑽̇ l/s 28.8 135 110 90[3] 135 85 110 146 140 140[7] 24 80 70 72…77 31 168 8.3…12.8 s 𝐩𝐩𝐩𝐩𝐩𝐩𝐩𝐩 𝐢𝐢𝐢𝐢𝐢𝐢 𝑸𝑸̇𝐩𝐩𝐨𝐨𝐨𝐨 𝑷𝑷𝐞𝐞𝐞𝐞 MW 𝑷𝑷𝐞𝐞𝐞𝐞 MW 𝜺𝜺 2.7 46…52 12.9 40.0[3] 43…48 13.7 40…44 34.4 43.0 35.5 n[9] 5.4 28.6 29.0 25…26 9.4 17 1.0…1.6 t 0.16 1.35 0.73 (0.76)[5]l 1.00 0.50 1.20 1.31 1.30 1.65[9]o 0.13 ? 0 0 0 0 0 0 0.142 0 0[9] 0 16 34…38 18 53 43…48 27 33…36 24 33 21 41 (0.80)p (0.60) p (0.21…0.31) q (0.17)[13]q 0.055 r 0.013…0.03 0 0 0 0 0.55 0.005…0.012 u 36 48 86…116 56 28 36…48 MW 𝜻𝜻𝟎𝟎 𝑻𝑻𝐚𝐚𝐚𝐚𝐚𝐚 = 𝟎𝟎 °𝐂𝐂 3.9 8.7…9.2 3.8 13.3 10.4…11.1 5.9 8.3…8.8 5.3 8.0 5.3 10.6 10.6 13.2 26.8…36.0 16.0 2.2 6.9…9.2 𝜻𝜻𝟐𝟐𝟎𝟎 = 𝟐𝟐𝟎𝟎 °𝐂𝐂 2.9 6.8…7.1 2.7 10.4 8.1…8.5 4.3 6.5…8.8 4.1 6.2 4.2 8.4 8.8 10.6 22.5…30.1 13.1 0.31 4.8…6.4 5 r k Assumption based on heating network return temperature of 55 °C Linear interpolation for indicated flow rate from nominal values 1.350 MW at 160 l/s6 m Calculated with eq. (10) n 38 MW is valid at 150 l/s, here scaled to actual production rate o Including losses in motor, cable and variable-frequency drive p Assumption made in the data source prod q Calculated as 𝐶𝐶el = 𝑉𝑉̇ 2 /𝐶𝐶𝑃𝑃/𝜂𝜂, where 𝜂𝜂prod = 0.7 and PI = 0.8 l/s (Soultz) or 23 2.8 … 3.5 l/s (Rittershofen) PI = II·10=73 according to (Eyerer et al. 2020), natural water level = sea level, surface at 9 m s Nominal volume flow rate of the two main circulation pumps vs. design flow rate of the plant. t Nominal power of main heat exchanger is given as 533 kW, while thermal power calculated from temperature difference and flow rate amounts to 1018 kW u No injection pump, includes compressor, pressure boosting pump block and heat exchanger circulation pump l 11 Version: 12.12.2021 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 List of references in Table 1: [1] (Schallenberg et al. 1999) [2] (Pietl, (Stadtwerke München) 2018) [3] (Agemar 2014a) [4] (Bauer et al. 2014) [5] ({Erdwärme Grünwald GmbH} 2013) [6] (Giese, (Grünwald Equity) 2018) [7] (Agemar 2014b) [8] (Eyerer et al. 2020) [9] (Geisinger, (Geothermie Unterhaching) 2018) [10](Schlagermann 2014) [11](Mouchot et al. 2018) [12](Baujard et al. 2017) [13](BINE Informationsdienst 2009) [14](Brehme et al. 2019) [15](GTN 2010) [16](TRIO-CORD KFT. 2012) 18 19 20 Table 1 lists the key parameters of the included geothermal sites as well as the calculated energy and exergy conversion factors. Where the input parameters were available as ranges or in several variants, they are listed as a value range of the resulting conversion factors. 21 22 23 24 Fig. 6 plots the energy conversion factors 𝜀𝜀, whereas Fig. 7 plots the exergy conversion factors 𝜁𝜁 for all included sites. The value range is indicated by the respective mean value with error bars. Fig. 8 shows the conversion factors plotted against the production temperature as well the Carnot factor as a reference representing the exergetic efficiency of electric heating working at the given temperature. energy conversion efficiency 𝜀𝜀 = Q̇out/Ẇel,in Baltic Basin 100 80 60 40 20 0 25 26 27 39° Pannonian Bsn. 89° 80° 95° Sth.German Bsn. 126° 97° 114° 123° Nth.German Bsn. 128° 130° Rhinegraben 170° 138° 140° 150° 160° 165° Fig. 6: Energy conversion factors (mean values with error bars indicating the value range) for selected geothermal sites, sorted and annotated by production temperature 12 Version: 12.12.2021 exergy conversion efficiency ζ = Ėout/Ẇel,in Baltic Basin 1 2 3 35 30 25 20 15 10 5 0 Pannonian Bsn. Sth.German Bsn. 89° 95° 97° 114° 123° 39° 80° 126° 128° Nth.German Bsn. 130° 138° 140° 150° Rhinegraben 170° 160° 165° Fig. 7: Exergy conversion factors (mean values with error bars indicating the value range) for selected geothermal sites at an ambient temperature of 0 °C., sorted by and annotated with production temperature. 35 30 85 25 65 20 15 45 10 25 5 20 8 9 10 11 𝜀𝜀 energy efficiencies ζ exergy efficiencies Baltic Basin Pannonian Basin South-German Basin North-German Basin Rhinegraben Carnot factor Baltic Basin 0 5 4 5 6 7 exergy conversion efficiency ζ= Ėout/Ẇel,in with T0=0 °C energy conversion efficiency 𝜀𝜀= Q̇out/Ẇel,in 105 40 60 80 100 120 140 160 180 production temperature Tprod / [°C] Fig. 8: GT plants: conversion factors vs. production temperature (exergy reference temperature T0 = 0°C, axis scaled to make ε plot match ζ plot for sites with min/max efficiencies), Carnot factor representing the exergetic efficiency of electric heating. Eqs. (5)-(15) describe a simple analytic model of a GT plant which can be used to explain the nonlinear influence of the production rate on the conversion factors and thus put the real-world values presented later into context. Applying this simple model to a range of production rates with a given set of parameters produces the data plotted in . 13 Version: 12.12.2021 Q̇out / [MW] (thermal output) 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 8 ε (energy conversion factor) 12 7 ζ (exergy conversion factor) 10 6 electricity consumption 5 net exergy output 8 net exergy maximum 4 6 3 4 2 2 1 0 conversion factor ε,ζ E*out , Pel,in, / [MW] 0.0 0 0 20 40 60 80 100 120 ṁ / [kg/s] (production rate) 1 2 3 4 Fig. 9: Conversion factors as a function of production rate for a fictional site (WHT=150 °C, ΔT=80 °C, PI = 5.4 m³/h/MPa, cp=4 kJ/kg/K, ϱ=1000 kg/m³) Discussion 5 6 7 8 9 10 11 12 According to the model equations visualized in Fig. 1Fig. 9, production rate grows proportionally with thermal output (indicated by the second abscissa), while pump effort increases quadratically, thus reducing the resulting conversion factors. Hence, the operating point yielding the highest net exergy output will be a tradeoff between thermal output and the energetic/exergetic conversion factor. As visualized by its broad maximum, there may be a point within the feasible operation range beyond which a further increase of production will cost more exergy in the form of electricity than is gained from additional provided heat. The flat curve, however, indicates a low sensitivity to a change of production rate around the maximum, which makes the choice less critical. 13 14 15 16 17 18 19 20 21 22 Before elaborating on the calculated conversion factors, note that this study aims at giving a first overview of the conversion factors realized in actually operating geothermal plants. The heterogeneity of the source data should be kept in mind when comparing the sites, especially when the sources are polished websites or optimistic press releases. Furthermore, a single datapoint per site can only be a snapshot or an average of variable quantities. On the other hand, off-design operation can decrease the conversion factor, as pump efficiency will be reduced outside of their design operation range or fluid friction in the well becomes relevant if the diameter is small. Finally, the calculated conversion factors are not meant as a rating of the plant design and construction as they are also a consequence of boundary conditions given by thermo-hydraulic aquifer properties as well as limitations imposed by the fluid chemistry. 23 24 25 26 The geothermal sites considered here show a wide range of energy conversion factors 𝜀𝜀 between 12 and 112 (Fig. 6). This matches roughly the theoretical values determined by Kastner et al.: between 6 and 21 for the Middle Buntsandstein and between 43 and 74 for the Rotliegend (Kastner et al. 2015) formation. 27 28 29 Similarly, in Fig. 7 the exergy conversion factors 𝜁𝜁 0°C show a broad distribution with values ranging from 1 to 36. Fig. 6 and Fig. 7 look quite alike except for the Klaipeda, where the low production temperature takes its toll on the exergy placing it lowest. 30 31 32 Before looking closer at single sites, comparing these ranges to the COPs and 𝜁𝜁 of compression heat pumps (CHP) with air/ground as low-temperature heat source yields a clear result: The energy conversion factors 𝜀𝜀 of GT plants are roughly one order of magnitude above COPs of common CHPs 14 Version: 12.12.2021 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (COP ≈ 2…6 when providing heat at 35 °C) (BAFA 2018),v, while delivering heat at usually higher temperatures. The 𝜁𝜁 of an ideal heat pump is by definition 1, which is 1…2 orders below the 𝜁𝜁 of the GT plants considered here. Let alone electrical heating with a 𝜁𝜁 < 1, given by the Carnot factor, which is plotted in Fig. 8 clearly below even the lowest plant 𝜁𝜁. This suggests that deep geothermal heat exploitation offers a highly efficient way of using electricity to provide heat. Before comparing the site performances, the thermal boundary conditions are worth a second look. Plotting the brine temperature differences between production and injection Δ𝑇𝑇 against 𝑇𝑇prod in Fig. 5 clearly shows a positive trend. So does plotting 𝑄𝑄̇out against 𝑇𝑇prod in Fig. 5, yet not over all sites, but within the two distinct regions that contain several sites. The sites in South-German Basin produce more heat than the ones in the Rhinegraben due to higher 𝑉𝑉̇ in spite of lower 𝑇𝑇prod . The small sample numbers do not allow conclusions for the other regions or with respect to different regions. Given the linear relation between 𝑄𝑄̇out and Δ𝑇𝑇 in eq. (10), the observed positive trend is expected (unless countered by a decrease of 𝑉𝑉̇, which is not the case here). Fig. 8 also shows a general positive trend w.r.t. 𝑇𝑇prod of both 𝜀𝜀 and 𝜁𝜁. Different from Fig. 5 however, the trend within the Rhinegraben region, is not clear. Again, a positive trend can be expected given the positive contribution from 𝑄𝑄̇out , and, in the case of 𝜁𝜁, from the Carnot factor. Exceptions from the positive trend are caused by either a high 𝑇𝑇inj , high electrical consumption due to a low productivity/injectivity or bad performance for other reasons. The offsets between the mid-range 𝜀𝜀 and 𝜁𝜁 are caused by the non-linear temperature dependence of the Carnot factor in eq. (14) and the logarithmic mean in eq. (15). 21 22 23 24 25 26 27 28 29 30 31 The 𝜁𝜁 listed and plotted above have been determined for the common yet arbitrarily chosen reference temperature 𝑇𝑇amb = 0 °C . Having the lowest production temperature, Klaipeda’s 𝜁𝜁 is most sensitive to a change of 𝑇𝑇amb of all sites. Recalculating Klaipeda’s 𝜁𝜁 with the reference 𝑇𝑇amb = 20 °C lowers it to 0.31, while all other sites remain well beyond 𝜁𝜁 = 1 with values ranging from 2.7 to 30. 𝜁𝜁 = 0.3 is in the lower range of what current CHPs achieve, as the exergetic efficiencies derived from a comprehensive list of CHP of different technologies available on the German market (BAFA 2018) show 𝜁𝜁 between 0.33 and 0.64 for 𝑇𝑇amb = 0 °C or 2 °C. Other sources give values between 0.28 and 0.5 (Russ et al. 2010). This assessment shows that the operation of the Klaipeda site would be exergetically reasonable if it were operating with the listed parameters. However, it is not, due to declining productivity caused by precipitation in spite of counter-measures, which is why the plant operation indefinitely ceased operation in March 2017 (Šliaupa et al. n.d.; Brehme et al. 2019). 32 33 34 35 36 37 38 39 40 The remarkably high conversion factors of the Rittershoffen site can be explained with high production temperature and low pumping demand to begin with, but possibly also with the fact that it is based on estimation rather than on operational measurements. Pump power for both this site and the one in Soultz have been estimated only from the well productivity, thus neglecting additional work caused by the static water table level, high well head pressure and friction within the brine circuit. Hence, the real conversion factors can be expected to be lower. Vice versa, the low factors of Klaipeda, Neustadt-Glewe and Freiham are a consequence of low production temperature and relatively high electricity consumption by the pumps. A detailed analysis of the individual reasons is out of the scope of this work 41 42 43 44 45 46 Applying the same method to the partly theoretical data compiled by (Banks et al. 2020) results in an even wider range of 𝜀𝜀 = 3…207 (𝜀𝜀̅ = 33), which corresponds to 𝜁𝜁 0°C = 0.6…50 (𝜁𝜁 0̅ °C =7.8). The high values result from very productive, nearly artesian wells in the gas field. For a real power plant, they would probably be operated at a higher production rate, thus yielding more output at lower conversion rates. Vice versa, the five wells with the lowest conversion rates would not be operated as assumed, because their pump power exceeds the produced electricity, as their temperatures are below 80 °C. v Note that, by definition, the heat pump COP does not include the auxiliary electric energy for heat provision, e.g. for circulating the fluid through the heat collector. Similarly, the conversion factors 𝜀𝜀 and 𝜁𝜁, due to limited data availability, in most cases also only consider the main consumers of auxiliary electric energy, namely the pumps for production and reinjection. 15 Version: 12.12.2021 1 2 3 Heat provision could still make sense energetically, even from the “worst” two wells at a supposedly suboptimal production rate, as, with an 𝜀𝜀 ={3.1,4.1} they are en par with common CHPs. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Geothermal heat is available independently of weather conditions. It may be considered as free of charge, but its exploitation certainly requires investment, not only of money, but also of energy. From the system perspective, geothermal plants are commonly only considered as heat sources. However, they require pumps to produce and/or reinject the geofluid, unless they operate under special circumstances (i.e. high-enthalpy steam producing reservoir and reinjection of condensed liquid or artesian production well and no reinjection). These pumps consume considerable amounts of valuable electricity, with their nominal powers often amounting to several hundreds of kW. Without formulating a complete energy balance, this study relates this electricity input to the thermal output in terms of energy and exergy based on the gathered production data of a selection of geothermal sites. The differing temperatures of the delivered heat are considered by calculating the respective exergy contents. The conversion factors calculated here show that extracting heat from the underground is a very efficient use of electricity, even though this ratio output/input varies by one order of magnitude among the sites considered in this study. Far more heat and exergy is provided than invested as electrical input, which, from the system perspective, makes GT plants basically very effective Power-to-Heat converters and as such, valuable links coupling the sectors of electricity and heat. 20 21 22 Hence, given the appropriate part-load capability and flexible heat demand and/or heat storage capacity, GT plants could serve as flexible electrical load that could be activated or shed depending on the availability of cheap surplus electricity. 23 24 25 26 27 The purpose of the definition of the conversion factors is to describe the efficiency of the system at a specific moment in time rather than over its lifetime. Such a lifetime assessment would require other much more extended investigations which would not significantly change the conclusions of this paper. Nevertheless, this may be a future topic for fine tuning the view on the efficiency of geothermal plant operation. 28 29 30 31 32 33 The exergetic conversion factor used in here can be helpful as a key parameter to characterize geothermal plants in strongly simplified energy system models. This allows a comparison to other methods of electrically powered heat provision such as electric heating or CHPs. For this and other purposes it would be beneficial to include the pump power consumption to overview tables and databases (DiPippo 2016; Agemar et al. 2014; Anon n.d.), which usually lack it w , compiling only other key figures such as thermal/electric power, production rate and temperature. 34 35 36 37 However, similar to the efficiency of a geothermal power cycle, the conversion factors are not the quantities to be maximized by varying mass flows, as this would lead to small thermal output. More reasonable is maximizing the net exergy output. Its maximum may help to identify the “sweet spot” with respect the production rate independent from economic parameters. Conclusion Outlook 38 39 40 41 Including more sites in this assessment would potentially allow to draw further conclusions, e.g. by correlating the conversion factors to plant design or operational parameters or clustering them by geologic setting. 42 43 44 45 Considering geothermal plants as sinks for surplus electricity raises the question of their part load performance and their part load ability, i.e. how far and how quickly can their output be reduced or increased. This should be quantified and be used as additional key parameters to describe geothermal plants from the perspective of the energy system. w The OpenEI online database(Anon n.d.) has a field for „parasitic load“, but no data 16 Version: 12.12.2021 1 2 3 4 5 6 7 8 9 10 11 12 The assessment method presented here could be extended from existing geothermal plants to existing boreholes or even to unexploited geothermal reservoirs, founding on existing data of geothermal potential (Wolfgramm et al. 2009). Following (Kastner et al. 2015), the energetic/exergetic conversion factor could be calculated based on the well productivity/injectivity, the water table and the reservoir temperature. Discarding the limitation in eq. (9), eq. (13) would then be adapted as follows: 𝜀𝜀 = 𝜚𝜚𝑐𝑐𝑝𝑝 �𝑇𝑇prod − 𝑇𝑇inj � . (PI −1 + II −1 )𝑉𝑉̇ (16) As described in the “state of the art”, this approach would yield rather high efficiencies at a small production rate (its maximum without an injection pump.) Another choice for the mass flow could be on the other end of the range: The maximum production rate limited by the maximum drawdown with the lowest possible production pump installation depth, which is reservoir depth. Besides the presence of an injection pump, that implies a dynamic water table lowered to the minimum height above the pump, which is the required net pump suction head NPSHR : ̇ = PI𝜚𝜚𝜚𝜚(𝑧𝑧wt − 𝑧𝑧res − NPSHR ) 𝑉𝑉max (17) 13 14 Consequentially, this approach would return rather high flowrates, low efficiencies, and, assuming PI = II, cancel the productivity from the equation only leaving the depths and the temperatures. 15 16 17 18 19 20 21 22 The practical optimum for production rate is somewhere between these two values, determined by a variety of boundary conditions, e.g. geologically motivated pressure limits, demand side requirements, financial deliberation or optimal net power output (Frick et al. 2010). With this information being unknown for non-existent plants, an educated guess for the design operating point could be made using the net exergy maximum as discussed before. The same may apply for an economic optimum as both electricity demand and heat production can be converted into cost and revenue. This economic optimum may, however, be offset w.r.t the net exergy output maximum, as it depends on the monetary parameters. 23 24 25 26 27 The presented conversion efficiencies can be calculated for any electrically driven heat provision technology, including geothermal sites operated as thermal storages (ATES, BTES, MTES x). Like the storage efficiency, the conversion factor could serve as key figure to assess different storage technologies or to compare storage to other heat/cold provision technologies. Eq. (13) should then be changed to include the energy invested for storing the heat/cold. 28 Nomenclature 29 𝐶𝐶𝐶𝐶𝐶𝐶 𝑐𝑐𝑝𝑝 30 ̇∗ 𝐸𝐸 31 32 g out ℎprod , ℎinj 33 34 35 39 exergy output N⁄kg gravitational acceleration J⁄kg specific enthalpy at production, injection well-head kg⁄s PI x W 𝑚𝑚̇ 𝐶𝐶el 38 J⁄(kg · K) specific heat capacity l/s/MPa Injectivity index 𝑝𝑝amb 37 Coefficient of Performance II Δ𝑝𝑝pump 36 1 mass flow rate Pa differential pump pressure Pa ambient pressure (= 1 bar) W electrical power consumption l/s/MPa productivity index Aquifer/Borehole/Mine Thermal Energy Storage 17 Version: 12.12.2021 1 2 3 4 5 6 7 8 9 10 11 12 13 prod inj 𝑝𝑝wh , 𝑝𝑝wh Pa 𝑇𝑇prod , 𝑇𝑇inj 𝑇𝑇amb 𝑇𝑇m 𝑇𝑇out 𝑊𝑊̇in 𝑄𝑄̇out 𝑉𝑉̇ 𝑋𝑋 𝑧𝑧𝑤𝑤𝑤𝑤 𝜀𝜀 𝜁𝜁 𝜂𝜂 well-head pressure K well-head temperature production, injection K ambient temperature K mean temperature of heat transfer K temperature of heat output W input power to the brine circuit W thermal power extracted from the brine circuit m3 ⁄𝑠𝑠 Flowrate, production rate kg⁄kg brine salinity m natural water table / hydraulic head 1 energetic conversion factor 1 exergetic conversion factor 1 kg⁄m energetic efficiency 3 15 𝜚𝜚 16 Availability of data and material 17 The collected data is provided as supplementary spreadsheet files 18 Competing interests 19 The author is employed by an institution conducting research in and advocating geothermal energy. 20 Funding 14 21 22 fluid density Declarations This work was funded by the Initiative and Networking Fund of the Helmholtz Association in the future topic “Energy Systems Integration” under grant number ZT-0002. 23 Authors' contributions 24 Not applicable. 25 Acknowledgements 26 The author is grateful to the plant operators that provided helpful information. 27 References 28 29 30 {Erdwärme Grünwald GmbH}. 2013. Die neue EWG-Tiefenpumpe geht in Betrieb. www.erdwaermegruenwald.de/Startseite/Informationen-Medien/Informationen/News-Archiv/E1120.htm [Accessed March 2, 2018]. 31 32 Agemar T (LIAG). 2014a. GeotIS - Geothermal Installation Grünwald (Laufzorn). geotis.de/geotisapp/templates/locationone.php?ID=70 [Accessed March 1, 2018]. 33 34 Agemar T (LIAG). 2014b. GeotIS - Geothermal Installation Unterhaching. geotis.de/geotisapp/templates/locationone.php?ID=103 [Accessed March 1, 2018]. 35 36 Agemar T (LIAG), Weber J & Schulz R. 2014. The Geothermal Information System for Germany - GeotIS. ZDGG, 165:129–144. doi:10.1127/1860-1804/2014/0060. 37 38 Anon. List of Geothermal Facilities \| Open Energy Information. openei.org/wiki/List_of_Geothermal_Facilities [Accessed February 14, 2021]. 18 Version: 12.12.2021 1 2 3 4 Aubele K, Baumann T, Dawo F et al. 2020. 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https://openalex.org/W2078353031	https://europepmc.org/articles/pmc3517311?pdf=render	English	null	3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development	BMC developmental biology	2,012	cc-by	14,216	Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 © 2012 Aguirre Lavin 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: Nathalie.Beaujean@jouy.inra.fr 1INRA, UMR1198 Biologie du Développement et Reproduction, F-78350, Jouy-en-Josas, France 2ENVA, F-94700, Maisons Alfort, France 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development Tiphaine Aguirre-Lavin1,2, Pierre Adenot1,2, Amélie Bonnet-Garnier1,2, Gaétan Lehmann1,2, Renaud Fleurot1,2, Claire Boulesteix1,2, Pascale Debey1,2 and Nathalie Beaujean1,2* RESEARCH ARTICLE Open Access Open Access Background with gene silencing [19,20]. Centromeric and pericentro- meric heterochromatic regions are highly important for chromosome stability and proper segregation [21]. How- ever, during interphase, these regions form higher-order chromatin structures – the so-called “chromocenter” clusters [22] – that act as transcriptionally repressive structures for genes spatially located in their vicinity [16,23]. Similarly, it has been found that silencing of rDNA genes is tightly linked to heterochromatin forma- tion [24]. Specific gene expression during cell differentiation results from the concerted effects of intermingled fac- tors: epigenetic modifications of DNA and histones, fix- ation of transcriptional factors, nuclear localization of genes, and the formation of higher-order chromatin structures. Indeed, over the past decade, the dynamic, temporal, and spatial organization of the eukaryotic cell nucleus has emerged as a central determinant of genome function [1-4]. When higher-order chromatin structures such as peri- centromeric heterochromatin were first analyzed in the mouse, a specific nuclear architecture exclusive to the first embryonic cleavages was observed [8,9]. Deconden- sation of pericentromeric heterochromatin seems to take place rapidly after fertilization, and it has been suggested that this maintains transcriptional silencing until EGA [10]. Thereafter, reorganization of the centromeric and pericentromeric heterochromatin into “chromocenters” occurs concomitantly with the major phase of EGA [8-10]. In fact, interference with the reprogramming of the pericentromeric structures significantly alters devel- opment; it has been shown that disruption of chromo- centers in mouse fertilized embryos results in developmental arrest [11,25] and that cloned embryos produced by nuclear transfer often show aberrant nu- clear architectures with remnants of somatic-like chro- mocenters, correlating with poor developmental rates [9,26,27]. When analyzing the correlation between nuclear organization and differentiation, early embryonic devel- opment offers a particularly interesting, although ex- tremely complex, system. Upon fertilization, the highly specialized male and female gametes must be repro- grammed to form a totipotent embryo that will then differentiate and give rise to all the tissues of a new individual [5,6]. In mammals, these events occur throughout the preimplantation period (in the female reproductive tract) and are thus accessible to detailed experimental investigations, especially in the mouse model. From large-scale transcriptomic analyses per- formed worldwide, it is now clear that this “reprogram- ming” process is dependent on finely tuned mechanisms of gene regulation [7]. However, few researchers have analyzed structural and functional genome organization during early embryonic development [8-11]. Background Many studies focusing on epigenetic modifications have shown that, immediately after fertilization, both parental genomes undergo extensive remodeling during early cell cycles that is correlated with major modifica- tions of gene expression [7]. However, while parental genomes are first transcriptionally silenced in zygotes after fertilization, the embryonic genome is progressively turned on: in the mouse, a "minor activation" occurs at the end of the 1-cell stage [12], followed by a "major ac- tivation" at the 2-cell stage [13]. This onset of embryonic gene expression (i.e. EGA, embryonic genome activa- tion) is characterized by a rapid increase in the synthesis of transcripts [14]. At the same time, transcription of ribosomal DNA (rDNA) is switched off in early mouse embryos and nucleoli are not present; instead, so-called nucleolar-precursor bodies (NPBs) are formed. The rein- itiation of rDNA transcription occurs at the end of the 2-cell stage, at the surface of the NPBs [15]. The first dif- ferentiation events take place later on with the physical and functional separation of the inner cell mass from the trophectoderm, at the blastocyst stage (day 3.5). Most of these results were acquired through the use of immuno-fluorescence and fluorescence in situ hybridization (FISH) to label compartments of interest in embryos. However, one important limitation of these studies is that the analysis of the corresponding fluores- cent images is mostly visual and focused on large-scale nuclear movements, which are easier to evaluate. Gen- ome wide approaches, especially chromosome conform- ation capture (3C), can provide more details to help decipher key nuclear events at the molecular level [4], but their use in embryos is limited due to the small size/number of the samples. Fluorescent imaging offers us the advantage of follow- ing several structures within each embryo, thanks to high-resolution microscopy and the combination of sev- eral color channels. However, most analyses are done either in two dimensions or on z-stack sections/projec- tions, and only rarely in three dimensions (3D) because they would be much more time-consuming. A promising approach to explore the embryonic nucleus in more detail is the use of computational imaging [28]. At present, we are still at the very beginning of this ap- proach, and the tools required to locate compartments of interest, to analyze their movements, and to measure physical distances still need improvement. Abstract Background: Embryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. Cells within this embryo will then differentiate and give rise to all the tissues of a new individual. Early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. When the organization of higher-order chromatin structures, such as pericentromeric heterochromatin, was first analyzed in mouse embryos, specific nuclear rearrangements were observed that correlated with embryonic genome activation at the 2-cell stage. However, most existing analyses have been conducted by visual observation of fluorescent images, in two dimensions or on z-stack sections/projections, but only rarely in three dimensions (3D). Results: In the present study, we used DNA fluorescent in situ hybridization (FISH) to localize centromeric (minor satellites), pericentromeric (major satellites), and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos (from the 1-cell to blastocyst stage). Their distribution was then analyzed in 3D on confocal image stacks, focusing on the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. We used computational imaging to quantify various nuclear parameters in the 3D-FISH images, to analyze the organization of compartments of interest, and to measure physical distances between these compartments. Conclusions: The results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. just after fertilization. We also found that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes other remarkable changes during preimplantation development, particularly at the 4-cell stage. Keywords: FISH, Heterochromatin, Centromeres, Telomeres, rDNA, Nucleolus, Nuclear organization, Embryo, Computational analysis Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 2 of 19 Page 2 of 19 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 3 of 19 pericentromeres organized rapidly around the NPBs in the female pronucleus (fPN; maternally inherited gen- ome) whereas in the male pronucleus (mPN; paternally inherited genome), they remained associated together in more or less unorganized masses located in the center (Additional file 1: Figure S1). Remarkably, at PN3, only ~3% of the NPBs were not associated with pericentro- meric signals in the fPN as opposed to almost 30% in the mPN (Table 1). We also noticed that the number of NPBs, while decreasing with time in both PNs, remained approximately twice as high in the mPN as compared to the fPN (Table 1). technique, however, Koehler and collaborators were re- cently able to describe, for the first time, 3D rearrange- ments of chromosome territories in preimplantation embryos [29]. We similarly analyzed major 3D nuclear rearrangements of centromeric and pericentromeric het- erochromatin in bovine and rabbit embryos with dedi- cated computational programs [30,31]. To obtain a more complete understanding of the nu- clear reorganization that takes place during the early de- velopmental stages in mouse, we analyzed, in detail, centromeric and pericentromeric chromatin local repro- gramming in preimplantation embryos with preserved 3D-shapes (from the 1-cell to blastocyst stage). We also developed new image analysis tools to quantify various nuclear parameters of the 3D-FISH images, i.e., the nu- clear volume, the number of NPBs/nucleoli, the nuclear polarity, the number and shape of pericentromeric het- erochromatin structures, and their proximity to NPBs/ nucleoli. It was only in the late 1-cell stage (PN4/PN5) that pericentromeric heterochromatin adopted the same dis- tribution in mPN and fPN, namely, more or less complete "shells" around the NPBs, in which the minor satellite centromeric signals were embedded (Figure 1A/ B/C). Pericentromeric heterochromatin (filaments or more compact foci) was also found at the nuclear per- iphery (in 74% of fPN and 96% of mPN), in association with centromeric spots. Pericentromeric heterochroma- tin formed other remarkable features such as “beaded” filaments extending from the nucleolar periphery to- wards the nuclear periphery (Figure 1, enlargement of B). In addition, as in earlier substages (PN0-PN3), the number of NPBs remained lower in the fPNs (3.9 ± 2.3, almost all associated with pericentromeric signals) than in the mPNs (5.7 ± 3, with on average 1 of them devoid of pericentromeric signals) (n = 42, Table 1). Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Our results highlight differences in nuclear organization in paternal and maternal inherited genomes at the 1-cell stage. We also find that the reprogramming of the embryonic genome, which starts at the 2-cell stage, under- goes several abrupt changes during preimplantation development. Background Using this Parallel, large-scale mapping studies in somatic cells have shown that chromatin is not randomly distributed within nuclei but forms higher-order chromatin struc- tures, some of which correlate with cell differentiation and gene activity [16-18]. For example, proximity to pericentromeric heterochromatin is generally associated Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Unique nuclear organization of zygotes BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 4 of 19 Page 4 of 19 Figure 1 Distribution of the pericentromeres, centromeres, and rDNA FISH signals in late 1-cell stage embryos. 1-cell embryos at the PN4 stage (collected at 27 hphCG) were processed with pericentromeric probes (red) and either centromeric (green, upper panel A/B/C) or rDNA probes (green, lower panel D/E/F). DNA was counterstained with Yopro-1 (grey). Upper panel: (A/B) In both PNs, pericentromeres form more or less complete shells around the NPBs, in which the centromeres are embedded. Pericentromeres are also found at the nuclear periphery, associated with centromeric signals (see enlargement of A), and can form filaments with a “beads on a string” appearance (see enlargement of B). (C) 3D reconstruction of the same nuclei. Lower panel: (D, E) Most of the rDNA signals are around the NPBs. However, there are occasionally some signals associated with pericentromeric filaments (extending from the NPBs towards the nuclear periphery) as well as rDNA signals joining two NPBs. (F) 3D reconstruction of E. Bar = 5 μm. tribution of the pericentromeres, centromeres, and rDNA FISH signals in late 1-cell stage embryos. 1-cell Figure 1 Distribution of the pericentromeres, centromeres, and rDNA FISH signals in late 1-cell stage embryos. 1-cell embryos at the PN4 stage (collected at 27 hphCG) were processed with pericentromeric probes (red) and either centromeric (green, upper panel A/B/C) or rDNA probes (green, lower panel D/E/F). DNA was counterstained with Yopro-1 (grey). Upper panel: (A/B) In both PNs, pericentromeres form more or less complete shells around the NPBs, in which the centromeres are embedded. Pericentromeres are also found at the nuclear periphery, associated with centromeric signals (see enlargement of A), and can form filaments with a “beads on a string” appearance (see enlargement of B). (C) 3D reconstruction of the same nuclei. Lower panel: (D, E) Most of the rDNA signals are around the NPBs. However, there are occasionally some signals associated with pericentromeric filaments (extending from the NPBs towards the nuclear periphery) as well as rDNA signals joining two NPBs. (F) 3D reconstruction of E. Bar = 5 μm. joining two NPBs (n = 4/66, Figure 1E). More surpris- ingly, we frequently observed rDNA foci at the nuclear periphery, associated with pericentromeric signals (Figure 1 lower panel). In fact, ~80% of the pericentro- meric signals at the nuclear periphery were flanked by rDNA foci. Unique nuclear organization of zygotes We first analyzed the distribution of centromeric (minor satellite) and pericentromeric (major satellite) hetero- chromatin in zygotes throughout the first cell cycle after fertilization (1-cell stage). At that stage, the parental genomes are separated in two haploid pronuclei (PN) containing nonfunctional NPBs, and zygotes can be clas- sified in substages from PN0 to PN5 [32,33]. As previ- ously described in the literature, we observed markedly different reorganizations within the male and female pronuclei from PN0 to PN5. Just after fertilization, Owing to the tight association we observed between pericentromeric heterochromatin and the NPBs, we next analyzed the localization of rDNA sequences also known to be structurally associated with NPBs [34]. For that purpose, we performed a dual FISH with major satellite and rDNA probes (n = 66). We found most of the rDNA signals associated with pericentromeric signals at the periphery of the NPBs or within the pericentromeric fila- ments (Figure 1D). We sometimes noticed rDNA signals Table 1 Distribution of pericentromeric signals in fPN and mPN during the 1-cell stage Female PN (fPN) Male PN (mPN) No. of NPBs No. of NPBs not associated with pericentromeric signals No. of NPBs No. of NPBs not associated with pericentromeric signals PN1/PN2 5.9 ± 2.6 a 1.0 ± 1.7 13.94 ± 4.6 a’ 8.8 ± 8 (n = 16) PN3 2.8 ± 2.0 b 0.08 ± 0.3 7.8 ± 4. 1 b’c 2.5 ± 2.0 (n = 18) PN4/PN5 3.9 ± 2.3 b 0.2 ± 0.8 5.7 ± 3 b’c 1.1 ± 1.1 (n = 42) Values with superscripts a and a’ are significantly different at p < 0.0001. Values with superscripts b and b’, and c and c’ are significantly different at p = 0.002. Values with superscripts a and b are significantly different at p < 0.001. Values with superscripts c are significantly different at p = 0.05. Table 1 Distribution of pericentromeric signals in fPN and mPN during the 1-cell stage Table 1 Distribution of pericentromeric signals in fPN and mPN during the 1-cell stage Female PN (fPN) Values with superscripts a and a’ are significantly different at p < 0.0001. Values with superscripts b and b’, and c and c’ are significantly different at p = 0.002. Values with superscripts a and b are significantly different at p < 0.001. Values with superscripts c are significantly different at p = 0.05. Aguirre-Lavin et al. Unique nuclear organization of zygotes It should also be mentioned that none of the NPBs devoid of pericentromeric signals were labeled with rDNA, and ~30% of the PNs contained NPBs bear- ing pericentromeric signals but no rDNA foci. It thus appears that the strong association of pericentromeric heterochromatin with NPBs is not restricted to chromo- somes bearing rDNA sequences, and that such chromo- somes are not exclusively associated with NPBs. in both PNs through a process equivalent to prometa- phase [35,36]; pericentromeres previously forming the peri-NPB shell condensed with their corresponding cen- tromeres and anchored the chromosomes to the NPB, whereas the rest of the chromosomal scaffold extended outwards, like a “cartwheel” (Figure 2C/D) [8]. One to three chromosomes seemed to escape from this radial organization and remained at the periphery of the cart- wheel. They most probably corresponded to the few centromeric/pericentromeric filaments and foci observed at the nuclear periphery from the PN3 to PN5 stages. Post-zygotic changes in nuclear organization After the zygotic stage, the embryonic genome under- goes structural and functional changes. For example, it is well-known that the compaction of pericentromeric heterochromatin that forms chromocenters starts at the 2-cell stage [8-10]. However, few data exist on the global nuclear morphological changes occurring during pre- implantation development, up to the blastocyst stage. We therefore performed systematic 3D-FISH with minor and major satellite probes (centromeric and pericentro- meric heterochromatin). We analyzed embryos at vari- ous time-points during the 2-cell/4-cell/8-cell/16-cell/ morula and blastocyst stages. Representative examples are shown in Figure 3. To gain deeper insight into chromatin higher-order organization within the pronuclei, we next analyzed the distribution of telomeres and performed triple-color FISH with major satellite, minor satellite, and telomeric probes (Figure 2). We could detect the same number of telomeric and centromeric spots in the fPN (35.0 ± 7.44 and 16.1 ± 2.0, respectively) and in the mPN (35.1 ± 6.1 and 15.7 ± 2.5, respectively) (n = 18, Table 2), which approached the expected numbers of 20 and 40, respect- ively. More interestingly, we found approximately half of the telomeres located around the NPBs or associated with extra nucleolar pericentromeric signals (Figure 2A) (17.7 ± 4.5 in the fPN versus 16.2 ± 5.5 in the mPN; Table 2), together with an equivalent number of centro- meres. The second half appeared to be free in the nu- cleoplasm or close to the nuclear envelope (Figure 2B). At the end of the 1-cell stage, chromosomes condensed We observed that remodelling of the embryonic gen- ome indeed started at the 2-cell stage. At the begin- ning of the second cell cycle, the major satellites were essentially associated with the NPBs, as in zygotes, Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 5 of 19 Page 5 of 19 Figure 2 Distribution of telomeres in late 1-cell stage embryos. 3D-FISH was performed with telomeric (blue), pericentromeric (red), and centromeric (green) probes; DNA is in grey. (A, B) Single confocal images of PN5 embryos (collected at 27 hphCG). Telomeres are found 1) at the NPBs’ periphery, 2) associated with extra nucleolar centromeric/pericentromeric signals (A insert), or 3) free in the nucleoplasm (B). (C, D) In embryos at prophase (collected at 30 hphCG), the “cartwheel” organization of condensing chromosomes can be observed in both the fPN (C) and mPN (D). In C/D, four successive confocal sections were merged to improve chromosome visualization. Post-zygotic changes in nuclear organization Bars = 5 μm. Figure 2 Distribution of telomeres in late 1-cell stage embryos. 3D-FISH was performed with telomeric (blue), pericentromeric (red), and centromeric (green) probes; DNA is in grey. (A, B) Single confocal images of PN5 embryos (collected at 27 hphCG). Telomeres are found 1) at the NPBs’ periphery, 2) associated with extra nucleolar centromeric/pericentromeric signals (A insert), or 3) free in the nucleoplasm (B). (C, D) In embryos at prophase (collected at 30 hphCG), the “cartwheel” organization of condensing chromosomes can be observed in both the fPN (C) and mPN (D). In C/D, four successive confocal sections were merged to improve chromosome visualization. Bars = 5 μm. forming either thick partial rims (58% of the NPBs; n = 20) or more spherical patches (5% NPBs) (Table 3 and Figure 3A). Centromeric spots were always asso- ciated with these rims and patches (Figure 3A/A’). The remaining NPBs (37%) were free of any FISH signal (Table 3). However, by the end of the second cell cycle, the percentage of NPBs associated with spherical patches of pericentromeric heterochromatin increased (37.6%, n = 19), whereas NPBs surrounded by rims tended to disappear (24.2%) (Table 3 and Figure 3B/ B’). In these embryos, most of the rDNA signals were located in close proximity to the NPBs and the peri- centromeric signals, as found in zygotes (data not shown & [34]). Remarkably, higher-order chromatin reorganization continued beyond the 2-cell stage. New structures con- taining centromeric and pericentromeric heterochromatin appeared at the 4-cell stage, forming structures very simi- lar to classical chromocenters, i.e., a compact mass of peri- centromeric heterochromatin surrounded by individual centromeres (Figure 3C/C’ and D/D’). During the same period, the number of nucleoli, which were now fully active [15], underwent an abrupt decrease between early 4-cell (11.4 ± 4.4; n = 55) and late 4-cell stages (3.0 ± 1.8; n = 94). Finally, by the blastocyst stage, the overall nuclear organization was very similar to that of somatic cell nuclei in terms of nucleoli numbers and chromocenter organization (Figure 3 from E/E’ to H/H’) [22,37]. Table 2 Distribution of centromeres, pericentromeric heterochromatin, and telomeres in late 1-cell embryos (PN4/PN5 stages) No. of NPBs No. of centromeric spots No. of centromeric spots not associated with NPBs Total no. of telomeric spots No. of telomeric spots associated with NPBs No. Post-zygotic changes in nuclear organization of telomeric spots not associated with NPBs fPN 3.4 ± 2.15 16.1 ± 2.0 1.15 ± 1.14 35.0 ± 7.4 17.7 ± 4.5 15.78 ± 5.55 (n = 30) (n = 13) (n = 13) (n = 18) (n = 18) (n = 18) mPN 4.9 ± 2.8 15.7 ± 2.5 1.08 ± 1.12 35.1 ± 6.1 16.2 ± 5.5 17.6 ± 5.27 (n = 30) (n = 13) (n = 13) (n = 18) (n = 18) (n = 18) f centromeres, pericentromeric heterochromatin, and telomeres in late 1-cell embryos (PN4/PN5 Table 2 Distribution of centromeres, pericentromeric heterochromatin, and telomeres in late 1-ce stages) Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 6 of 19 However, we scanned more than 1000 embryos in total, making the image analysis tedious. In these condi- tions, only the most obvious large-scale nuclear move- t ld b l t d b i l l i W therefore configured semi-automated image analysis tools particularly adapted to the size and geometry of the embryonic nuclei, describing quantitative morpho- t i f t f th l i d th NPB / l li W Figure 3 Distribution of pericentromeres and centromeres at different stages of mouse preimplantation development. Single confocal sections (A-H) of each preimplantation stage with pericentromeric (major satellite, red), centromeric (minor satellite, green), and DNA (grey) labeling are presented here, as well as the corresponding 3D reconstructions (A'-H'). (A, A') Example of an embryo at the early 2-cell stage. Pericentromeres and centromeres are essentially located at the periphery of NPBs, but some NPBs remain devoid of any signal. (B, B') In embryos at the late 2-cell stage, pericentromeric signals are now forming more spherical patches mostly associated to NPBs. The following images show embryos cultured up to the 4-cell (early C, C’ versus late D, D'), 8-cell (early E, E' versus late F, F’), 16-cell (G, G') and blastocyst (H, H’) stage. Pericentromeric and centromeric heterochromatin now forms chromocenters of heterogenous sizes and shapes. Note that at the blastocyst stage we randomly analyzed the trophectoderm cells and the inner cell mass. Bars = 5 μm. Figure 3 Distribution of pericentromeres and centromeres at different stages of mouse preimplantation development. Single confocal sections (A-H) of each preimplantation stage with pericentromeric (major satellite, red), centromeric (minor satellite, green), and DNA (grey) labeling are presented here, as well as the corresponding 3D reconstructions (A'-H'). Morphometric features of nuclei and NPBs/nucleoli p DNA labeling was used to delineate the embryonic nu- clei from the confocal 3D-stacks and to calculate nuclear volumes (as described in Material and Methods). It should be mentioned that, for early stages, we distin- guished early and late time points. However, at later stages, cellular divisions were no longer synchronous and such an analysis could not be performed; we thus pooled the data within each stage. Figure 4 shows that the nuclear volume decreased progressively from the 2-cell stage (3484 μm3 ± 480 μm3; n = 275) to the blastocyst stage (389 μm3 ± 193 μm3; n = 73) by a factor of 10, with the most marked decrease occurring between the 2- and 4-cell stages (twofold). In the following stage, the number of NPBs decreased drastically, as expected, from ~11 NPBs in early 4-cell (n = 100) to ~3 in late 4-cell embryos (n = 87, p < 0.0001, Table 4 and Figure 5). Remarkably, the me- dian value of the NPB volume reached 64.5 μm3 by the end of the 4-cell stage (n = 275, Table 4), suggesting that the number of NPBs decreases via NPB fusion. Controls performed on representative 2-cell and 4-cell embryos showed a slight divergence in the results obtained from manual counting of the NPBs and those from the computerized image analysis, with 8% to 10% divergence in late 2-cell (n = 101), early 2-cell (n = 39), 4-cell (n = 104) stages. However, we observed that this difference was related to the smallest NPBs only (volume < 5 μm3). We next performed a quantitative automated analysis of NPB/nucleolus numbers and volumes. As shown in Figure 5 and Table 4, the number of NPBs decreased slightly but significantly between the early and late 2-cell stage (mean values 12.2 ± 3.90, n = 72 versus 9.9 ± 3.07, n = 211; p < 0.0001). This decrease during the 2-cell stage was accompanied by a marked Figure 4 Quantitative analysis of nuclear volume in preimplantation mouse embryos. Box plots of the nuclear volume at each developmental stage are presented, indicating the smallest observation (sample minimum), the lower quartile, the median value, the upper quartile, and the largest observation. The number of nuclei analyzed at each stage is indicated in brackets above the box plots. At the 2-cell, 4-cell, and 8-cell stages, early (E) and late (L) embryos have been analyzed separately. Post-zygotic changes in nuclear organization (A, A') Example of an embryo at the early 2-cell stage. Pericentromeres and centromeres are essentially located at the periphery of NPBs, but some NPBs remain devoid of any signal. (B, B') In embryos at the late 2-cell stage, pericentromeric signals are now forming more spherical patches mostly associated to NPBs. The following images show embryos cultured up to the 4-cell (early C, C’ versus late D, D'), 8-cell (early E, E' versus late F, F’), 16-cell (G, G') and blastocyst (H, H’) stage. Pericentromeric and centromeric heterochromatin now forms chromocenters of heterogenous sizes and shapes. Note that at the blastocyst stage we randomly analyzed the trophectoderm cells and the inner cell mass. Bars = 5 μm. However, we scanned more than 1000 embryos in total, making the image analysis tedious. In these condi- tions, only the most obvious large-scale nuclear move- ments could be evaluated by visual analysis. We therefore configured semi-automated image analysis tools particularly adapted to the size and geometry of the embryonic nuclei, describing quantitative morpho- metric features of the nuclei and the NPBs/nucleoli. We Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 7 of 19 Table 3 Organization of pericentric heterochromatin in early and late 2-cell embryos No. of NPBs No. of NPBs not associated with pericentromeric signals No. of NPBs associated with pericentromeric rims No. of NPBs associated with condensed pericentromeric patches No. of “free” cytoplasmic pericentromeric patches early 2-cell (n = 20) 13.75 ± 3.78 4.95 ± 1.76 (37%) b 8.10 ± 3.26 (58%) a 0.7 ± 0.57 (5%) a 2.7 ± 1.7 b late 2-cell (n = 19) 11.78 ± 3.47 3.47 ± 1.54 (29.6%) b’ 2.67 ± 1.64 (24.16%) a’ 4.83 ± 2.4 (37.6%) a’ 4.05 ± 2 b’ Values with superscripts a and a’ are significantly different at p < 0.0001. Values with superscripts b and b’ differ at p = 0.02. Table 3 Organization of pericentric heterochromatin in early and late 2-cell embryos also analyzed, in detail, heterochromatin behavior in the context of such morphological changes. modification (p < 0.0001) in the distribution of NPB volumes (Table 4): the median value increased from 28.7 μm3 (n = 887) to 41.5 μm3 (n = 1959). Interestingly, NPBs associated with pericentromeric heterochromatin were larger than those not associated with pericentro- meric heterochromatin, both at early (p < 0.01) and late (p < 0.0001) stages. Post-zygotic changes in nuclear organization At 8 cell, the changes in nucleolar number and size distribution are much smaller, suggest- ing that this fusion process is less prominent. Morphometric features of nuclei and NPBs/nucleoli Differences in mean values between each stage are highly significant, with p < 0.0001 (no subscripts) or p < 0.001 (subscript a, early and late 2-cell stage). Figure 4 Quantitative analysis of nuclear volume in preimplantation mouse embryos. Box plots of the nuclear volume at each developmental stage are presented, indicating the smallest observation (sample minimum), the lower quartile, the median value, the upper quartile, and the largest observation. The number of nuclei analyzed at each stage is indicated in brackets above the box plots. At the 2-cell, 4-cell, and 8-cell stages, early (E) and late (L) embryos have been analyzed separately. Differences in mean values between each stage are highly significant, with p < 0.0001 (no subscripts) or p < 0.001 (subscript a, early and late 2-cell stage). Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 8 of 19 Page 8 of 19 Figure 5 Quantitative automated analysis of NPBs/nucleoli numbers in preimplantation mouse embryos. Box plots of the number of NPBs/nucleoli in 2-cell through 32-cell stages (early (E) and late (L) embryos have been analyzed separately at the 2-cell, 4-cell, and 8-cell stages). The number of nuclei analyzed at each stage is indicated in brackets above the box plots. Differences in mean values between each stage are highly significant, with p < 0.0001; significant between late 4-cell and 16-cell stages (subscript a), with p = 0.00125; and less significant between early 2-cell and early 4-cell stages (subscript b), with p = 0.0103. The difference between late 2-cell and early 4-cell stages as well as between 16- cell and 32-cell stages is not significant (p > 0.05). Figure 5 Quantitative automated analysis of NPBs/nucleoli numbers in preimplantation mouse embryos. Box plots of the number of NPBs/nucleoli in 2-cell through 32-cell stages (early (E) and late (L) embryos have been analyzed separately at the 2-cell, 4-cell, and 8-cell stages). The number of nuclei analyzed at each stage is indicated in brackets above the box plots. Differences in mean values between each stage are highly significant, with p < 0.0001; significant between late 4-cell and 16-cell stages (subscript a), with p = 0.00125; and less significant between early 2-cell and early 4-cell stages (subscript b), with p = 0.0103. The difference between late 2-cell and early 4-cell stages as well as between 16- cell and 32-cell stages is not significant (p > 0.05). Morphometric features of nuclei and NPBs/nucleoli During later development, the number of nucleoli remained quite low (Figure 5), with a slight increase at the 8-cell stage (4.1 ± 2.61, n = 89 at early 8-cell and 4.7 ± 1.91, n = 184 at late 8-cell). However, we noticed that the total nucleolar volume relative to the nuclear volume decreased continuously from 15% at the 2-cell stage (n = 287) to approximately 10% at the 16-cell (n = 158) and 32-cell stages (n = 117) (Table 4). Within each column, values with different subscripts are significantly different at p < 0.01. Maintenance of nuclear polarity of embryonic nuclei during preimplantation development We next performed systematic 3D-FISH with telomeric and pericentromeric probes on embryos from the 2-cell stage until blastocyst. Representative examples are shown in Figure 7. Whereas we observed a peculiar ra- dial distribution of the telomeres versus the centro- meres/pericentromeres in the 1-cell stage, their spatial distribution at the 2-cell stage was completely different (Figure 7A/B). At the later stage, centromeres/pericen- tromeres and their corresponding telomeres were con- fined to one part of the nuclei (whether associated to NPBs or as free chromocenters), while the remaining tel- omeres were clustered in the other part (Figure 7B). This polarity, known as Rabl-like configuration, has already been revealed in nuclei of 2-cell stage embryos via the staining of centromeres/pericentromeres [8,9,38] and in some 8-cell embryos [11]. It is believed to reflect the anaphase orientation of chromosomes and, as such, is usually lost in interphase, except in rare cases [39]. Strik- ingly, it seemed to be maintained in mouse preimplanta- tion embryos during the 2-cell stage and in the following developmental stages (Figure 7D-F). As polarity is quite difficult to analyze visually in toto after the 4-cell stage, essentially because of the small nuclear sizes, we devel- oped a quantitative method to evaluate polarity over a large collection of nuclei (see Material and Methods). As shown in Figure 8, centromere distribution within the nuclei highlights the presence of polarity in all stages that were examined. Curiously, we noticed that this po- larity is lower in the late 4-cell stage as compared to any other stage. Polarity increases again in early 8-cell embryos, then continuously decreases up to the blasto- cyst stage (Figure 8). We immediately noticed that pericentromeres partially surrounding NPBs usually had a roundness that was less than 0.5. We therefore created another tool to analyze the relationship between elongated pericentromeres and NPBs/nucleoli. In brief, we measured the volume of pericentromeric signal interactions with NPBs/nucleoli within a three-pixel distance from the NBPs/nucleoli (see Material and Methods for details). We then deter- mined five categories: Null or Close for those without clear interactions versus Low, Medium, and Strong for those with pericentromere and NPB/nucleolus interac- tions. Figure 6B shows that the proportion of elongated pericentromeres with a strong NPB/nucleolus inter- action was higher in early 2-cell than in late 2-cell embryos. Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 9 of 19 and 4-cell stages, when it reached 90%, a value that did not vary much thereafter (Figure 6E). the “roundness” of the pericentromeric 3D-FISH signals: in brief, the surface of the object undergoing segmenta- tion was divided by the surface of a sphere with an equivalent volume (see Material and Methods for details). We could thereafter classify the pericentromeres either as “compact”, when their roundness was greater than 0.8 and their size larger than 0.47 μm3, or “elon- gated”, when their roundness was less than 0.8 and their size larger than 0.47 μm3. Pericentromeric signals of less than 0.47 μm3 were “not analyzed” (NA); these repre- sented less than 5% of the total volume of pericentro- meres at 2-cell and 4-cell stages and less than 10% in later stages. Figure 6A/A’/A” illustrates the segmentation and classification of the signals obtained using 3D-FISH with pericentromeric probes on a 2-cell stage embryo. Maintenance of nuclear polarity of embryonic nuclei during preimplantation development It then decreased dramatically between the 2- and 4-cell stages, suggesting that the dissociation of peri- centromeric heterochromatin from NBPs/nucleoli begins at the 2-cell stage and finishes at the 4-cell stage. Inter- estingly, in some late 2-cell nuclei, we noticed apparently “compact” pericentromeres with a less intense “core”. However, these pericentromeres were classified as “elon- gated” due to their crescent shape after segmentation (Figure 6C). We believe these pericentromeres represent intermediate configurations between elongated and com- pact heterochromatin structures. Discussion I Important structural remodeling and functional repro- gramming affect the parental genomes during the critical preimplantation developmental period, which encom- passes the transition from totipotency to differentiation. In this study, we used FISH with various genomic probes to analyze higher-order chromatin reorganization in de- tail on large numbers of mouse embryos with 3D pre- served nuclei. Both elongated and compact heterochromatin struc- tures were present in 2-cell stage embryos. Figure 6D/D’ shows the percentage of each structure in nuclei of early 2-cell and late 2-cell stage embryos. Although these per- centages vary from nucleus to nucleus, we could already observe important changes during the 2-cell stage: the percentage of compact pericentromeres was below 50% in the majority of the early 2-cell nuclei and above 50% in the majority of late 2-cell nuclei. When we analyzed a larger number of nuclei from different experiments, we could still see that the percentage of “compact” pericen- tromeres increased between early and late 2-cell stages (p = 0.160; Figure 6E). However, it clearly appears that this number increased more sharply between the 2-cell Pericentromeric heterochromatin structure and organization One of the major events affecting centromeres and peri- centromeres during preimplantation development is their relocation from the periphery of the NPBs towards the nucleoplasm, where they form structures resembling the chromocenters in somatic mouse nuclei. In order to gain deeper insight into this phenomenon, we analyzed Table 4 Characterization of NPBs/nucleoli in mouse embryonic nuclei Stage No. of nuclei No. of NPBs/nucleoli Mean no. of NPBs/nucleoli per nuclei Relative volume of NPBs/nucleoli (% of nuclear volume) Median volumes of NPBs/nucleoli (μm3) [1st quartile; 3rd quartile] Early 2-cell 72 887 12.2 ± 3.90 a 14.9 ± 3.93 ab 28.7 a [16.0; 50.2] Late 2-cell 211 1959 9.9 ± 3.07 b 15.6 ± 3.68 a 41.5 b [16.9; 78.9] Early 4-cell 100 1091 10.9 ± 4.19 ab 14.1 ± 4.47 bc 12.1 cd [6.2; 24.4] Late 4-cell 87 275 2.7 ± 1.9 c 14.3 ± 3.06 c 64.5 e [13.4; 203.9] Early 8-cell 89 364 4.1 ± 2.62 d 13.3 ± 2.32 cd 13.4 cf [5.5; 58.3] Late 8-cell 184 883 4.7 ± 2.65 d 12.6 ± 2.39 d 11.1 d [4.3; 33.0] 16-cell 158 283 2.2 ± 2.13 e 10.8 ± 2.93 e 26.1 af [4.9; 81.0] 32-cell 117 220 1.9 ± 1.41 e 9.6 ± 2.87 f 19.6 cd [3.4; 50.9] Within each column, values with different subscripts are significantly different at p < 0.01. Table 4 Characterization of NPBs/nucleoli in mouse embryonic nuclei Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Peculiar features of zygote nuclear organization Just after fertilization and during the 1-cell stage, the two parental genomes are still separated in two pro- nuclei. This allows the observation of their different behaviors; for example, the differences in terms of epi- genetic marks have already been well-documented Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 10 of 19 Figure 6 (See legend on next page.) Fi 6 (S l d ) Page 11 of 19 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 (See figure on previous page.) Figure 6 Computerized analysis of pericentromere structures and organization in preimplantation mouse embryos. Panel A-A”: Segmentation and classification of the pericentromeric signals in a late 2-cell nucleus. (A) Original confocal section of the 3D-FISH analysis with the pericentromeric probe; (A’) Same confocal section after segmentation and classification into “compact” (red), “elongated” (green), or “non analyzed” (blue) signals; (A”) 3D reconstruction of the pericentromeric signals after segmentation and classification. Panel B: Proximity between elongated pericentromeres and NPBs/nucleoli was analyzed, and five different categories were distinguished: Null, Close, Low, Medium, and Strong. The graph represents the percentage of each group at 2-cell, 4-cell, and 8-cell stages. Panel C: Example of a pericentromeric signal classified as “elongated” through computerized analysis, although it would be classified as “compact” by visual analysis. Note the less intense “core” of this pericentromeric signal. Panel D-D’: Percentage of “compact” (blue) and “elongated” pericentromeres in several nuclei from early versus late 2-cell stage embryos. Panel E: Box plots representing the ratio of “compact” pericentromeres relative to the total observed pericentromeric signals from 2-cell to 32-cell stages (early (E) and late (L) embryos have been analyzed separately at the 2-cell, 4-cell, and 8-cell stages). The number of nuclei analyzed at each stage is indicated in brackets above the box plots. Differences in mean values between stages with different subscripts are highly significant (p < 0.0001) to significant (p = 0.0079). [6,40]. Similarly, higher-order chromatin structures such as pericentromeric heterochromatin have also already been analyzed in mouse embryos [8-10]. As described in the Results section, we observed marked reorganizations within both pronuclei, male (mPN) and female (fPN), during the 1-cell stage. Just after fertilization, pericentro- meres organize rapidly around the NPBs in the fPN, but remain associated in more or less unorganized masses in the mPN. Peculiar features of zygote nuclear organization Figure 8 Computerized analysis of nuclear polarity in preimplantation mouse embryos. Box plots represent the nuclear polarity evaluation at each developmental stage from 2-cell to blastocyst (early (E) and late (L) embryos have been analyzed separately at the 2-cell, 4-cell, and 8-cell stages). The number of nuclei analyzed at each stage is indicated in brackets above the box plots. observed a tight association of this type of heterochro- matin with NPBs/nucleoli, as previously described [8-10]. This tight association does not however hold for all chromosomes, since pericentromeric heterochroma- tin foci were also found at the nuclear periphery in inter- phasic 1-cell embryos (martin et al., 2006, this work) and escaping “peripheral” chromosomes are observed at condensation (Figure 2D). Whether these are specific chromosomes remains unknown; this could be analyzed by chromosome territories painting. It was, however, quite surprising to find that, whenever pericentromeres were located at the nuclear periphery, rDNA signals were almost always associated with them. This confirms that rDNA genomic sequences are not automatically associated with NPBs [34]. It also suggests that, at least in early stages, NPBs are not basic nucleolar precursors, but may have another role and/or function. This hypoth- esis is in agreement with the fact that oocyte nucleolar components are necessary for the reassembly of newly formed NPBs in both pronuclei after fertilization and for further embryonic development [46]. and the transcriptional activation of the corresponding genomic sequences. The highly decondensed state of pericentromeres at the 1-cell stage has also been observed by electron spec- troscopic imaging [44]. In our study, it is highlighted by the fact that “filaments” of pericentromeric signals could be observed escaping the periphery of the NPBs towards that of the nucleus. This result is quite surprising when compared to previous analyses performed by the immuno-staining of HP1β, the associated heterochroma- tin protein [8,9]. We can infer from our results that HP1β is not associated to the totality of the pericentro- meric heterochromatin and is absent from the radial fila- ments. We believe this highly decondensed state of pericentromeres participates to the onset of pericentric satellites expression that starts in late 1-cell stage embryos [11]. It is interesting to note that a similar “dispersion” of pericentromeric heterochromatin followed by a sequen- tial reassembly was observed upon dedifferentiation- redifferentiation of Arabidopsis leaf cells [45] and in nu- clear transfer experiments (upon reprogramming of somatic cell nuclei by recipient oocytes) [22,27]. Peculiar features of zygote nuclear organization Through the detailed analysis of our 3D-FISH images, we show here that paternal pericentromeric het- erochromatin remains aggregated in a central mass up to the PN3 stage, and is only later dispersed to become associated with NPBs. This difference between the two parental genomes may be related to 1) the specific higher-order structure of sperm heterochromatin; 2) the progressive replacement of sperm protamines by his- tones; and/or 3) the specific epigenetic marks present only in male chromatin [40-43]. Regardless of their initial differences, by the end of the first cell cycle, maternal and paternal pericentromeric heterochromatin experience very similar decondensation states, together with a significant tendency to surround NPBs. This decondensation of pericentromeric hetero- chromatin takes place at the time of minor genome acti- vation [12], suggesting a direct functional link between the decondensation of pericentromeric heterochromatin Figure 7 Distribution of pericentromeres and telomeres at different stages of mouse preimplantation development. 3D-FISH was performed with telomeric (blue) and pericentromeric (red) probes; DNA is in grey. Representative single plane confocal images are presented for: early and late 2-cell embryos (A, B) early and late 4-cell (C, D), early and late 8-cell (E, F), 16-cell (G), and blastocysts (H). As expected, only half of the telomeres are associated with pericentromeric signals. . Note that at the blastocyst stage we randomly analyzed the trophectoderm cells and the inner cell mass. B. Bars = 5μm. Figure 7 Distribution of pericentromeres and telomeres at different stages of mouse preimplantation development. 3D-FISH was performed with telomeric (blue) and pericentromeric (red) probes; DNA is in grey. Representative single plane confocal images are presented for: early and late 2-cell embryos (A, B) early and late 4-cell (C, D), early and late 8-cell (E, F), 16-cell (G), and blastocysts (H). As expected, only half of the telomeres are associated with pericentromeric signals. . Note that at the blastocyst stage we randomly analyzed the trophectoderm cells and the inner cell mass. B. Bars = 5μm. Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 12 of 19 Figure 8 Computerized analysis of nuclear polarity in preimplantation mouse embryos. Box plots represent the nuclear polarity evaluation at each developmental stage from 2-cell to blastocyst (early (E) and late (L) embryos have been analyzed separately at the 2-cell, 4-cell, and 8-cell stages). The number of nuclei analyzed at each stage is indicated in brackets above the box plots. Peculiar features of zygote nuclear organization Taken together, these results suggest that this specific re- arrangement of pericentromeric heterochromatin could be one of the features of totipotency. However, the exact composition of these prominent compact fibrillar structures, which are present in fully grown oocytes and early embryos, is far from being com- pletely deciphered. Different approaches have shown that they do not contain DNA, but rather RNA, nucle- olar proteins (fibrillarin, nucleolin, nucleophosmin B23), and non-nucleolar spliceosomal factors [47-51]. It is only during the first half of the 2-cell stage that components of the rDNA synthesis machinery are progressively assembled at the NPB surface, where the first rRNAs are synthesized at the mid/late-2-cell stage [15]. Remarkably, while a small but significant cell-cycle-dependent de- crease in NPB number is observed at 1-cell and 2-cell Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 13 of 19 polarity is conserved up to the 32-cell stage but decreases in blastocysts, as previously suggested by 2D-FISH on centromeric repeats [38]. stages, the decrease is more drastic in 4-cell embryos and is accompanied by a large increase in the median NPB volume. This might reflect a rapid transition in the NPBs’ function. Indeed, if the onset of rRNA synthesis was previously precisely timed [15], nothing is known concerning the dynamics of the other steps of rRNA maturation and pre-ribosomal particles assembly. Unexpectedly, we also found that the 4-cell stage represents a major step in preimplantation develop- ment. When we classified the pericentromeres as either “compact” or “elongated”, we observed that the propor- tion of elongated pericentromeres with a strong NPB/ nucleolus interaction was higher in the early 2-cell than in the late 2-cell stage. This proportion then decreased dramatically between the 2- and 4-cell stages, while the percentage of “compact” pericentromeres increased drastically to reach 90%. Altogether, this suggests that dissociation of pericentromeric heterochromatin from NBPs/nucleoli begins at the 2-cell stage but finishes at the 4-cell stage. The factors or mechanisms that first favor pericentromeric/centromeric association to NPBs and then initiate the formation of chromocenters re- main largely unknown. From a more structural point of view, the fact that the decrease in NPB number is associated with an increase in the median NPB volume, without a significant reduction in the overall volume, suggests the existence of a fusion process in early embryos. A similar fusion process could explain the slight decrease in NPB number at late 1-cell, as suggested by the rDNA bridges sometimes observed between 2 NPBs (Figure 1). Remarkably, the increase of the NPB volume stops at the 4-cell stage and is not observed anymore at the 8-cell stage. This would fit with the fact that active polymerase I transcription and related processing machineries are functionally organized at the NPB surface starting from the end of the 4-cell stage [15]. This fusion process of the NPB could reflect the entropy- driven nonspecific self-organizing forces (“depletion-at- traction”) proposed by some authors to underlie the prin- ciples of nuclear organization [52-55]. Recent computer simulation of chromatin dynamics [56] suggests that these “depletion-attraction” forces are sufficient to explain the position of chromocenters and nucleoli in interphasic Arabidopsis nuclei. However, one such factor could be the HP1β protein. Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 In somatic cells, the presence of HP1β in fibrillarin-rich regions of nucleoli has already been reported [57]. In mouse 1- and 2-cell embryos, we previously showed that fibrillarin is located at the NPB surface [15] and could therefore represent an anchoring protein for HP1β and pericentromeric heterochromatin. Indeed, in 1-cell stage embryos, HP1β accumulations are detectable in the fPN mainly around NPBs and have been also detected, in lower amounts, in association with the paternal pericen- tromeres [8,10,58,59]. This hypothesis is supported by recent data showing HP1 mislocalization, abnormal nu- clear organization, and developmental arrest in H3.3 K27R mutant embryos [25]. Importance of NPBs/nucleoli in global nuclear organization In the present study, we analyzed the distribution of centromeric/pericentromeric heterochromatin with re- spect to the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first develop- mental stages. In 1-cell and 2-cell stage embryos, we Page 13 of 19 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Structural features of the centromeres/pericentromeres in post-zygotic embryonic stages Because of the highly decondensed state of pericentro- meric heterochromatin in zygotes, we were not able to segment the FISH signals in these embryos with suffi- cient precision to perform further computational ana- lysis. On the other hand, as reorganization of the centromeric and pericentromeric heterochromatin into “chromocenters” occurs post-zygotically, in subsequent stages we were able to more precisely analyze hetero- chromatin reorganization as well as various nuclear parameters using the 3D-FISH images. Unique image analysis tools developed for large objects such as individ- ual embryos in toto were specifically adapted to analyze nuclear elements of highly different and complex sizes and shapes, especially the pericentromeric signals. Fi- nally, as all the segmented signals/objects bore labels, we were able to analyze their relationships and measure interaction volumes. Thanks to these computational tools, we were able to analyze, for the first time, a large number of embryos (more than one thousand) covering the whole preimplantation period. Modifications of epigenetic marks could also be involved in the reorganization of pericentromeric/centromeric chro- matin. Complex and asymmetric histone/DNA modifica- tions occur continuously throughout early development in both parental genomes and may regulate the balance between pericentromeric “elongated” versus “compact” structures [6,40]. Finally, regarding other factors that might play a role in heterochromatin assembly and organization, it must be recalled that an unidentified RNA is an integral com- ponent of pericentromeric heterochromatin in humans [60] and is necessary for the accumulation of HP1α on pericentromeric heterochromatin [61]. Moreover, small centromeric RNAs are involved in murine centromeric heterochromatin assembly [62], and in mouse embryos, small RNAs seem to participate in the formation of chromocenters as shown by the use of locked nucleic acid (LNA)-DNA gapmers to interfere with the tran- scription of major satellites in early embryos. This induces developmental arrest before the completion of chromocenter formation [11]. These methods also allowed us to statistically document development-dependent modifications of embryonic gen- ome organization. In particular, we show here that nuclear Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 14 of 19 Ethics statement Animal care and handling were carried out according to European regulations on animal welfare. NB has the authorization to work with laboratory animals from the departmental veterinary regulatory service (license N° 78–95) and from the local ethics committee (Comethea Jouy-en-Josas/AgroParisTech). 3D-FISH Unless otherwise specified, all steps were performed at room temperature. The zona pellucida of embryos was first removed through two rapid incubations in acidic tyrode (Sigma). The embryos were then rinsed in M2 medium, fixed in 4% paraformaldehyde (PFA) for 30 min, rinsed in PBS, and gently deposited with a mini- mum amount of PBS on microscope slides to allow ad- herence. They were then fixed again in 4% PFA for 30 min, permeabilized for 30 min in 0.5% Triton X-100, and rinsed once for 5 min in 2x saline-sodium citrate (SSC), pH 6.3. RNA digestion was performed by incuba- tion in 200 μg/ml RNase (Sigma) in 2x SSC for 30 min at 37°C. After two rinses of 5 min each in 2x SSC at room temperature, the slide was equilibrated in the hybridization buffer (50% formamide, SSC 2X, Denhardt 1X, 40 mM NaH2PO4, 10% dextran sulfate) for 1–2 h. The probes and the slide were separately denatured for 10 min at 85°C in the hybridization buffer. We deposited the probes onto the slide, which was then placed at 37°C for 24 h in a humidified chamber. After two rinses in 2x SSC at 42°C, samples were either directly post-fixed in 2% PFA for 15 min, or further processed for immunode- tection of the telomeric probes: permeabilization for 10 min in 0.5 Triton X-100, blocking for 15 min in 4x SSC containing 1% bovine serum albumin (BSA), and in- cubation with the secondary antibody for 45 min. DNA was counterstained with YoproI (Molecular probes, 1 μM) or propidium iodide (Sigma, 1 μg/ml). We also demonstrated that the 4-cell stage represents a major step in preimplantation development, especially with regards to pericentromeric structures. Pericentro- meric structures may impact the regulation of develop- mental genes, particularly on heterochromatin-dependent gene silencing. As in somatic cells, the role of these rear- rangements during preimplantation development may be to bring different nuclear compartments (chromocenters, nuclear periphery) in close proximity in order to activate/ repress specific genes yet to be identified. Mouse embryo collection and culture Embryos were produced by natural fertilization of C57/ CBAF1 mice. Superovulation was induced by injection of pregnant mare serum gonadotropin (PMSG, Intervet, 5 UI) followed, 48 h later, by injection of human chori- onic gonadotropin (hCG, Intervet, 5 UI). Female mice were then mated with C57/CBAF1 males. Fertilization occurred at about 12 hours after hCG injection, which was used as the reference point for embryonic develop- ment (hours post-hCG i.e. hphCG). Fertilized eggs were collected at the 1-cell stage from the ampulla in M2 medium (Sigma) after a brief treatment with 1 mg/ml of hyaluronidase in phosphate-buffered saline (PBS, pH 7.5) to separate them from the surrounding follicular cells. In vivo developed 2-cell stage embryos were col- lected from the mice oviducts at 38 hphCG, 40 hphCG, and 48 hphCG, and immediately processed by FISH (see Conclusions below). Later stages were obtained from embryos col- lected at the 1-cell stage and cultured in vitro in M16 medium (Sigma) at 37°C in a humidified atmosphere enriched to 5% CO2. They were processed at 53 hphCG (early 4-cell), 62 hphCG (late 4-cell), 64hphCG (early 8- cell), 72 hphCG (late 8-cell), 82 hphCG (16-cell), and 110 hphCG (blastocysts). Altogether, our results show that significant genome re- structuring occurs during the entire preimplantation period. Just after fertilization, zygotes have a very peculiar nuclear organization with highly decondensed pericentro- meric heterochromatin structures. During the next cell cycle (at the 2-cell stage), nucleolar precursor bodies (NPBs) and pericentromeric heterochromatin undergo important reorganization, as previously described in the literature. However, thanks to new computational tools, we were able to analyze these elements for the first time in a large number of embryos, all the way up to the blasto- cyst stage. We believe that these tools could now be used to enable detailed analyses of fluorescent 3D images in other models/organisms. Specifically, we consider compu- tational imaging a promising approach to explore large- scale nuclear movements. Microscopy and image acquisition All specimens were mounted in Vectashield (Vector La- boratories, Burlingame, CA, USA). To preserve the three-dimensional nuclear structure as much as possible, a thin spacer was drawn with a Dako-pen around the embryos before covering them with a 170-μm thick coverslip. Imaging was performed with an inverted Zeiss LSM 510 confocal microscope (MIMA2 platform, INRA) with a 63X oil immersion objective (Plan-Apochromat, N.A.1.4). The z-stacks were acquired using a frame size of 512 × 512, a pixel depth of 8 bits, and 0.371 μm z- steps, with sequential multitrack scanning using the 488-, 543-, and 633-nm wavelengths of the lasers. Segmentation of centromeric and pericentromeric signals Segmentation of centromeric (minor satellite) and peri- centromeric (major satellite) signals obtained by FISH was performed with two different, but similar, proce- dures. In the minor satellite 3D data sets, centromeric signals appeared quite spherical and could be extracted with a one-scale procedure defined to find spots. In the major satellite 3D data sets, pericentromeric signals appeared as different shapes and a multiscale extraction was therefore required. However, these two procedures followed common rules: 1) a preliminary step was required to prepare the cropped images for segmenta- tion, then we had to 2) produce binary masks containing these structures, 3) label connected binary voxels in order to generate independent objects, and 4) remove some of the objects that were not biologically pertinent. Computational image analysis All embryos were first visually analyzed with the LSM510 software, step-by-step through the confocal z- stacks, and with the help of 3D reconstructions using AMIRA software. Except for the 1-cell stage embryos, which presented a peculiar nuclear organization, we then analyzed all the preimplantation embryos with the semi- automated image processing and analysis tools described hereafter. These tools are based on the ITK library (http://www.itk.org) interfaced with Python scripting language [65]. In each case, the LSM image files were first imported with the Bio-Formats library (http://www. loci.wisc.edu), then the color channels were split into separate 3D data sets and upsampled to get a threefold increase in the number of pixels along the z-axis with an isotropic voxel size [0.1236 × 0.1236 × 0.1236 μm3]. Images were then processed to get segmented, labeled objects. To check the efficiency of the segmentation pro- cedures, segmented images were superimposed on their original grayscale image using either macros developed with the ImageJ software (http://imagej.nih.gov/ij/) or the 3D object analysis from Fiji software (http://fiji.sc). In the pre-segmentation step, the noise was eliminated from cropped images using a 2D median filter (radius = 1 pixel). The histogram of gray values was then normal- ized to a mean value of zero and a standard deviation equal to 1. The resulting image was rescaled between 0 and 255 before subsequent treatments. Next, we decreased the local background around the intensity peaks with a morphological top-hat transformation to produce binary masks using an intensity threshold filter set as [mean + 3.3sigma]. Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Britton-Davidian (ISEM - UMR 5554). The 28S and 18S rDNA were purified with PROMEGA Pure Yield Plasmid Miniprep System, labeled separately with Digoxigenin- 11-dUTP by nick translation according to the Roche Protocol, then mixed together in the hybridization buffer. mask, the weighting factor f was used as the signal-to- noise ratio (SNR) in the embryos. This SNR value was generally between 1 and 2 under our image acquisition conditions; when it was outside this range, we used the closest limit of this interval. Next, nuclei were extracted from the binary mask with an a priori method based on their size and shape: a combination of 2D and 3D attribute opening transfor- mations was applied to remove the smallest objects. Connected voxels representing nuclei were then identi- fied with label object representation and manipulation filters [67]; 3D morphological opening and closing trans- formations were applied to fill and smooth the rough la- beled objects. Finally, the bounding box of each nucleus was used to crop smaller 3D data sets in the three color channels, allowing both faster processing and lower computer memory requirements. FISH genomic probes F h d i For the detection of major satellites (pericentromeric heterochromatin), we used a probe prepared by PCR on genomic mouse DNA with the primers 5’-CATATTC CAGGTCCTTCAGTGTGC-3’ and 5’-CACTTTAGGAC GTGAAATATGGCG-3’, and Cy3- or Cy5-labeling by random priming (Invitrogen Kit, Ref 18095–011). Simi- larly, for minor satellite detection (centromeric hetero- chromatin), we used the following two primers: 5’-ACT CATCTAATGTTCTACAGTG-3’ and 5’-AAAACACAT TCGTTGGAAACGCG-3’. For telomere detection, we used the mixmer tTaGgGtTaGgGtTaGgG [3'] Biotine, a kind gift of C. Escudé (MNHN). The plasmids containing the cloned gene fragments of the mouse 28S rDNA (BE-2-pSP64, 1.5kb) [63] and 18S rDNA (SalC-pSP64, 2kb) [64] were provided by Pr. J. Page 15 of 19 Page 15 of 19 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Page 16 of 19 Page 16 of 19 because nucleoli were no longer spherical at that stage. The 2D Otsu method was replaced by a 3D Otsu method and the nuclear contour was then removed by searching for the minimum values between the 3D Otsu image and the nuclear binary mask. combined through an OR bit-wise filter to obtain one bin- ary mask containing the intensity peaks of different sizes. The binary mask of the intensity peaks was then filtered 1) by the binary mask of the brightest regions to remove those in the darkest areas, and 2) by the ROI of the nu- cleus to keep only those in the nucleus. Finally, a 3D shape attribute opening transformation was applied to remove binary structures smaller than 0.123 μm3, i.e. a spherical volume of 5 voxels diameter. Thereafter we used the label representation filters to identify connected voxels as inde- pendent objects, and we kept only the labeled objects cor- responding to “true” heterochromatin signals. Subsequently, we performed a preliminary manual analysis using the 3D object analysis of the Fiji software to determine criteria that could be used to remove objects showing features incompatible with nucleoli structures. These criteria were essentially based on a combination of the flatness and the elongation of the la- beled objects. Different combinations were used depend- ing whether the value for the labeled object’s volume was lower or higher than 94.5 μm3. In the first case, objects showing roundness (surface of the object divided by the surface of a sphere with an equivalent volume) lower than 0.5 were removed. In the second case, we noticed that, most of the time, the objects were formed by two very close nucleoli. Therefore, we applied a morphological erosion with a mild structuring element (3 × 3 × 3 voxels), and after labeling of connected voxels, a dilation with the same structuring element usually enabled us to recover two separate labeled objects. p g g The top-hat transformation applied to the centromeric data set used a local neighborhood of 3 × 3 × 1 voxels. However, preliminary manual analysis performed with the Fiji software showed that the largest labeled objects sometimes represented the juxtaposition of two centro- meric spots, and that some of the smallest labeled objects corresponded to background values. Segmentation of NPBs and nucleoli Segmentation of NPBs and nucleoli In early embryonic nuclei, NPBs and nucleoli are com- pact structures that appear as black round areas after DNA staining. Therefore, segmentation of NPBs/nucleoli was done by searching for dark regions within the DNA images. First, binary objects were obtained using a 2D Otsu threshold method on the cropped DNA image in which the contour of black round areas had been ampli- fied by the addition of the gradient filtered image (sigma = 0.62 μm). Since the nuclear contour was also extracted by the Otsu method, it was necessary to discriminate binary objects connected to this nuclear contour by sub- tracting the nuclear contour obtained from the nuclear mask. Then we applied a morphological opening trans- formations that smoothed rough objects’ contours, sepa- rated collapsed objects, and removed objects smaller than 0.23 μm3. A modification of the segmentation workflow was necessary for 32-cell stage embryos Segmentation of nuclei Since top-hat transformation is a filtering method that generates peaks, we needed to determine which peaks really represented pericentromeric signals (“true” values). To identify the brightest regions where the structures should be present, we applied a Gaussian filter with a wide sigma value (1.24 μm) followed by an intensity threshold set as [mean + 3.3sigma]. We then used three different structuring elements (3 × 3 × 1, 8 × 8 × 2, and 15 × 15 × 1 voxels) to find the pericentromeric signals. The binary masks created by these top-hat transformations were Segmentation of the nuclei in the DNA channel was a critical step because it defined the regions of interest (ROIs) where we looked for centromeric and pericentro- meric structures in our 3D data sets. Since most voxels corresponded to the background of the images, a 3D binary mask was first determined by a threshold method largely used in astronomy: it analyzes only the back- ground intensities and assigns the intensity value [mean + f * sigma] as the lower threshold [66]. To get the best Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 To deter- mine parameters that could be used to improve the automatic selection, we manually analyzed several 2-cell and 4-cell nuclei with the R statistics software (http:// www.r-project.org). Consequently, three selection pro- cesses were used: first, when the labeled objects were bigger than 1.90 μm3, we applied morphological erosion filtering with a small isotropic structuring element (1x1x1 voxels). After the labeling of connected voxels, a dilation with the same structuring element was applied in order to recover two labeled objects when possible. Second, labeled objects with a flatness greater than 3 were removed when their volume was smaller than 1 μm3, and third, when the number of labeled objects was greater than 40 (i.e. the number of chromosomes in the mouse nucleus) all labeled objects smaller than 1% of the overall spots were removed. However, when we checked our procedure using Ima- geJ macros to superimpose the contour of objects onto grayscale images, we noticed that NPBs/nucleoli were sometimes missing after segmentation, especially when they were too small and located on the nuclear periph- ery, while others were not correctly segmented. We therefore added one step to check the segmentation results manually and remove improper NPBs/nucleoli. To add missing NPBs/nucleoli, we drew a new image containing circles corresponding to the midsection of the missing NPBs/nucleoli on the superimposed image. This provided centroid positions and ellipsoidal dimen- sions of the NPBs/nucleoli on which to perform asym- metrical reconstruction by morphological dilation and to insert newly labeled objects using the ITK software li- brary. Finally, when nucleolar reconstruction was not ef- ficient enough, we discarded the corresponding nucleus from our analysis. Nuclear polarity Th d The segmented images of the nuclei and the centro- meres were first spatially normalized to make their prin- cipal moments equivalent. Polarity of centromere distribution was defined as the distance between the center of mass of the previously extracted centromeres and the center of mass of the nucleus and measured using the ITK software library. This distance was then normalized using the radius of a sphere of equivalent volume, and we tested whether this distance was signifi- cantly different from the value that could be obtained for a random distribution of centomeres. Random pat- terns were generated for each nucleus with the same number of centromeres as detected in the nucleus. The distances for the random distribution were measured for 500 independent patterns, and we were able to calculate a p-value for each nucleus using the proportion of ran- dom patterns with a distance equal to or above that observed. Under the hypothesis that centromeres are randomly distributed, the p-value within a population is uniformly distributed between 0 and 1. This was tested using a two-sided Kolmogorov-Smirnov test (α = 1%) in the R statistical software package. 3. Schneider R, Grosschedl R: Dynamics and interplay of nuclear architecture, genome organization, and gene expression. Genes Dev 2007, 21:3027–3043. 4. Trinkle-Mulcahy L, Lamond AI: Nuclear functions in space and time: gene expression in a dynamic, constrained environment. FEBS Lett 2008, 582:1960–1970. 4. Trinkle-Mulcahy L, Lamond AI: Nuclear functions in space and time: gene expression in a dynamic, constrained environment. FEBS Lett 2008, 582:1960–1970. 5. Hajkova P: Epigenetic reprogramming–taking a lesson from the embryo. Curr Opin Cell Biol 2010, 22:342–350. 5. Hajkova P: Epigenetic reprogramming–taking a lesson from the embryo. Curr Opin Cell Biol 2010, 22:342–350. 6. Fulka H, St John JC, Fulka J, Hozák P: Chromatin in early mammalian embryos: achieving the pluripotent state. Differ Res Biol Divers 2008, 76:3–14. 7. Sirard Ma: Activation of the embryonic genome. Soc Reprod Fertil Suppl 2010, 67:145–158. 7. Sirard Ma: Activation of the embryonic genome. Soc Reprod Fertil Suppl 2010, 67:145–158. 8. Martin C, Beaujean N, Brochard V, Audouard C, Zink D, Debey P: Genome restructuring in mouse embryos during reprogramming and early development. Dev Biol 2006, 292:317–332. 8. Martin C, Beaujean N, Brochard V, Audouard C, Zink D, Debey P: Genome restructuring in mouse embryos during reprogramming and early development. Dev Biol 2006, 292:317–332. 9. Nuclear polarity Th d Merico V, Barbieri J, Zuccotti M, Joffe B, Cremer T, Redi CA, Solovei I, Garagna S: Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos. Chromosome Res 2007, 15:341–360. 10. Probst AV, Santos F, Reik W, Almouzni G, Dean W: Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote. Chromosoma 2007, 116:403–415. 11. Probst AV, Okamoto I, Casanova M, Elmarjou F, Le P, Almouzni G, Marjou FE, Baccon PL: A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development. Dev Cell 2010, 1:625–638. 12. Bouniol C, Nguyen E, Debey P: Endogenous transcription occurs at the 1- cell stage in the mouse embryo. Exp Cell Res 1995, 218:57–62. 12. Bouniol C, Nguyen E, Debey P: Endogenous transcription occurs at the 1- cell stage in the mouse embryo. Exp Cell Res 1995, 218:57–62. 13. Nothias JY, Majumder S, Kaneko KJ, DePamphilis ML: Regulation of gene i h b i i f li d l J B l Ch 13. Nothias JY, Majumder S, Kaneko KJ, DePamphilis ML: Regulation of gene expression at the beginning of mammalian development. J Biol Chem 1995, 270:22077–22080. expression at the beginning of mammalian development. J Biol Chem 1995, 270:22077–22080. Acknowledgements W h k Cl F We thank Claire Francastel, Christophe Escudé, Britton-Davidian Janice and Frédéric Catez for their help with 3D FISH probes. We are grateful to Juliette Salvaing and Laetitia Herrzog for their support and assistance. We also acknowledge the platform MIMA2 (Microscopie et Imagerie des Microorganismes, Animaux et Elements) for confocal microscopy and UEIERP for animal care (Unité Expérimentale d'Infectiologie Expérimentale des Rongeurs et Poissons). The present work was supported by INRA « Jeune Equipe » funding and by ANR - programme d’Investissements d’Avenir REVIVE. Received: 11 June 2012 Accepted: 11 October 2012 Published: 24 October 2012 Received: 11 June 2012 Accepted: 11 October 2012 Published: 24 October 2012 Statistical analysis and boxplot representations were per- formed with the R statistical software. The data from dif- ferent cell stages were compared using a Wilcoxon test. Authors' contributions TAG and ABG conducted most of the experimental part of the work, especially the FISH experiments. RF and CB participated in embryo collection and FISH experiments. TAG, PA, ABG, and NB performed the confocal observations. PA and GL performed most of the data analysis. NB and PD coordinated the work. TAG, ABG, PA, PD, and NB co-wrote the manuscript. All authors read and approved the final manuscript. References 1. Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T: Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet 2007, 8:104–115. 1. Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T: Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet 2007, 8:104–115. 2. Misteli T: Beyond the sequence: cellular organization of genome function. Cell 2007, 128:787–800. 2. Misteli T: Beyond the sequence: cellular organization of genome function. Cell 2007, 128:787–800. Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 For example, to know which pericentromeres were connected with NPBs/nucleoli and to analyze their de- gree of proximity, we applied a 3D morphological dila- tion to the binary mask of NPBs/nucleoli with a small structuring element (3 × 3 × 3 voxels). Then we deter- mined the labels of intersected objects with this new mask and we measured the interaction surface of peri- centromeres with nucleoli on the new binary mask. To analyze the degree of interaction between elongated pericentromeres and NPBs/nucleoli, we compared the observed interaction surface with the theoretical inter- action surface of a sphere with a volume equivalent to the pericentromeres’ volume. Ratios smaller than 1 cor- responded to elongated pericentromeres located close to NPBs/nucleoli or showing only a weak interaction. Ratios higher than 1 indicated that these elongated peri- centromeres interacted more strongly with nucleoli. 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Raynal F, Michot B, Bachellerie JP: Complete nucleotide sequence of mouse 18 S rRNA gene: comparison with other available homologs. FEBS Lett 1984, 167:263–268. 65. Lehmann G, Pincus Z, Regrain B: WrapITK: Enhanced languages support for the Insight Toolkit. Insight J 2006, http://hdl.handle.net/1926/188. 66. Lehmann G: Kappa Sigma Clipping. Insight J 2006, http://hdl.handle.net/ 1926/367. 67. Lehmann G: Label object representation and manipulation with ITK. Insight J 2007, http://hdl.handle.net/1926/584. doi:10.1186/1471-213X-12-30 Cite this article as: Aguirre-Lavin et al.: 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development. BMC Developmental Biology 2012 12:30. Aguirre-Lavin et al. BMC Developmental Biology 2012, 12:30 http://www.biomedcentral.com/1471-213X/12/30 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
https://openalex.org/W2743877347	https://europepmc.org/articles/pmc5933481?pdf=render	English	null	C-Reactive Protein/Albumin Ratio and Prognostic Nutritional Index Are Strong Prognostic Indicators of Survival in Resected Pancreatic Ductal Adenocarcinoma	Journal of pancreatic cancer	2,017	cc-by	4,453	Journal of Pancreatic Cancer Journal of Pancreatic Cancer Journal of Pancreatic Cancer Journal of Pancreatic Cancer Volume 3.1, 2017 DOI: 10.1089/pancan.2017.0006 Open Access ORIGINAL ARTICLE C-Reactive Protein/Albumin Ratio and Prognostic Nutritional Index Are Strong Prognostic Indicators of Survival in Resected Pancreatic Ductal Adenocarcinoma Masahide Ikeguchi,* Takehiko Hanaki, Kanenori Endo, Kazunori Suzuki, Seiichi Nakamura, Takashi Sawata, and Tetsu Shimizu Abstract Purpose: We evaluated the clinical importance, such as the occurrence of postoperative pancreatic ﬁstula (POPF) or prognosis, of preoperative serum markers of chronic inﬂammation, nutrition, and immunity, as well as that of serum tumor markers after curative resection of pancreatic ductal adenocarcinomas (PDACs). Methods: Between 2006 and 2015, 43 patients with PDACs underwent curative resection at Tottori Prefectural Central Hospital. We analyzed which preoperative indicators (i.e., C-reactive protein/albumin ratio [CAR], neutro- phil/lymphocyte ratio [NLR], prognostic nutritional index [PNI], carcinoembryonic antigen [CEA], and carbohy- drate antigen 19-9 [CA 19-9]) were the most relevant risk factors for occurrence of POPF and poor patient survival. Results: POPF was detected in 8/43 (18.6%) patients. One patient died of pancreatic ﬁstula at 2 months post- operatively. Among nine candidate factors (operative procedure, operation time, tumor stage, preoperative serum amylase, preoperative CAR, NLR, PNI, CEA, and CA 19-9), we did not identify any signiﬁcant risk factor for the occurrence of POPF. The 5-year overall survival (OS) rate of the 43 patients was 22.4%, and the overall median survival time was 21 months. The multivariate OS analysis demonstrated that high CAR and low PNI were strong preoperative markers of poor prognosis independently of tumor stage. Preoperative CAR and PNI are useful prognostic markers for patients with operable PDACs. Conclusions: Preoperative CAR and PNI are useful prognostic markers for patients with operable Keywords: C-reactive protein/albumin ratio; overall survival; pancreatic ductal adenocarcinoma; postoperative pancreatic ﬁstula; prognostic nutritional index ª Masahide Ikeguchi et al. 2017; Published by Mary Ann Liebert, Inc. 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 work is properly cited. Address correspondence to: Masahide Ikeguchi, MD, Department of Surgery, Tottori Prefectural Central Hospital, 730Ezu, Tottori 680-0901, Japan, E-mail: ikeguchim@ pref tottori lg jp Department of Surgery, Tottori Prefectural Central Hospital, Tottori, Japan. Address correspondence to: Masahide Ikeguchi, MD, Department of Surgery, Tottori Prefectural Central Hospital, 730Ezu, Tottori 680-0 pref.tottori.lg.jp asahide Ikeguchi, MD, Department of Surgery, Tottori Prefectural Central Hospital, 730Ezu, Tottori 680-0901, Japan, E-mail: ikeguchim@ p g y p p *Address correspondence to: Masahide Ikeguchi, MD, Department of Surgery, Tottori Prefectural Central Hospital, 730Ezu, Tottori 680-0901, Japan, E-mail: ikeguchim@ pref.tottori.lg.jp Surgical procedure All patients underwent open laparotomy. According to the tumor location, we performed DP with splenectomy in 14 patients, subtotal stomach-preserving pancreatico- duodenectomy (SSPPD) in 24 patients, SSPPD combined with portal vein partial resection in 4 patients, and total pancreatectomy with splenectomy in 1 patient. After DP, we closed the proximal pancreas stump by suturing with 4-0 prolene in 12 patients and by linear stapler in 2 patients. Reconstruction after SSPPD or SSPPD with par- tial resection of the portal vein was performed by Child’s method (e.g., Braun’s anastomosis after pancreaticojeju- Patients Between 2006 and 2015, we enrolled 43 patients who were diagnosed with operable PDAC and underwent curative resection (no residual tumors macroscopically) at Tottori Prefectural Central Hospital. The average age of the 30 male and 13 female patients was 71.6 (range: 37– 88) years. Clinical and pathological staging of PDACs was performed using the American Joint Committee on Cancer 6th edition tumor-node-metastasis (TNM) stag- ing system for pancreatic cancer.18 All patients were fol- lowed up at Tottori Prefectural Central Hospital until the end of 2016. The mean follow-up period was 25.2 (range: 1–80) months. No patients received chemotherapy and radiation therapy before the operation. Informed consent for medical treatment and use of clinical data from med- ical records were obtained from all patients. Introduction speciﬁc survival rate was 23.1%, and the disease-free survival (DFS) rate was 16.8%. Moreover, it has been reported that after resection of the pancreas, postoper- ative complications occur very frequently. Anastomotic leakages between the pancreatic duct and the jejunum have been observed in 14–17% of patients after pan- creatoduodenectomy (PD).6–8 In addition, pancreatic ﬁstula is frequently detected (16–26%) after distal pan- createctomy (DP).9–11 Such anastomotic leakages or pancreatic ﬁstula are often fatal. The prognosis of patients with pancreatic ductal adeno- carcinomas (PDACs) is extremely poor. Even though curative resection is performed, many patients die of cancer recurrence within a few months.1–3 Yamamoto et al.4 reported that the median survival of 195 patients who underwent pancreatic resection was 27.1 months, and the 5-year actuarial survival rate was 34.5%. In ad- dition, Sato et al.5 reported that in patients with resected PDACs in Japan, the estimated 5-year disease- 31 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 32 nostomy, choledochojejunostomy, and gastrojejunos- tomy).19 An internal short stent was placed across a pancreaticojejunostomy, but no stent was placed follow- ing choledochojejunostomy. Recently, several studies found a strong correlation be- tween poor survival and preoperative chronic inﬂamma- tion, poor nutrition, and low immunity among patients with various cancers.12–14 Patient preoperative C-reactive protein/albumin ratio (CAR) has been used in combina- tion with other parameters not only to diagnose chronic inﬂammation but also to assess the nutritional status of patients with cancer. Neutrophil/lymphocyte ratio (NLR) and prognostic nutritional index (PNI) are markers of chronic systemic inﬂammation and the patients’ immune status and nutritional condition.15,16 Also, serum carci- noembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) levels are known to be good progression indicators among patients with PDACs, including follow- up after resection of tumors.1,17 Postoperative complications Postoperative complications were analyzed by patient clinical record data. The severity of postoperative com- plications was graded according to the Clavien–Dindo classiﬁcation.20 In addition, a Clavien–Dindo score of III or higher was considered a major complication. Study design This study was a retrospective study. We recorded clin- ical parameters, including operation time, postopera- tive hospital stay, and occurrence of postoperative complications. Blood samples were taken from each patient routinely at the time of admission to our hospi- tal. C-reactive protein, serum albumin, serum amylase, total lymphocyte count, and tumor markers, (including CEA and CA 19-9), were recorded. In addition, CAR, NLR, and PNI were calculated. The PNI was calculated using the following formula: 10· serum albumin con- centration (g/dL)+0.005·lymphocyte counts (no./mm3) in peripheral blood.15 The reference ranges of CAR, NLR, and PNI were determined by investigating the pre- operative CAR, NLR, and PNI in 147 patients who un- derwent radical inguinal hernia operations from 2012 to 2015 at our hospital. None of these patients had either chronic or acute inﬂammation. The average age of the 147 control patients was 69 years (range: 27–93 years). Ninety-two were male, and 55 were female. The mean CAR, NLR, and PNI among of the 147 control patients were 0.037 (range: 0.002–0.464), 2.5 (range: 0.9–19.4), and 49.9 (range: 35.2–61.7), respectively. To determine the cutoff values corresponding to the CAR, NLR, and PNI, we plotted receiver operating characteristic (ROC) curves for the 147 control patients and 43 patients with PDACs. The Youden index (sensitivity + speciﬁcity 1) was used, and the highest Youden index values were con- sidered the cutoff levels.21 According to a previous report, the normal range of serum amylase, CEA, and CA 19-9 levels were 37–125U/L, 0–5ng/mL, and 0–37U/mL, re- spectively.22 This study was approved by the Ethical Review Board of Tottori Prefectural Central Hospital (ap- proval number: 2016-49). In the present study, we evaluated the prognostic and clinical importance of serum markers of chronic inﬂammation, nutrition, and immunity combined with tumor markers of patients with PDACs. Results The clinical characteristics, operative procedure, oper- ation time, postoperative complications, and postoper- ative hospital stay of all 43 patients with PDACs are presented in Table 1. Postoperative complications were detected in 34.9% of cases. POPF secondary to pancre- atic juice leakage at the anastomotic site or at the pancreas stump was frequently detected (8/43, 18.6%). The mean length of postoperative hospital stay of the 15 patients with postoperative complications (61.4 days) was signiﬁcantly longer than that of the 28 patients without postoperative complications (22.2 days, p <0.001). One patient died from postoperative peritoni- tis secondary to POPF 2 months postoperatively. Thus, the operative mortality and morbidity in our series were 2.3% and 34.9%, respectively. The 5-year overall survival (OS) rate of the 43 pa- tients was 22.4%, and the overall median survival time (MST) was 21 months. In addition, the 5-year DFS rate of the 43 patients was 9.1%, and the disease- free MST was 9 months. The correlation between the 10 candidate factors (operative procedure, operation time, tumor stage, occurrence of postoperative compli- cations, preoperative serum amylase, preoperative CAR, NLR, PNI, CEA, and CA 19-9) and the overall MST and disease-free MST of 43 patients are shown in Table 2. We found that low preoperative PNI was important risk factor of poor survival in patients with PDACs and, we found that there was even no statistical signiﬁcance, high preoperative CAR was also risk factor of poor survival. Figure 1 shows the OS curves of 28 pa- tients with high PNI (solid line) and 15 patients with low PNI (dotted line). The 5-year OS of patients with low PNI (4%) was signiﬁcantly poorer than that of pa- tients with high PNI (35%, p = 0.001). The multivariate analysis of OS showed that high CAR and low PNI were strong prognostic preoperative markers indepen- dently of tumor stage (Table 3). In addition, in multi- variate analysis of DFS, we found that high CAR was a signiﬁcant poor survival and recurrence preoperative marker (CAR: p = 0.018, odds ratio = 2.948, 95% conﬁ- dence interval = 1.203–7.222), but low PNI was not (PNI: p = 0.124, odds ratio = 2.247, 95% conﬁdence in- terval = 0.802–6.289). The mean and median (range) of preoperative serum amylase, CAR, NLR, PNI, CEA, and CA 19-9 of all 43 patients with PDACs were as follows: serum amylase Table 1. DP, distal pancreatectomy; SSPPD, subtotal stomach-preserving pan- creaticoduodenectomy. Statistical analysis Differences between two normally distributed parame- ters were compared using the v2 and Fisher’s exact Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 33 (111.7, 76, [42–399] U/L), CAR (0.11, 0.05, [0.01– 0.57]), NLR (2.3, 2.2, [0.9–5.2]), PNI (47.7, 48.8, [29.4–63.4]), CEA (7.7, 4.5, [1.1–48.5] ng/mL), and CA 19-9 (369.7, 165, [1–2640] U/mL). According to the cutoff levels of the six parameters, abnormal levels of serum amylase, CAR, NLR, PNI, CEA, and CA 19-9 were detected in 11 (25.6%), 23 (53.5%), 18 (41.9%), 15 (34.9%), 19 (44.2%), and 31 (72.1%) of the 43 patients. probability tests. The Mann–Whitney U test was used to compare differences between two parameters with nonnormal distribution. Correlations between the pa- rameters and occurrence of postoperative pancreatic ﬁstula (POPF) were estimated using logistic-regression analysis. Long-term OS was calculated using the Kaplan–Meier method and the prognostic difference between the two groups was compared using the log- rank test. All data were analyzed by StatView software (Abacus Concepts, Inc., Berkeley, CA). p < 0.05 was regarded as indicating statistical signiﬁcance. We analyzed factors that were thought to inﬂuence the occurrence of POPF. The nine candidate factors were operative procedure, operation time, tumor stage, preoperative serum amylase, preoperative CAR, NLR, PNI, CEA, and CA 19-9. We divided the patients into two subgroups (long and short) according to the median values of operation time, but we could not ﬁnd any signiﬁcant risk factor for the occurrence of POPF (data not shown). Results Sample Characterizations (n = 43) Parameters Age (years, mean, range) 71.6 (37–88) Gender (male, %) 30 (69.8) Operative procedures (%) DP 14 (32.6) SSPPD 24 (55.8) SSPPD combined with portal vein partial resection 4 (9.3) Total pancreatectomy with splenectomy 1 (2.3) Operation time: min (mean, range) 324.7 (150–568) Pathological tumor stages (%) IA 3 (7) IB 2 (4.7) IIA 7 (16.3) IIB 25 (58.1) III 6 (14) Postoperative complications None 28 Yes (pancreatic fistula, passage disorder, others) 15 (8/3/4) Postoperative hospital stay: day (mean, range) 35.9 (10–240) DP, distal pancreatectomy; SSPPD, subtotal stomach-preserving pan- creaticoduodenectomy. Table 1. Sample Characterizations (n = 43) Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Discussion The incidences of severe POPF (Clavien–Dindo score of III or higher) were reported in 14–23% of patients after PD23 and in 10–30% of patients after DP.24 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 34 Table 2. Univariate Survival Analyses of 43 Patients Table 2. Univariate Survival Analyses of 43 Patients N Overall MST (months) p Disease-free MST (months) p Operative procedures DP or total pancreatectomy 15 29 0.266 12 0.614 SSPPD with or without portal vein resection 28 15 8 Operation time Long >297 min 21 15 0.083 7 0.115 Short £297 min 22 28 13 Tumor stages Stage I and IIA 12 61 0.247 33 0.132 Stage IIB and III 31 18 8 Postoperative complication Yes 15 23 0.658 9 0.67 No 28 19 8 Preoperative amylase level High >125 U/L 11 15 0.481 10 0.994 Low £125 U/L 32 24 8 Preoperative CAR High >0.04 23 19 0.06 6 0.085 Low £0.04 20 29 12 Preoperative NLR High >2.5 18 19 0.248 8 0.311 Low £2.5 25 23 10 Preoperative PNI High >44.7 28 26 0.001 13 0.003 Low £44.7 15 9 7 Preoperative CEA High >5 ng/mL 19 24 0.89 9 0.485 Low £5 ng/mL 24 21 10 Preoperative CA 19-9 High >37 U/mL 31 15 0.207 8 0.13 Low £37 U/mL 12 38 23 CA 19-9, carbohydrate antigen 19-9; CAR, C-reactive protein/albumin ratio; CEA, carcinoembryonic antigen; NLR, neutrophil/lymphocyte ratio; PNI, prognostic nutritional index. CA 19-9, carbohydrate antigen 19-9; CAR, C-reactive protein/albumin ratio; CEA, carcinoembryonic antigen; NLR, neutrophil/lymphocyte ratio; PNI, prognostic nutritional index. FIG. 1. Overall survival curves of patients. Twenty-eight patients with high PNI (solid line) had signiﬁcantly better survival than 15 patients with low PNI (dotted line, p = 0.001). PNI, prognostic nutritional index. Also, some cases of POPF can be fatal. Soft pancreas or pancreatic thickness has been thought to be risk factors for occurrence of POPF.25,26 In our series, we found POPF in eight cases (18.6%, PD: 5/28, 17.9% and DP: 3/15, 20%), however, we found no signiﬁcant correla- tion between POPF and poor survival. p The preoperative estimation of risk factors for poor prognosis of patients with PDACs has been studied. References In addition, pretreatment CAR was shown to be a signiﬁcant prognostic indicator in various carcinomas.27–29 PNI is based on albumin and absolute lymphocyte count, which are measured routinely in clin- ical practice. This index is designed to assess nutritional and immunological status, which may be useful in pre- dicting prognosis. Moreover, a signiﬁcant correlation between low PNI and poor survival has been reported in various cancers.16,30,31 In PDACs, Geng et al.31 reported that low PNI correlated signiﬁcantly with shorter OS in patients with advanced pancreatic cancer. In this study, multivariate analysis identiﬁed PNI as an independent prognostic factor for OS. Thus, we recom- mended the preoperative assessment of CAR and PNI before planning the treatment strategies for patients with PDACs. Also, it may be possible that improvement of nutrition before surgery may improve patient’s post- operative prognosis in PDACs. 5. Sato D, Tsuchikawa T, Mitsuhashi T, et al. Stromal palladin expression is an independent prognostic factor in pancreatic ductal adenocarcinoma. PLoS One. 2016;11:e0152523. 6. Arnaud JP, Tuech JJ, Cervi C, et al. Pancreaticogastrostomy compared with pancreaticojejunostomy after pancreaticoduodenectomy. Eur J Surg. 1999;165:357–362. 7. Tabatabei SA, Hashemi SM. Pancreatic anastomosis leakage management following pancreaticoduodenectomy how could be manage the anasto- mosis leakage after pancreaticoduodenectomy? J Res Med Sci. 2015;20:161–164. 7. Tabatabei SA, Hashemi SM. Pancreatic anastomosis leakage management following pancreaticoduodenectomy how could be manage the anasto- mosis leakage after pancreaticoduodenectomy? J Res Med Sci. 2015;20:161–164. 8. Pugalenthi A, Protic M, Gonen M, et al. Postoperative complications and overall survival after pancreaticoduodenectomy for pancreatic ductal adenocarcinoma. J Surg Oncol. 2016;113:188–193. 9. Ceppa EP, McCurdy RM, Becerra DC, et al. Does pancreatic stump closure method inﬂuence distal pancreatectomy outcomes? J Gastrointest Surg. 2015;19:1449–1456. 10. Akita H, Takahashi H, Gotoh K, et al. Closure method for thick pancreas stump after distal pancreatectomy: soft coagulation and polyglycolic acid felt with ﬁbrin glue. Langenbecks Arch Surg. 2015;400:843–848. 11. Yamashita S, Ishizawa T, Ichida A, et al. Advantages and disadvantages of prophylactic abdominal drainage in distal pancreatectomy. World J Surg. 2016;40:1226–1235. 12. Ubukata H, Motohashi G, Tabuchi T, et al. Evaluation of interferon-/ interleukin-4 ratio and neutrophil/lymphocyte ratio as prognostic indi- cators in gastric cancer patients. J Surg Oncol. 2010;102:742–747. 13. Chua W, Charles KA, Baracos VE, et al. Neutrophil/lymphocyte ratio pre- dicts chemotherapy outcomes in patients with advanced colorectal cancer. Br J Cancer 2011;104:1288–1295. 14. Ikeguchi M. Author Disclosure Statement No competing ﬁnancial interests exist. References 1. Distler M, Pilarsky E, Kersting S, et al. Preoperative CEA and CA 19-9 are prognostic markers for survival after curative resection for ductal ade- nocarcinoma of the pancreas—a retrospective tumor marker prognostic study. Int J Surg. 2013;11:1067–1072. 2. Ben Q, An W, Wang L, et al. Validation of the pretreatment neutrophil- lymphocyte ratio as a predictor of overall survival in a cohort of patients with pancreatic ductal adenocarcinoma. Pancreas. 2015;44:471–477. 3. Marsoner K, Kornprat P, Sodeck G, et al. Pancreas cancer surgery in oc- togenarians—should we or should we not? Anticancer Res. 2016;36:1979–1984. 4. Yamamoto T, Yagi S, Kinoshita H, et al. Long-term survival after resection of pancreatic cancer: a single-center retrospective analysis. World J Gas- troenterol. 2015;21:262–268. at a single center. In doing so, we found that high CAR and low PNI were strong prognostic preoperative markers independently of tumor stage. Also, we found that postoperative complications did not inﬂu- ence the survival of the patients. Two markers of sys- temic inﬂammatory response, CRP and albumin, have been used in combination to diagnose not only chronic inﬂammation but also the nutritional status of cancer patients. In addition, pretreatment CAR was shown to be a signiﬁcant prognostic indicator in various carcinomas.27–29 PNI is based on albumin and absolute lymphocyte count, which are measured routinely in clin- ical practice. This index is designed to assess nutritional and immunological status, which may be useful in pre- dicting prognosis. Moreover, a signiﬁcant correlation between low PNI and poor survival has been reported in various cancers.16,30,31 In PDACs, Geng et al.31 reported that low PNI correlated signiﬁcantly with shorter OS in patients with advanced pancreatic cancer. In this study, multivariate analysis identiﬁed PNI as an independent prognostic factor for OS. Thus, we recom- mended the preoperative assessment of CAR and PNI before planning the treatment strategies for patients with PDACs. Also, it may be possible that improvement of nutrition before surgery may improve patient’s post- operative prognosis in PDACs. at a single center. In doing so, we found that high CAR and low PNI were strong prognostic preoperative markers independently of tumor stage. Also, we found that postoperative complications did not inﬂu- ence the survival of the patients. Two markers of sys- temic inﬂammatory response, CRP and albumin, have been used in combination to diagnose not only chronic inﬂammation but also the nutritional status of cancer patients. Conclusion High preoperative CAR and low PNI levels strongly correlated with poor survival in patients with PDACs. These markers should be assessed when selecting the treatment strategies for patients with PDACs. We specially thank Dr. Hiroaki Saito (Department of Surgery, Division of Surgical Oncology, Faculty of Med- icine, Tottori University, Yonago 683-8504, Japan) for analyzing the ROC curves. Discussion Pathological tumor stage was reported as a strong prog- nostic factor for patients with PDACs, but preoperative serum tumor markers, such as CA 19-9 and pancreatic cancer-associated antigen (DUPAN-2), were not.4 However, clinicians may change the treatment strategy for patients with PDACs based on strong preoperative prognostic makers. For patients who have poor prog- nosis based on preoperative prognostic markers, we recommend chemoradiotherapy before radical surgery, or perhaps even avoiding the surgical procedure alto- gether because of the high incidence of postoperative complications. In this study, we retrospectively ana- lyzed the prognostic markers of patients with PDACs FIG. 1. Overall survival curves of patients. Twenty-eight patients with high PNI (solid line) had signiﬁcantly better survival than 15 patients with low PNI (dotted line, p = 0.001). PNI, prognostic nutritional index. Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 35 Table 3. Multivariate Overall Survival Analysis of 43 Patients p Odds ratio 95% Confidence interval Operative procedures DP or total pancreatectomy vs. SSPPD with or without portal vein resection 0.995 0.996 0.325–3.053 Operation time Long >297 min vs. short £297 min 0.062 2.625 0.954–7.194 Tumor stages Stage I and IIA vs. Stage IIB and III 0.148 2.1 0.768–5.747 Postoperative complication Yes vs. no 0.083 0.419 0.157–1.118 Preoperative amylase level High >125 U/L vs. low £125 U/L 0.4 0.617 0.201–1.899 Preoperative CAR High >0.04 vs. low £0.04 0.025 2.895 1.142–7.339 Preoperative NLR High >2.5 vs. low £2.5 0.103 0.373 0.114–1.22 Preoperative PNI High >44.7 vs. low £44.7 0.003 6.803 1.919–24.39 Preoperative CEA High >5 ng/mL vs. low £5 ng/mL 0.829 0.914 0.403–2.072 Preoperative CA 19-9 High >37 U/mL vs. low £37 U/mL 0.746 1.231 0.35–4.335 Table 3. Multivariate Overall Survival Analysis of 43 Patients Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 36 17. Kanda M, Fujii T, Takami H, et al. Combination of the serum carbohydrate antigen 19-9 and carcinoembryonic antigen is a simple and accurate predictor of mortality in pancreatic cancer patients. Surg Today. 2014;44:1692–1701. 29. Chen Z, Shao Y, Fan M, et al. Prognostic signiﬁcance of preoperative C- reactive protein: albumin ratio in patients with clear cell renal cell carci- noma. Int J Clin Exp Pathol. 2015;8:14893–14900. 30. Miyazaki T, Sakai M, Sohda M, et al. Prognostic signiﬁcance of inﬂam- matory and nutritional parameters in patients with esophageal cancer. Anticancer Res. 2016;36:6557–6562. 18. Wasif N, Ko CY, Farrell J, et al. Impact of tumor grade on prognosis in pancreatic cancer: should we include grade in AJCC staging? Ann Surg Oncol. 2010;17:2312–2320. 31. Geng Y, Qi Q, Sun M, et al. Prognostic nutritional index predicts survival and correlates with systemic inﬂammatory response in advanced pan- creatic cancer. Eur J Surg Oncol. 2015;41:1508–1514. 31. Geng Y, Qi Q, Sun M, et al. Prognostic nutritional index predicts survival and correlates with systemic inﬂammatory response in advanced pan- creatic cancer. Eur J Surg Oncol. 2015;41:1508–1514. 19. Oida T, Kano H, Mimastu K, et al. Reconstruction following pancreatico- duodenectomy for patients who have previously undergone partial gastrectomy. Hepatogastroenterology. 2009;56:1199–1202. 20. Miki Y, Toyokawa T, Kubo N, et al. C-reactive protein indicates early stage of postoperative infectious complications in patients following minimally invasive esophagectomy. World J Surg. 2017;41:796–803. Cite this article as: Ikeguchi M, Hanaki T, Endo K, Suzuki K, Nakamura S, Sawata T, Shimizu T (2017) C-reactive protein/albumin ratio and prognostic nutritional index are strong prognostic indicators of survival in resected pancreatic ductal adenocarcinoma, Journal of Pancreatic Cancer 3:1, 31–36, DOI: 10.1089/pancan.2017.0006. Cite this article as: Ikeguchi M, Hanaki T, Endo K, Suzuki K, Nakamura S, Sawata T, Shimizu T (2017) C-reactive protein/albumin ratio and prognostic nutritional index are strong prognostic indicators of survival in resected pancreatic ductal adenocarcinoma, Journal of Pancreatic Cancer 3:1, 31–36, DOI: 10.1089/pancan.2017.0006. 21. Kawahara T, Yokomizo Y, Ito Y, et al. Pretreatment neutrophil-to- lymphocyte ratio predicts the prognosis in patients with metastatic prostate cancer. BMC Cancer. 2016;16:111. 22. Chiaravalloti A, Fiorentini A, Palombo E, et al. Evaluation of recurrent disease in the re-staging of colorectal cancer by 18F-FDG PET/CT: use of CEA and CA 19-9 in patient selection. Oncol Lett. 2016;12:4209–4213. 23. Kinaci E, Sevinc MM, Ozakay A, et al. References Glasgow prognostic score and neutrophil-lymphocyte ratio are good prognostic indicators after radical neck dissection for advanced squamous cell carcinoma in the hypopharynx. Langenbeck’s Arch Surg. 2016;401:861–866. 15. Nozoe T, Kohno M, Iguchi T, et al. The prognostic nutritional index can be a prognostic indicator in colorectal carcinoma. Surg Today. 2012;42:532–535. 16. Ikeguchi M, Ashida K, Saito H. New prognostic indicator is useful for predicting the survival of patients with unresectable advanced colorectal cancer. Hepatogastroenterology. 2015;62:61–64. Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Ikeguchi et al.; Journal of Pancreatic Cancer 2017, 3.1 http://online.liebertpub.com/doi/10.1089/pancan.2017.0006 Intraoperative acidosis is a new predictor for postoperative pancreatic ﬁstula after pancreaticoduode- nectomy. Hepatobiliary Pancreat Dis Int. 2016;15:302–309. Abbreviations Used CA 19-9 ¼ carbohydrate antigen 19-9 CAR ¼ C-reactive protein/albumin ratio CEA ¼ carcinoembryonic antigen DFS ¼ disease-free survival DP ¼ distal pancreatectomy MST ¼ median survival time MST ¼ median survival time NLR ¼ neutrophil/lymphocyte ratio OS ¼ overall survival PD ¼ pancreatoduodenectomy PDAC ¼ pancreatic ductal adenocarcinoma PNI ¼ prognostic nutritional index POPF ¼ postoperative pancreatic ﬁstula ROC ¼ receiver operating characteristic SSPPD ¼ stomach-preserving pancreaticoduodenectomy Abbreviations Used CA 19-9 ¼ carbohydrate antigen 19-9 CAR ¼ C-reactive protein/albumin ratio CEA ¼ carcinoembryonic antigen DFS ¼ disease-free survival DP ¼ distal pancreatectomy MST ¼ median survival time MST ¼ median survival time NLR ¼ neutrophil/lymphocyte ratio OS ¼ overall survival PD ¼ pancreatoduodenectomy PDAC ¼ pancreatic ductal adenocarcinoma PNI ¼ prognostic nutritional index POPF ¼ postoperative pancreatic ﬁstula ROC ¼ receiver operating characteristic SSPPD ¼ stomach-preserving pancreaticoduodenectomy y p y 24. Fujino Y. Perioperative management of distal pancreatectomy. World J Gastroenterol. 2015;21:3166–3169. 25. Sugimoto M, Takahashi S, Kojima M, et al. In patients with a soft pancreas, a thick parenchyma, a small duct, and fatty inﬁltration are signiﬁcant risks for pancreatic ﬁstula after pancreaticoduodenectomy. J Gastrointest Surg. 2017. [Epub ahead of print]; DOI: 10.1007/s11605-017-3356-7. urg. 2017. [Epub ahead of print]; DOI: 10.1007/s11605-017-3356 26. Chang YR, Kang JS, Jang JY, et al. Prediction of pancreatic ﬁstula after distal pancreatectomy based on cross-sectional images. World J Surg. 2017. [Epub ahead of print]; DOI: 10.1007/s00268-017-3872-3. 27. Gockel I, Dirksen K, Messow CM, et al. Signiﬁcance of preoperative C-reactive protein as a parameter of the perioperative course and long- term prognosis in squamous cell carcinoma and adenocarcinoma of the oesophagus. World J Gastroenterol. 2006;12:3746–3750. 28. Hwang JE, Kim HN, Kim DE, et al. Prognostic signiﬁcance of a systemic inﬂammatory response in patients receiving ﬁrst-line palliative chemo- therapy for recurred or metastatic gastric cancer. BMC Cancer. 2011;11:489. Publish in Journal of Pancreatic Cancer -Immediate, unrestricted online access -Rigorous peer review -Compliance with open access mandates -Authors retain copyright -Highly indexed -Targeted email marketing liebertpub.com/pancan Publish in Journal of Pancreatic Cancer -Immediate, unrestricted online access -Rigorous peer review -Compliance with open access mandates -Authors retain copyright -Highly indexed -Targeted email marketing liebertpub.com/pancan liebertpub.com/pancan
https://openalex.org/W4362340804	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S2_from_PP2A_Inhibitor_PME-1_Drives_Kinase_Inhibitor_Resistance_in_Glioma_Cells/22413807/1/files/39859827.pdf	English	null	Supplementary Figure S4 from PP2A Inhibitor PME-1 Drives Kinase Inhibitor Resistance in Glioma Cells	null	2,023	cc-by	547	Supplementary Figure S2 Supplementary Figure S2 pp y g siRNA: PME-1 Scr. Control U87MG-luc STS (50 nM) UCN-01 (0.5 µM) PKC412 (2.5 µM) K252a (2.5 µM) CEP-701 (1.5 µM) 0 10 20 30 40 50 60 70 Colony growth (Area %) Control UCN-01 (0.5 µM) PKC412 (2.5 µM) K252a (2.5 µM) CEP-701 (1.5 µM) STS (50 nM) U87MG-luc Scr. siRNA PME-1 siRNA * * * * * b-actin U87MG-luc PME-1 1 2 3 7 10 1 2 3 7 10 Scr siRNA PME-1 siRNA Time (days): E siScr tumor siPME-1 tumor UCN-01 0.00E+00 1.00E+10 2.00E+10 3.00E+10 4.00E+10 5.00E+10 6.00E+10 7.00E+10 1 3 5 7 9 11 13 15 17 19 Time (days after tumor cell injection) siScr tumors siPME-1 tumors * siRNA Tumor-associated bioluminescence, fux (p/sec) B 0 0.2 0.6 1 1.4 * siScr tumor siPME-1 tumor Average PME-1 staining (fold change) F CD133 CD15 Nestin Olig2 PME-1 PP2AC SOX2 GAPDH Bulk GSC Bulk GSC Bulk GSC Bulk GSC Bulk GSC E2 G7 R15 R10 R24 D C A siRNA: PME-1 PME-1 Scr GAPDH U251 siRNA: PME-1 PME-1 Scr. GAPDH E2 (GSC) siRNA: PME-1 PME-1 Scr. GAPDH R10 (GSC) G H A siRNA: PME-1 PME-1 Scr GAPDH U251 siRNA: PME-1 Scr. Control U87MG-luc STS (50 nM) UCN-01 (0.5 µM) PKC412 (2.5 µM) K252a (2.5 µM) CEP-701 (1.5 µM) 0 10 20 30 40 50 60 70 Colony growth (Area %) Control UCN-01 (0.5 µM) PKC412 (2.5 µM) K252a (2.5 µM) CEP-701 (1.5 µM) STS (50 nM) U87MG-luc Scr. Supplementary Figure S2 siRNA PME-1 siRNA * * * * * b-actin U87MG-luc PME-1 1 2 3 7 10 1 2 3 7 10 Scr siRNA PME-1 siRNA Time (days): B C A siRNA: PME-1 PME-1 Scr GAPDH U251 B A PKC412 (2.5 µM) K252a (2.5 µM) CEP-701 (1.5 µM) 0 10 Col Control UCN-01 (0.5 µM) PKC412 (2.5 µM) K252a (2.5 µM) CEP-701 (1.5 µM) STS (50 nM) b-actin U87MG-luc PME-1 1 2 3 7 10 1 2 3 7 10 Scr siRNA PME-1 siRNA Time (days): C C E siScr tumor siPME-1 tumor 0 0.2 0.6 1 1.4 * siScr tumor siPME-1 tumor Average PME-1 staining (fold change) E siScr tumor siPME-1 tumor UCN-01 0.00E+00 1.00E+10 2.00E+10 3.00E+10 4.00E+10 5.00E+10 6.00E+10 7.00E+10 1 3 5 7 9 11 13 15 17 19 Time (days after tumor cell injection) siScr tumors siPME-1 tumors * siRNA Tumor-associated bioluminescence, fux (p/sec) 0.6 1 1.4 * ME-1 staining change) D E si UCN-01 0.00E+00 1.00E+10 2.00E+10 3.00E+10 4.00E+10 5.00E+10 6.00E+10 7.00E+10 1 3 5 7 9 11 13 15 17 19 Time (days after tumor cell injection) siScr tumors siPME-1 tumors * siRNA Tumor-associated bioluminescence, fux (p/sec) ME-1 staining D E D Time (days after tumor cell injection) 0 0.2 siScr tumor siPME-1 tumor Average PM (fold c F CD133 CD15 Nestin Olig2 PME-1 PP2AC SOX2 GAPDH Bulk GSC Bulk GSC Bulk GSC Bulk GSC Bulk GSC E2 G7 R15 R10 R24 siRNA: PME-1 PME-1 Scr. GAPDH E2 (GSC) siRNA: PME-1 PME-1 Scr. GAPDH R10 (GSC) G H F CD133 CD15 Nestin Olig2 PME-1 PP2AC SOX2 GAPDH Bulk GSC Bulk GSC Bulk GSC Bulk GSC Bulk GSC E2 G7 R15 R10 R24 F siRNA: PME-1 PME-1 Scr. GAPDH E2 (GSC) siRNA: PME-1 PME-1 Scr. GAPDH R10 (GSC) G H G H
https://openalex.org/W2074350410	https://zenodo.org/records/2501526/files/article.pdf	German	null	LXXVII. Über einige Chinazoline aus o‐Amido‐m‐Xylyl‐p‐Toluidin	Journal für praktische Chemie	1,906	public-domain	6,311	') Dies. Journ. [2] 71, 153. ,) In der Folge einfach ,,Dihydrochinazolin" genannt. ,) In der Folge einfach ,,Dihydrochinazolin" genannt. Journal f. prakt. Chemie r2] Rd. 78. v. Walther u. Bamberg: Ober einige Chinazoline. v. Walther u. Bamberg: Ober einige Chinazoline. 209 ') Dies. Journ. [2] 71, 153. ,) In der Folge einfach ,,Dihydrochinazolin" genannt. Journal f. prakt. Chemie r2] Rd. 78. 14 LXXVII. Uber einige Chinazoline an8 o-Amido-m-Xylyl- p-Tolnidin ; von R. von Walther uiid R. Bamberg. In einer friiheren Abhandlung l) haben wir mehrere Derivate des o-Amido- m-Xylyl-p-Toluidins beschrieben; es wurde dabei erwahnt, daB mit Hilfe von Amylnitrit ziemlich leicht ein heterocyklischer TriaziDkSrper entsteht. Die Reaktionsfahigkeit des erwahnten Ausgangsmaterials ist ganz die eines monalky- lierten o- Amidobenzylamins; es waren deswegen bei der Acy- lierung Chinazolinderivate zu erwarten. Bei den dahinzielendeii Versuchen zeigte es sich, daB wohl relativ leicht die Um- setzungen rnit Chlorkohlenoxyd, Schwefelkohlenstoff und auch mit Ameisensaure resp. o-Ameisensaureather durchfiihrbar sind, daB dagegen schon bei der Verwendung von Essigsaure Eemmungen auftreten, die nur durch Benutzung des reaktions- fahigen Imidoathers zu uberwinden sind. Von den zu er- wartenden Chinazolinen sind vorl'aufig folgende gewonnen worden. z (6) -Met h y 1- (3 n)- p - To 1 y 1- (3.4)- D ih y d r o chin az olin, 2) CH N CH/B\ /;\CH c H s . & ~ \,N.c6H4.cHs 2 l CH CH, 10 g o- Amido-m-Xylyl-p-Toluidin wurden mit 15 g Ortho- ameisensaureather (($5 g berechnet) auf dem Wasserbad am RiickfluBkuhler eine Stunde lang erwarmt. Nach dem Ab- kiihlen hatten sich Kristalle ausgeschieden, denen nach dem 14 210 l h B b Ob i i 210 v. Wttlther u. Bamberg: Ober einige Chinazoline. Verdunsten des Orthoameisensiiureathers noch Schmieren an- hafteten. Dieselben wurden durch Absaugen entfernt und es resultierten 9 g Rohprodukt = BOO/, der Theorie. Bus Alkohol erhielt man sechsseitige, weiBe Blattchen vom Schmelzp. 158O. Sehr leicht liislich in Benzol und Chloroform, loslich in Alkohol und in Ather, weniger leicht in Ligroin und Petroliitber, unloslich in Wasser. Die Realrtion vollzieht sich nach folgender Qleichung: ,NIT H, C.C,H, + HC.(OC,H,), = \CH,.NH.C,B,.CH, N ,NIT H, C.C,H, + HC.(OC,H,), = \CH,.NH.C,B,.CH, N Da es zweifelhaft erschien, ob RingschuS oder Amidin- H,C.C,H,/ \CH,.NH.C,H,.CR, eingetreten war - was aus den Analysenresultaten allein nicht hervorgeht - wurde der neue Korper einer Molekulargewichts- bestimmung unterworfen, deren Resultat die erstere Annahme bestatigte. eingetreten war - was aus den Analysenresultaten allein nicht hervorgeht - wurde der neue Korper einer Molekulargewichts- bestimmung unterworfen, deren Resultat die erstere Annahme bestatigte. Diese M 01 e kul a r g e wi cht s b e s t imm un g wurde mit B e c km ann s Gefrierpunkts-Erniedrigungs- Apparat ausgefiihrt und zwar mit Benzol als LGsungsmittel. Das Moleltnlargewieht M berechnet sich gemal3 der Gleichung: iv = K . - ..'- A . LXXVII. Uber einige Chinazoline an8 o-Amido-m-Xylyl- p-Tolnidin ; L aus aus aus K = Konstante fur Benzol (4900), s = Abgewogene Substanzmenge (0,2006 g), A = Gefrierpunktsdepression in OC (0,165 i. M.)) L = Menge des Losungsmittels: 25,25 g. 0 2006 0,165.25,25 J4 = 4900. -L- = 235,9. 211 8. Walther 11. Barnberg; Ober einige Chinazoline. 8. Walther 11. Barnberg; Ober einige Chinazoline. Aus der Forinel C,,H,,N, ergibt sich: Aus der Forinel C,,H,,N, ergibt sich: I. 11. 0,1324 g Substanz ergaben 0,3930 g CO, und 0,0836 g H,O. 0,1002 g Subst. gaben bei 22 und 749 mm Druck = 11,6 ccm N Berechnet auf Gefunden: C,,H,,N, = 236: I. 11. c 81,36 80,96 - O i , H 6,lS 1,07 - ,I K 11,86 - 11,86 ,, . Nach den Ergebnissen der Molekulargewichtsbestimmung sowie der Analysen verlauft die Kodensation des o -8mido-m- Xylyl-p-Toluidins mit Orthoameisensaureather unter Austritt von 3 Molekulen Alkohol und zwar unter RingschuB: Es hat sich also das erwartete Dihydrochinazolin-Derivat gebildet. Zu demselben Dihydrochinazolin-Derivat gelangt man auch, wenn man statt des Orthoameisensaureathers Ameisensaure selbs verwendet; jedoch verlauft die Umsetzung nicht so hoch- prozentig. 10 g o-Amido-m-Xylyl-p-Toluidin wurden in 10 g 80°/,iger Ameisensaure, dem Dreifachen der berechneten Menge, auf- gelost, wobei Warmeentwicklung und Braunfarbung eintrat. Die Losung wurde eine Stunde lang am RiickfluDkiihler er- warmt, dann mit Tierkohle gekocht, filtriert und nach dem Erkalten die BaRe durch Ammoniak frei gemacht. Ausbeute 5 g = 50°/, der Theorie. Die Base erwies sich als identisch mit der durch Ortho- ameisensaureather erhaltenen und zeigte densclben Schmelz- punkt 153O. 0,1119 g Substanz gaben bci 22O und 750 mm Druck 11,6 ccm N. 0,1119 g Substanz gaben bci 22O und 750 mm Druck 11,6 ccm N. Bcrechnct auf C,,,II,,N, = 236: N = 11,86 Gefunden: 11,59 O i 0 . Bcrechnct auf C,,,II,,N, = 236: N = 11,86 Gefunden: 11,59 O i 0 . Das als Zwischenstufe zu erwartende Formylderivat wurde nich t gefa €it. Salzsaures Salz. 2 v. Walther ti. Barnberg: Uber einige Chinazoline. 3 Molekiile Wasser enthalten. Schmelzp. des wasserfreiera Salzes 212O. 3 Molekiile Wasser enthalten. Schmelzp. des wasserfreiera Salzes 212O. K r i s t all wasser - B e s ti m mung. I. Salzsaures Salz, luftrrockeu gewogen und bei 130° bis zur Ge- wichtskonstanz getrocknet. Es ergaben dann 0,1107 g Substanz einan Gewichtsverlust von g 0,0181 g. Gefunden : g Berechnet auf C,,H,,N,.HCl i- 3H,O: 20 16,54 g Berechnet auf C,,H,,N,.HCl i- 3H,O: H20 16,54 C,,H,,N,.HCl i- 3H,O: 16,89 Das s c h w e f el saur e S alz kritallisierte in prachtigen, weiWen Kristallnadeln vom Schmelzp. 132O. Salpetersaures Saiz. Beim Versetzen der Base mit verdiinnter Salpetersaure wurde ein Teil derselben angegriffen. Es bildeten sich braune Schmieren; aus dem Eiltrat schied sich dann nach langerem Stehen das salpetersaure Salz des Chinazolinderivsts in langen, schwach gelblichen Nadeln ab. Schmelzp. 95 O. Zersetzung bei 170O. Salzsaures Salz. Die Base, in verdiinnter Salzsaure unter Erwarmen geliist, ergibt beim Erkalten das salzsaure Salz in weiBen Nadeln, die * 14 * 212 212 v. Walther ti. Barnberg: Uber einige Chinazoline. V. Walther 11. Barnberg: nber einige Chinaxoline. V. Walther 11. Barnberg: nber einige Chinaxoline 213 P i k r i ns aur e s S alz. Die Base wurde in Eisessig gelost und mit einer kon- zentrierten Losung von Pikrinsaure in Eisessig versetzt, Nach kurzer Zeit schied sich das Pikrat in prachtigen, orange- gelben Nadeln aus, die aus Eisessig umkristallisiert wurden. Schrnelzp. 201 O. 0,1134 g Substanz gaben bei 22O und 746 mm Druck. 14,8 ccm N. Berechnet auf Gefunden : C,,H,9N50, = 465: N 15,05 14,50 "/". P 1 a t inc hl ori d -D o p p el s alz. Man 1Sst die Base in Eisessig, sauert etwas mit Salzsaure an und versetzt mit Platinchloridlosung ; das Salz scheidet sich ahbald in gelblichen, zu Biischeln vereinigten Nadeln vom Schmelzp. 202O aus. 0,2028 g Substanz ergaben 0,0455 g Pt. Berechnet auf (C,,,H,sN,€I),PtCI, = 882: Pt 22,09 0,2028 g Substanz ergaben 0,0455 g Pt. Berechnet auf (C,,,H,sN,€I),PtCI, = 882: Pt 22,09 Gefunden : 22,43 O,@. l) In der Folge; ,,Tetrahydrochinazolin"-Base. Br o m i e r un g s v e r su ch d e s D i h y d r o c hin a z olins. Br o m i e r un g s v e r su ch d e s D i h y d r o c hin a z olins. Zu 2 g der in absolutem Alkohol geliisten Dihydrochinazolin- Base fugt man allmahlich 2 Atome Brom (d. i. 1,3 g oder 0,4 ccm). Es tritt neben starker Erwarmung Geruch nach Aldehyd auf und nach einiger Zeit scheidet sich ein fester Eorper in roten Nadeln ab, der aus Alkohol umkristallisiert wurde. B r o m-B e s t immun g. Es ergaben I. 0,1685 g Substanz 0,1584 g AgBr. 11. 0,1543 g Substanz 0,1470 g AgBr. Berechnet auf Gefunden: C,,H,,N,Br, = 395,9 : I. 11. Br 40,39 40,Ol 40,54 '/,,. Berechnet auf C,,H,,N,Br, = 395,9 : Br 40,39 Br Entgegen der Erwartung ergibt sich aus den Analysen- resultaten und den sonstigen Eigenschaften des Korpers, daB keine Substitution, sondern nur eine einfache Addition des Broms an den 3 wertigen Stickstoff unter Ammonsalzbildung erfolgt ist : Rr Br DaB das Brom sich nur an den Stickstoff angelagert hat und nicht substituierend in den Chinazolinkern eingedrungen ist, beweist das Verhalten des Korpers gegen schweflige Saure. Die rotliche, alkoholische Liisung wurde sofort durch schweflige Saure entfarbt und aus dieser Losung auf Zusatz von Alkali die ursprungliche Dihydrochinazolin-Base erhalten. Verhalten des Dihydrochinazolins gegen Jod. 2 g Dihydrochinazolin-Base in Alkohol wurden mit 2 g Jod, ebenfalls in Alkohol gelost, versetzt, wobei Geruch nach Aldehyd wahrnehmbar war. Es schieden sich bei langerem Stehen Kristalle von rijtlich metallischem Glanz aus, die aus Alkohol umkritallisiert wurden. Die Analyse ergab aber, daB nicht, wie beim Brom- 4 v. W a l t h e r u. Bamberg: nber einige Chinazoline. 14 v. W a l t h e r u. Bamberg: nber einige Chinazoline. 214 Additionsprodukt, nur 2, sondern 3 Atome Halogen einge- treten waren, yon denen eins als Jodwasserstoff, der hierbe in Nebenreaktion auftritt, sich an das andere N-Atom an- lagert : J J J J Auch hier fand nur eine Addition des Jods und des Jod- wasserstoffes an die N-Atome statt. Die mit schwefliger SBure entfarbte Losung schied auf Zusatz von Alkali wieder das Dihydrochinazolin aus , kenntlich an Kristallform und Schmelzpunkt. Es ergaben: I. 0,1506 g Substanz 0,1701 g AgJ. 11. 0,1520 g Substanz 0,1714 g AgJ. Berechnet auf Gefunden: C,,H,,N,J, = 617,5: I. 11. J 61,65 61,03 60,93 yo. Es ergaben: I. 0,1506 g Substanz 0,1701 g AgJ. Br o m i e r un g s v e r su ch d e s D i h y d r o c hin a z olins. 11. 0,1520 g Substanz 0,1714 g AgJ. Berechnet auf Gefunden: C,,H,,N,J, = 617,5: I. 11. J 61,65 61,03 60,93 yo. B z- (6) M e t h y 1- (3 n) p - T o 1 y 1-(1,2,3,4) T e t r a h y d r o chin - a z ol in l), N €1 Die Annahme des Dihydrochinazolin-Charakters der soeben behandelten Verbindung findet eine weitere Bestatigung in den Ergebnissen der Reduktion. Die im Dihydrochinazolin anzunehmende Doppelbindung mu6 den Korper befahigen, 2 Atome Wasserstoff aufzunehmen und dabei in ein Tetra- h y dr ochinaz olin iib erzu ge h en. In einem mit Ruckflubkiihler versehenen Kol'ben wurden 3 g Dihydrochinazolin-Base in absolutem Alkohol gelost, und 215 Y. Walther ti. Bamberg: ober einige Chinazoline. das Vierfache der berechneten Menge Natrium in kleinen Stiickchen allmahlich zugegeben. Gibt man nach Beendigung der Beaktion, nachdem alles Natrium aufgelost ist, Wasser zum Kolbeninhalt, so fallt ein Kijrper in feinen, weiten Nadeln aus. Das Reduktions- produkt kristallisiert in weiBen Nadeln vom Schmelzp. 1 YS O. Dieselben sind leicht loslich in Benzol und Chloroform, etwas schwerer in Alkohol und in Ather, unloslich in Wasser. Der ProzeB verYauft beinahe quantitativ. I. 0,1510 g Substanz ergaben 0,4464 g CO, und 0,1090 g H,O. 11. 0,1544 g Substanz gaben bei 16O und 748 mm Druck 15,O ccm N. I. 0,1510 g Substanz ergaben 0,4464 g CO, und 0,1090 g H,O. 11. 0,1544 g Substanz gaben bei 16O und 748 mm Druck 15,O ccm N. Berechnet auf Gefunden : C,,H,,N, = 238: I. 11. C 80,67 80,62 - o/o N 11,76 - 11,14 ,,. €I 7,56 8,09 - ,, Dsls s alzs aur e Salx kristallisiert in weiBen Wiirfeln, das salpetersaure in gelblicher; 1angenNadeln und das schwefel- saure in kleinen weiBen Nadeln. Das Platinchloriddoppel- salz kristallisiert aus Eisessig in derben, gelben Nadeln, aus Alkohol in mikroskopisch kleinen, braunen Kristallen. Schmelzp. 203 O-205'. 0,2083 g Substanz ergaben 0,0468 g Pt. Berechnet auf Gefunden: (Cl,HI,N,H),PtCI, = 648,5: Pt 22,Ol 22,46 o/G. Gefunden: Zur Darstellung des pikrinsauren Salzes wurde die Tetrahydrobase in der Warme in Eisessig gelost und mit einer konzentrierten Losung von Pikrinsiiure in Eisessig ver- setzt, worauf nach dem Erkalten das Pikrat ausfiel und aus Eisessig umkristallisiert wurde. Orangegelbe Kristallnadeln, die beim Liegen an der Luft sich rotlich farben. Schme'iz- punkt 200 O. 0,0792 g Substanz gaben bei 1 5 O und 752 rnm Druck 10,O ccm N. 0,0792 g Substanz gaben bei 1 5 O und 752 rnm Druck 10,O ccm N. Berechnet auf C,,H,lN,O, = 467: N 14,99 Gefunden : 14,64 Ole. Br o m i e r un g s v e r su ch d e s D i h y d r o c hin a z olins. Berechnet auf C,,H,lN,O, = 467: N 14,99 Gefunden : 14,64 Ole. 0,0792 g Substanz gaben bei 1 5 O und 752 rnm Druck 10,O ccm N. 0,0792 g Substanz gaben bei 1 5 O und 752 rnm Druck 10,O ccm N. Berechnet auf C,,H,lN,O, = 467: N 14,99 Gefunden : 14,64 Ole. Berechnet auf C,,H,lN,O, = 467: N 14,99 Gefunden : 14,64 Ole. 216 v. Walther u. Bamberg: 'ifber einige Chinazoline. 216 v. Walther u. Bamberg: 'ifber einige Chinazoline. 216 v. Walther u. Bamberg: 'ifber einige Chinazoline. 216 Bz- (6)Me thyl-( 1 n) Me t h yl- (3n)p-Tolyl-Tetra hy d r oc hin- azolin Bz- (6)Me thyl-( 1 n) Me t h yl- (3n)p-Tolyl-Tetra hy d r oc hin- azolin, N.CH, N.CH, /\CH, C6H3 IN. CeH4. cw, * /. \/ CH3 CII, IN 2 g Tetrahydrobase wurden mit 4 g Jodmethyl in methyl- alkoholivcher Losung irn Druckrohr 3 Stunden lang auf 100° erhitzt, nach dern Erkalten das iiberschiissige Jodmethyl und der Methylalkohol auf dem Wasserbade verjagt, der braune Iiiickstand mit heiBem Wasser aufgenommen und heil3 filtriert. Aus dem Filtrat schied sich schon beim Erkalten das jodwasser- stoffsseure 9alz der Base in Prismen aus. Dasselbe wurde in vie1 Wasser gelost und mit Atznatron die freie Base gefallt, die aus Ligroin und wenig Alkohol umkristallisiert, rhombische Tafelri vom Schmelep. 155O darstellt. 0,0977 g Substanz gaben bei 15O und 759 mm Druck 9 ccm N 0,0977 g Substanz gaben bei 15O und 759 mm Druck 9 ccm N. Berechnet auf Gefunden : C,,H,,N, = 252: N 11,ll io,m 01,. 0,0977 g Substanz gaben bei 15O und 759 mm Druck 9 ccm N. Berechnet auf Gefunden : C,,H,,N, = 252: N 11,ll io,m 01,. Gefunden : Berechnet auf io,m 01,. Das salzsaure Salz dieser n(1)-methylierten Tetrahydro- base kristallisiert in quadratischen Tafeln, das P1 a tind oppel- s :ilz in strahligen Kristallbiischeln. Die Acylierung der n(1)- Imidogruppe des T e t r a - hydro c hi n az o 1 in - D e riva t e s wurde erfolglos, und zwar sowohl mit Benzoedure- als auch niit Essigsaure-Anhydrid versucht. Es resultierten stets nur stark verschmierte Produkte, aus denen kein kristallinischer Iiorper erhalten werden konnte. Versuche zur Gewinnung von Homologen des Di- hydrochinazolins. Versuche zur Gewinnung von Homologen des Di- hydrochinazolins. Durch Eondensation des o-Amido-m-Xylyl-p-Toluidins mit Ameisensaure erhielt man das schon beschriebene Dihydrochin- azolin-Derivat. Es lag nun nahe, urn zu einem (2) methylierten Di- hydrochinazolin-Derivat zu gelangen, an Stelle von Ameisensaure Essigsaure zu verwenden. v. Walther u. Bamberg: Ober einige Chinazoline. 217 v. Walther u. Bamberg: Ober einige Chinazoline. Jedoch wurden bei der Einwikung von Essigsaure auf das 0- Amido-m-Xylyl-p-Toluidin nur ganz geringe Mengen eines fagbaren Produktes isoliert, das sich mit dem gleich zu be- schreibenden, aus Essigsaure-Anhydrid dargestellten Konden- sationsprodukt als identisch erwies. p Dabei erhielt man zwar nuch nicht, wie erwrtrtet, unter RingschluB das (2) - methylierte Dihydrochinazolin - Derivat, sondern es bildet sich, wie die Analyse ergab, unter Austritt von 1 Molekul Wasser ein Diacetyl-Derivrtt des o-Amido-m- Xylyl-p-Toluidins. o- Acetylamido-m-Xylyl-p-acettoluidid, NH.CO.CH, CII.N.C,H,.CH, H, C. C, H,( I CO.CH, NH.CO.CH, CII.N.C,H,.CH, H, C. C, H,( I CO.CH, 10 g o- Amido-m-Xylyl-p-Toluidin wurden mit 10 g Essig- saureanhydrid kurze Zeit lang auf dem Wasserbad erwarmt. Beim Erkalten schieden sich Kristalle aus, die, aus verdiinntem Alkohol umkristallisiert, sechsseitige Blattchen vom Schmelzp. 135O darstellen; die Ausbeute an Rohprodukt betragt 10,4 g oder ?Sol0 der Theorie. I. 0,1464 g Substauz ergaben 0,3924 g CO, und 0,0973 g H,O. 11. 0,1202 g Substanz gaben bei 21* und 755 rnm Druck 10,1 ern N. Berechuet auf Gefuuden : C,BH,,N,O, = 310: I. I I. C i3,55 73,10 - O/il H 7,09 i,45 - 9949 1, * N 9)03 - Auch bei Anwendung von Chlorzink als Kondensations- mittel erzielt man keinen RingschuB , sondern ebenfalls obiges Diacetyl-Derivat. Ebenso erfolglos war es, das o-Amido-m-Xylyl-p-Toluidin mit Eisessig im Druckrohr auf 180° zu erhitzen. g Bei einem weiteren Versuche wurden 0,5 g o-Amido- m-Xylyl-p-Toluidin mit 4 ccm Essigester 5 Stunden lang im Druckrohr auf 180° erhitzt. Die entstandenen Eristalle wurden durch Aufpressen auf einen Tonteller vom gr6Bten Teil der Schmieren befreit und dann mit Ligro’in gedeckt. v. Walther u. Bamberg: cber einige Chinazoline. 218 v. Walther u. Bamberg: cber einige Chinazoline. v. Walther u. Bamberg: cber einige Chinazoline. Nach dem Um kristallisieren aus LigroIn erhielt man flache Prismen von Schmelzpunkt 86 O, also das Ausgangsmaterial wieder. Nach dem Um kristallisieren aus LigroIn erhielt man flache Prismen von Schmelzpunkt 86 O, also das Ausgangsmaterial wieder. Ve r h a 1 t e n d e s o -A c e t y 1 a mid o - m -X y 1 y 1 - p -A c e t t o 1 ui d i d s gegenuber verdiinnten SBuren in der Warme. Bei diesen Versuchen sollten die verdunnten Sauren durch partielle Verseifung eine Acetylgruppe als Essigsaure abspalten und das so entstandene Produkt unter Wasseraustritt Ring- schlu6 bilden. v. Walther u. Bamberg: Ober einige Chinwzoline Der Versuch zeigt also, daB zwar die beiden Acetyl- gruppen sich sehr leicht durch Kochen mit verdunnten Sauren abspalten lassen, ein partielles Verseifungsprodukt aber nicht zu isolieren ist. v. Walther u. Bamberg: Ober einige Chinazoline. 0,5 g des aus Essigsaureanhydrid nach obiger Beschreibung gewonnenenDiacety1-Derivates wurden ungefahr 1 Stunde lang am Steigrohr mit verdunnter Salzsaure gekocht, wobei es sich auf- loste. Bus dem Piltrat wurde dann die Base in Freiheit ge- setzt, die aber zu stark verschmiert war, um weiter verarbeitet werden zu konnen. Ebensowenig Erfolg hatte ein 5 stundiges Erhitzen des Diacetyl -Derivates mit verdunnter Salzsaure im Druckrohr auf 160O. DaB bei diesem Versuch tatsachlich Essigsaure abgespalten war, bewies der nach Zusatz voh Alkohol stark auftretende Geruch nach Essigester. Allzu reichliche Schmieren machten auch hier ein weitei es Arheiten unmoglich. Anwendung verdunnter Schwefelsaure, statt Salzsaure, anderte das ungunstige Resultat nicht. Kocht man 3 g des Diacetyl- o-Amido-m-Xylyl-p-Toluidin langere Zeit mit verdunnter Schwefelsaure am RuckfluBkiihler, so scheidet sich beim Erkalten ein Korper in weiBen Flocken aus, der von der Losung abfiltriert wurde. Derselbe loste sich in vie1 Wasser; diese Losung schied auf Zusatz von Alkali einen Korper aus, der sich nach dem Umkristallisieren und Reinigen nach Kristallform (prismntische Blattchen) und Schmelzp. 87 O als das o-Amido-m-Xylyl-p-Toluidin erwies. Im Filtrat der Losung war noch das schwefelsaure Salz der Base, und konnte daraus ebenfalls die Base mit Atznatron in Freiheit gesetzt und mit Ather ausgeschuttelt werden. Nach dem Verdunsten des Auszuges hinterblieb ebenfalls ein Korper vom Schmelzp. 87 0, also das o- Amido-m-Xylyl-p-Toluidin. 219 v. Walther u. Bamberg: Ober einige Chinwzoline. o - Amid o - m - X yl y 1 - p - To 1 ui d i n u n d Ace t amid. Versuche, durch Kondensation des o-Amido-m-Xyly1-p- Toluidins mit Acetamid unter Austritt von Ammoniak und Wasser zu dem erwunschten (2)-methylierten Dihydrochinazolin- Derivat zu gelangen, waren ebensowenig erfolgreich. 0,5 g o- Amido-m-Xylyl-p-Toluidin wurden mit 4 g Acetamid 5 Stunden lang auf 160° im Druckrohr erhitzt. Nach dem Erkalten hatten sich starke Kristallmassen ausgeschieden, welche sich unter Auftreten von Ammoniakgeruch in Wasser losten. Nach Auslthern der Losung und Verdunsten des Athers hinterblieben jedoch ebenfalls nur dicke Schmieren, die keine Neigung zur Kristallisation zeigten. B z - (6) 1cI e t h y 1- (2) M e t h y 1 - (3) - p -To 1 y1(3,4) - Di h y d r o chi n - az o lin, CH, -C.CH, / C,", , ) N . C,H,.CH, * CHa CH, B z - (6) 1cI e t h y 1- (2) M e t h y 1 - (3) - p -To 1 y1(3,4) - Di h y d r o chi n - az o lin, CH, -C.CH, / C,", , ) N . C,H,.CH, * CHa CH, B z - (6) 1cI e t h y 1- (2) M e t h y 1 - (3) - p -To 1 y1(3,4) - Di h y d r o chi n - az o lin, CH, -C.CH, / C,", , ) N . C,H,.CH, * CHa CH, Aus dem Acetylderivat war die Abspaltung von Wasser unter ChinazolinringschluB nicht zu erzielen. Von dem Gedanken geleitet, daB die Chinazoline intra- molekulare Amidinbindungen aufweisen , mrde versucht , eine derjenigen Methoden zu benutzen, die sonst fur die Darstellung von Amidinen von gutem Erfolge sind. Betrachtet man die Moglichkeiten, um aus Aminbasen zu Amidinen zu gelangen, so liegen deren zwei nahe. Erstens die Bernthsensche au8 salzsaurem Amin und Nitrilen: CH,.CN f RH, .R = CHa-C(NH)(NH.R) CH,.CN f RH, .R = CHa-C(NH)(NH.R) und zweitens aus Aminbasen und Imidoathern. Beide Methoden wurden angewandt. Die Kondensation wiirde dann nach fol- gender Gleichung verlaufen: und zweitens aus Aminbasen und Imidoathern. Beide Methoden wurden angewandt. Die Kondensation wiirde dann nach fol- gender Gleichung verlaufen: 220 v. Walther u. Bamberg: Ober einige Chinazoline. 220 v. Walther u. Bamberg: Ober einige Chinazoline. + CH,.CN = 2 g salzsaures o-Amido-m-Xylyl-p-Toluidin wurden mit 4 g Acetonitril 5 Stunden lang im Druckrohr auf 180° er- hitzt. Nach dem Erkalten fand sich in der Rohre eine reich- liche Menge von Chlorammonium ausgeschieden, welches von der braunen Fliissigkeit abfiltriert wurde. Die braune Losung, mit verdiinnter Schwefelsaure unter Zusatz von etwas Tierkohle gekocht und he% filtriert, schied beim Zusatz von Atznatron die Base aus, die beim Schiitteln mit Ather in diesen uberging. Nach dem Verdunsten desselben erhielt man jedoch wiedernm nur Schmieren. J edenfalls Yatlt aber das Auftreten des Chlorammoniums bei diesem Versuch auf einen teilweisen Verlauf des Prozesses in der erwarteteu Richtung schlietlen. g Erfolgreich waren schlieBlich die Kondensationsversuche des o- Amido-m-Xylyl-p-Toluidins mit salzsaurem Acetimidoather. 5 g 0- Amido-m-Xylyl-p-Toluidin wurden mit 4 g salz- saurem Acetimidoather in einer wasserfreien Benzollosung kurze Zeit erwarmt. Es schied sich alsbald aus der klaren Losung eine weiBe Kristallmasse ab, die neben Chlorammonium, welches nachgewiesen werden konnte, noch das salzsaure Salz der er- warteten Base enthielt. Diese Kristallmasse wurde in Wasser gelijst und die filtrierte Losung mit Atznatron versetzt, wodurch die Base in Ereiheit gesetzt und ausgeathert werden konnte. Der Ather-Ruckstand erwies Rich zuerst als harzartiges, durchsichtiges 01, das keine Neigung zur Kristallisation zeigte. Durch wiederholtes Reiben mit einem Glasstabe erstarrte es aber ganz allmahlich zu einer gelbweiBen Kristallmasse, die in der Warme in Ligro'in gelost wurde. v. Walther u. Barnberg: Dber einige Chinazoline. 221 Nach dem Erkalten schied sich aus der Losung langsam die freie Base in groBen, derben Kristalldrusen aus. Sie ist leicht lijslich in Ather, Benzol, Alkohol und Chloroform, schwerer in Ligro'in und unloslich in Wasser. Schmelzp. 89O bis 93O. I. 0,0976 g Substanz ergaben 0,2933 g CO, und 0,0674 g H,O. g g g 11. 0,1546 g Substanz gaben bei 11 O und 745 mm Druck 15,2 ccm N Berechnet fur Gefunden : C,,H,,N, = 250: I. 11. C S1,60 S1,95 - H 7,20 1,74 - N 11,20 - 11,48 ,,. Wie die Analyse bestatigt, liegt also hier das (2)-methylierte Dihydrochinttzolin-Derivat vor. Das bei der Reaktion intermediar auftretende Amidin konnte nicht gefaBt werden. D as salzsaure Salz I) Ber. 25, 2851. salzsaure Salz kristallisiert aus der Losung der Base in konzentrierter Salz- saure in quadratischen, durchsichtigen Blattchen vom Schmelz- punkt 26 1 O. Das Platindoppelsalz erhalt man durch Zusatz von Platinchlorid zur waljrigen Losung des salzsauren Salzes. Es kristallisiert in schonen, orangegelben Nadelbiischeln, die sich spater in Rhomben verwandeln. Schmelzp. 235 9 Bei nochmaliger Darstellung des Platindoppelsalzes ist noch eine zweite Kristallform beobachtet worden ; es sind quadratische Blattchen, die bei 207 O unter Zersetzung schmelzen. I. Platinsalz (Schmelzp. 235 O). I. Platinsalz (Schmelzp. 235 O). Ee ergaben 0,1178 g Substanz 0,0248 g Pt. Ee ergaben 0,1178 g Substanz 0,0248 g Pt. 11. Platinsalz (Schmelzp. 207 "). 11. Platinsalz (Schmelzp. 207 ). Es ergaben 0,1023 g Substaua 0,0224 g Pt. Es ergaben 0,1023 g Substaua 0,0224 g Pt. Berechriet auf (C,,H,,N,),H,PtCI, = 909: Pt = 21,42 Gefunden : I. 11. 21,05 'Ll,rj9 Oi0. Gefunden : I. 21,05 v. Walther u. Bamberg: aber einige Chinazoline. 2 v. Walther u. Bamberg: aber einige Chinazoline. 222 B a - (6) M e thy 1 - (2) K e t o - (3 n) - p -.T o 1 y 1 - T e t r ah y d r o chin - azolin. NH NH I n einem Reagenzrohr wurden 8 g o-Amido-m-Xyly1-p- Toluidin mit 2 g Harnstoff (berechnete Menge) zusammenge- schmolzen und auf ca. 1500 erhitzt, wobei eine starke Ammoniak- Entwicklung auftrat. Die braune, dickfiiissige Masse erstarrte beim Erhitzen allmahlich zu einem festen Kijrper, der nach dem Erkalten aus Eisessig und etwas Wasser umkristallisert wurde ; nach nochmaligem Umkristallisieren war derselbe rein. Die Auvbeute betrug 5 g oder 62O/, der Theorie. Uer Vorgang verlauft nach folgender Gleichung: Der Kijrper, nach den Andysenergebnissen das erwartete Ketochinazolin- Derivat, kristallisierte in weiI3en Nadeln vom Schmelzp. 238 O-240 O. Dicselben sind leicht loslich in heiBem Alkohol, Essigester und Benzol, schwerer in Ather und Ligro'in und unloslich in Wasser. 0,0992 g Substanz gaben bei 1 5 0 und 746 mm Druck I0 ccrn N. 0,0992 g Substanz gaben bei 1 5 0 und 746 mm Druck I0 ccrn N. Berechnet auf CleH,eN,O = 252: N 11J1 Gefunden : 11,58 Gefunden : Berechnet auf CleH,eN,O = 252: N 11J1 11,58 Bereits Paal und Bu%chl) haben Studien iiber analoge Umsetzungen des o-Amidobenzamids mit Harnstoff und dessen Abkijmmlingen unternommen. Beinahe obenso glatt wie mit Rarnstoff vedauft die 223 v. Walther u. Baraberg: Uber einige Chinazoline. Kondensation des o-Amido-m-Xylyl-p-Toluidins mit Chlor- ko hlenoxy d. salzsaure Salz Kondensation des o-Amido-m-Xylyl-p-Toluidins mit Chlor- ko hlenoxy d. Kondensation des o-Amido-m-Xylyl-p-Toluidins mit Chlor- ko hlenoxy d. Das 0-Amido-m-Xylyl-p-Toluidin wurde in Benzol gelost und bis zur Sattigung Chlorkohlenoxyd eingeleitet. Nach kurzer Zeit fie1 ein Korper in gelben Plocken aus, die sich bald zu dicken Schmieren vereinigten, bei weiterem Einleiten von Chlor- kolenoxyd aber wieder auflosten. Nach dem Verdunsten der filtrierten Losung hinterblieb ein Riickstand, der aus Eisessig unter Zusatz von etwas Wasser umkristallisiert wurde und sich nach Kristallform und Rchmelzpunkt als das erwertete Keto- tetrahydrochinazolin-Derivat erwies. Er kristallisierte in weiBen Nadeln vom Schmelzp. 240'. 0,1257 g Substanz ergaben 0,3503 g CO, und 0,0749 g H,O. Berechnet auf CI,H,,N,O = 252: C 76,19 H 6,35 Gefunden: o-Amido-m-Xylyl-p-Toluidin und Cyansaure. Der basische Charakter des o-Amido-m-Xylyl-p-Toluidins lie8 es erwarten, da8 Cyansaure glatt reagieren, dann sekundar womijglich RingschluB unter Austritt von Ammoniak erfolgen wiirde. Es ergab sich aber im Verlauf des Versuches, da8 sich hierbei nicht sofort das erwartete Ketotetrahydrochinazolin- Derivat bildet, sondern intermedik ein Doppelharnstoffderivat, das dann unter Austritt von 1 Molekiil Barnstoff in das er- wartete (2)-Ketotetrahydrochinazolin-Derivat ubergefuhrt werden kann: H,C. C,H,<NHz + SHOCN = CH, . NH . C,H, . CH, - NH. CO .NH, H,C. c,H,/ - \cH,.N. C~H,.CH, I I CO . NH, NH H,C. C,H,<NHz + SHOCN = CH, . NH . C,H, . CH, - NH. CO .NH, H,C. c,H,/ - \cH,.N. C~H,.CH, I I CO . NH, NH 224 v. Walther u. Bamberg: Ober einige Chinnzoline. v. Walther u. Bamberg: Ober einige Chinnzoline. Das o-Amido-m-Xylyl-p-Toluidin wurde in verdiinnter Salzsaure gelost und mit der berechneten Uenge cyansaurem Kali versetzt. Es fie1 ein braunweikier Korper aus, der sich in allen angewandten Losungsmitteln als unloslich erwies. Er wurde deshalb durch Auskochen mit Benzol, Alkohol und Ather von den Verunreinigungen bsfreit und stellt dann ein weikies Pulver dar, das bei 219O unter Zersetzung schmilzt. Durch die Stickstoff-Bestimmung wurde die Bildung des Doppelharnstoff-Derivates erwiesen. 0,1117 g Substanz gaben bei 14O und 743 mm Druck 17,8 ccm N. Berectinet auf C,,E-I,"N,O, = 312: N l7,95 Gcfunden : 18,31 %. Berectinet auf C,,E-I,"N,O, = 312: N l7,95 18,31 %. Beim Erhitzen des Doppelharnstoff-Derivates geht dasselbe, wie oben bemerkt, unter Harnstoff- Abspaltung bezw. dessen Zersetzungsprodukten in das Ketochinazolin uber. B z - (6)M e t h y 1 - (2) T h i o - (3n) p -!I! o 1 y 1 t e t r a h y d r o c h in az o 1 in a u s o - Amido-m-Xylyl-p-Toluidin und Schwefel- kohlenstoff, NH B z - (6)M e t h y 1 - (2) T h i o - (3n) p -!I! o 1 y 1 t e t r a h y d r o c h in az o 1 in a u s o - Amido-m-Xylyl-p-Toluidin und Schwefel- kohlenstoff, kohlenstoff, NH Eine Lijsung von 1 g o-Amido-m-Xylyl-p-Toluidin in 3 g absolutem Alkohol wurde mit 3 g Schwefelkohlenstoff und etwas Schwefelblume im Druckrohr 4 Stunden lang auf 130O bis 140O erwarmt. Bald nach dem Zuschmelzen des Rohres schieden sich aus der vollkommen klaren Fliissigkeit allmahlich derbe Kristallmassen ab , wahrscheinlich ein dithiocarbamin- saures Salz. Nach beendetem Erhitzen war im Rohr starker Geruch nach Schwefelwasserstoff wahrzunehnien und es hatten sich schijn ausgebildete, silberweikie Blattchen ausgeschieden, die rnit Schwefelkohlenstoff gewaschen wurden. Die Ausbeute betrug 1 g oder 83O/,, der Theorie. Der Korper, der sofort analysenrein ist, wird bei 2000 gelb und schmilzt zwischen 258O und 260°. Er kristallisert v. Walther u. Bamberg: Ober einige Chinazoline. 225 in sechseckigen, durchsichtigen Blattchen und ist unl6slich in Natronlauge; er bildet ziemlich schwer losliche Salze, von denen einige erwahnt sein mogen: in sechseckigen, durchsichtigen Blattchen und ist unl6slich in Natronlauge; er bildet ziemlich schwer losliche Salze, von denen einige erwahnt sein mogen: Das schwefelsaure Salz, kleine verzweigte Nadeln, die bei 275O noch nicht geschmolzen sind; das salzsaure Salz, strahlige Kristallblattchen, Schmelzp. Journal f. prakt. Chemie [2] Bd. 73. v. Walther u. Bamberg: Ober einige Chinnzoline. B L - (6) M e t h y 1 - (2) J o d m e t h y 1 - T h io - (3 TI) p - T 01 y 1 t e t r a - ahy d r oc hin az olin, B L - (6) M e t h y 1 - (2) J o d m e t h y 1 - T h io - (3 TI) p - T 01 y 1 t e t r a - ahy d r oc hin az olin, NH NH 1 g p-Tolylthiotetrahydrochinazolin-Derivat wurde mit 2 g Jodrnethyl und 30 ccm Methylalkohol im Druckrohr 4 Stunden lang auf 130° erhitzt. Nach dem Erkalten hatten sich im Rohr prachtige, gelbe Kristalle ausgeschieden, die sich aus 70 prozent. Alkohol umkristallisieren liel3en. Man erhielt auf diese Weise einen Korper in Form gelber Stabchen, die bei 255O anfangen sich zu braunen und bei 260" unter Zersetzung schmelzen 5 g p-Tolylthiotetrahydrochinazolia-Derivat ergaben 6 g des neuen Korpers, was einer Ausbeute von 77O/, der Theorie entspricht. I. 0,1090 g Substanz gaben bei 1 7 " urid 748 mm Druclr 6,4 ccm N. 11. 0,2001 g Substane gabeii 0,115'2 g AgJ. I. 0,1090 g Substanz gaben bei 1 7 " urid 748 mm Druclr 6,4 ccm N. I. 0,1090 g Substanz gaben bei 1 7 " urid 748 mm Druclr 6,4 ccm N. 11. 0,2001 g Substane gabeii 0,115'2 g AgJ. . 0, 090g Substa gabe be u d 7 8 uc 6, cc N. 11. 0,2001 g Substane gabeii 0,115'2 g AgJ. 11. 0,2001 g Substane gabeii 0,115'2 g AgJ. 11. 0,2001 g Substane gabeii 0,115'2 g AgJ. N J Berechnet auf Befunden: C,,H,,N,S. J = 410: I. 11. v. Walther u. Bamberg: Ober einige Chinnzoline. 220 O-225 0; das Platindoppelsalz, niikroskopisch kleine, orangegelbe Kristalle, die bei 250 O unter Zersetzung schmelzen; das Pikrat, sechseckige, durchsichtige gelbe Tafeln, Schmelzp. 240O; das Acetat, welches in sechs- eckigen Tafeln vom Schmelzp. 257O und das Oxalat, das in langen, weiBen Nadeln vom Schmelzpunkt 247O- 2520 kri- stallisiert. Die Thio -Verbindung selbst ist schwer loslich in Ather, Alkohol und Schwefelkohlensoff , leichter in Benzol, Chloroform und heiBem Eisessig, am besten aus Alkohol um- kristallisierbar. Es ergaben 0,1252 g Substanz 0,3305 g CO, und 0,0709 g H,O. Berechnet suf C,,H,,N,S = 268: C 71,64 H 5,97 Oefunden : Wie weitere Versuche ergeben haben, ist ein Erhitzen im Druckrohr nicht unbedingt erforderlich. Die in absolutem Alkohol geliiste Base geht schon beim einstundigen Kochen mit Schwefelkohlenstoff und etwas Schwefelblume in das p-Tolylthiotetrahydrochinazolin-Derivat iiber. Bei diesem letzten Versuch wurden 10 g o- Amido-m-Xylyl-p-Toluidin verwandt uud 9,6 g Thio-Verbindung erhalten, was einer Ausbeute von P t i 0 l O der Theorie entspricht. Die Entschweflung der Thioverbindung erfolgt nur bei hoherer Temperatur im Druckrohr gut, sie ergibt dann aller- dings ziemlich glatt das Ketotetrahydrochinazolin vom Schmelzp. 238O in Form weiBer Nadeln. Gut verlauft auch die Reduktion des Thiochinazolins mit Natrium in alkoholischer LGsung zur Tetrahydrobase vom Schmelzp. 138O, die sich beim Erkalten des Ansatzes direkt als Kristallbrei absetzt. 15 Journal f. prakt. Chemie [2] Bd. 73. 26 v. Walther ti. Baniberg: Cber einip Chinazoline. 226 E in w i r k un g v o 11 J o d m e t h y 1 a u f d as p - To 1 y I t hi o t e t r a - h y d r o hinaz o 1 in- D e r i v s t. E in w i r k un g v o 11 J o d m e t h y 1 a u f d as p - To 1 y I t hi o t e t r a - h y d r o hinaz o 1 in- D e r i v s t. v. Walther u. Barnberg: Uber einige Chiunzoline. v. Walther u. Barnberg: Uber einige Chiunzoline. kalten fie1 ein Korper in feinen weiBen Blattchen aus, der aus verdunntem Alkohol in kleinen durchsicbtigen Prismen vom Schrnelzp, 87 kristallisiert. I. 0,1055 g Substanz ergaben 0,2810 g CO, und 0,0666 g H,O. 11. 0,0965 g Substanz gaben bei 18O und 768 mm Druck 8 ccm N. Berechnet suf Gefunden: C,,TI,,~,S = 282: I. 11. c 7434 72,64 - ' I0 9,68 1) ' N 9,93 - Ii 6,33 7,07 - ,, Diese freie Base ist leicht loslich in Ather, Benzol, Essig- athcr, weniger leicht in verdiinntem Alkohol. Von den Salzen dieser Methylthio-Base seien erwahnt: das schwefelsaure, lange, weikie feine Nadeln vom Schmelzp. 208"; das salzsaure, derbe, lanzettfijrmige SCtbchen vom Schmelzp. 258"; das Patindoppelsalz, mikroskopisch kleine, derbe Kristalle vom Schmelzp. 222" und das Pikrat, feine, diinne, gelbc Nadeln vom Schmelzp. 168O. Durch Anlagerung der Methyl- an die CS-Gruppe ist der Charakter der Verbindung ganz verandert worden. Wahrend das p-Tolylthiotetrahydrochinazolin-Derivat sich a19 vollstandig neutral erwies, ist die neue Verbindung stark basisch geworden, sie gibt init Mineralsauren vollkornmen bestandige, gut kristalli- sierende Salze und der Schmelzpunkt ist urn ca. 170° herunter- gedriickt und wahrend das CS-Derivat sich in den meisten Losungamitteln als urildslich erwies , ist die neue Methylthio- Base in den meisten Lijsurigsmitteln BuBerst leicht liislich. v. Walther u. Bamberg: Ober einige Chinnzoline. 6,83 6,70 - '/,, 50,9$ - 31,10 ,, B z - ( ~ ) e t h yl- ( 2 ) M e t h y 1 t h i o - (3 n) p -To 1 y 1 - (3,4) ) i h y d r o - chinazolin, B z - ( ~ ) e t h yl- ( 2 ) M e t h y 1 t h i o - (3 n) p -To 1 y 1 - (3,4) ) i h y d r o - chinazolin, N B z - ( ~ ) e t h yl- ( 2 ) M e t h y 1 t h i o - (3 n) p -To 1 y 1 - (3,4) ) i h y d r o - chinazolin, Urn zunachst aus der Thionium-Verbindung die freie Base darzustellen, wurden 6 g der Verbindung in 7Oprozent. Alkohol in der Wiirnie geltist, und die heiB filtrierte Losung mit Atz- natron bis zur alkalischen Reaktion versetzt. Nach dem Er- 227 v. Walther u. Barnberg: Uber einige Chiunzoline. Mitteilungen am dem chemischen Universitat Heidel berg. Institnt der Mitteilungen am dem chemischen Universitat Heidel berg. Institnt der Beduktion der Alethylthio-Base mit Zirikstaub und v e r d ii 11 n t e r S c h we f e 1s a u r e. Eine kleine Mcnge dieser Methylthio-Base wurde in ver- dunnter Schwefelsaure in der Warme gelost und etwas Zink- staub zugegeben. Sofort trat starker Qeruch cach Mercaptan auf. Es wurde nun so lange auf dem Wasserbade erwarmt, bis der Geruch nach Mercaptan vollstandig verschwunden war, voin iiberschiissigen Zinkrtaub abfiltriert, das Filtrat mit Atz- natron und Ammoniak bis zur alkalischen Reaktion versetzt, 15* 228 Mohr: 1II.Mitteilung ub.dieHofmannscheReaktion. 8 Mohr: 1II.Mitteilung ub.dieHofmannscheReaktion. und die ausgeschiedene Base ausgeathert. Das erhaltene Produkt, aus Alkohol umkristallisiert, stellte das schon wieder- bolt erwahnte p-Tolyltetrahydrochinazolinderivat vom Schmelzp. 138O vor. Durch entsprechende Modifizierung lBBt sich eine dem Methylderivat vollig analoge Athylthioverbindung gewinnen. Dresden, im Januar 1906. Dresden, im Januar 1906. g g g a) Chemiker-Zeitung 1903, 1009 11. 1108; Zeitsctir. f. angew. Chem. 1003, 1061; Chem. Centralbl. 19OG, I, 1227. l) Bei der Redaktion eingegangen am 8. Januar 1906. E. Mohr. von E. Mohr. von E. Mohr. von Nachweis der Carbarninate als Zwischenprodukte hei der Hofmannschen Reaktion. Die alteste Angabe uber diesen Nachweis enthalt das deutsche Reichspatent No. 127 138 (Hiichster B'arbwerke, 1901). Nach den Angaben dieses Patents kann man die Einwirkung von Natriumhypochlorit suf Phtalimid so leiten, da8 saures isatosaures Natrium Die alteste Angabe uber diesen Nachweis enthalt das deutsche Reichspatent No. 127 138 (Hiichster B'arbwerke, 1901). Nach den Angaben dieses Patents kann man die Einwirkung von Natriumhypochlorit suf Phtalimid so leiten, da8 saures isatosaures Natrium CO, H C&/ \NH.CO,N~ auskristallisiert; in der Patentschrift wird hinzugefiigt: ,,Dieses Resultat ist insofern uberraschend, als die Kofmannsche Reaktion sonst immer unter gleicbzeitiger Abspaltung von Kohlensaure verlauft." Auch S c h es t a k o w s benzalbydrazin- carbonsaures Natrium z, ist hier anzufuhren, wenn auch vielleicht noch Zweifel dariiber mijglich sind, ob die 'Jberfiihrung von Harnstoff in Hydrazin eiii wahres Analogon der €I ofmannschen
https://openalex.org/W4382467095	https://link.springer.com/content/pdf/10.1007/978-3-031-31678-4_21.pdf	English	null	Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know and How to Move Forward?	Springer eBooks	2,023	cc-by	8,002	A. Bautista (*) · J. Yu · K. Lee · J. Sun Department of Early Childhood Education, Centre for Educational and Developmental Sciences, The Education University of Hong Kong, Hong Kong, China e-mail: abautista@eduhk.hk Keywords Early childhood education · Play · Policy and practice · Child outcomes · Asia © The Author(s) 2023 R. Maulana et al. (eds.), Effective Teaching Around the World, https://doi.org/10.1007/978-3-031-31678-4_21 1 Introduction As a result of glocalization, Early Childhood Education (ECE) policies in Asia might have been influenced by Western theories and pedagogies (Gupta, 2018; Yang & Li, 2019). One clear instance is the widespread presence of the notion of play in official curriculum guidelines, licensing and accreditation frameworks, and other high stakes policies, where play is commonly regarded as an essential strategy for child development and learning (Bautista, Yu, et al., 2021b; Grieshaber, 2016; Gupta, 2014). Following international trends (Organisation for Economic Co-operation and Development [OECD], 2004; The LEGO Foundation & UNICEF, 2018), Asian ECE teachers are encouraged to implement play-based pedagogies that are child-centered and process-oriented, providing children with actively engaging, meaningful, socially interactive, and joyful learning opportunities. Environments that promote play, exploration, and hands-on experiences are under stood to be the core of effective ECE programs across Asia (Bautista et al., 2019; Cheung et al., 2015; Fujisawa et al., 2008; Gupta, 2014). g j p ) However, research in support of the inclusion of play in ECE settings has been primarily conducted in Western societies (Lai et al., 2018). Given what we know about significant differences in many aspects of human psychology across cultures, and particularly about the unique characteristics of the Asian learner (King & Bernardo, 2016; Li, 2010), it is imperative to examine the state of the art on play research conducted with Asian children. In view of the lack of systematic reviews in Asia, this chapter brings together the available literature on the impact of play-based pedagogies across four specific Asian contexts: Mainland China, Hong Kong, Singapore, and Japan. The chapter aims to describe the types of play impact studies conducted in these jurisdictions and their overall findings, as well as suggest future research agendas for play researchers within the Asian continent. g p y The chapter is structured into five sections. First, we provide a brief overview of Western research on play within ECE settings and its impact on children’s develop ment and learning. The second section describes socio-cultural beliefs about ECE in the four Asian jurisdictions considered, as well as the visions of play articulated in their official curriculum policy frameworks. In the third section, we provide an overview of the research conducted in these regions to analyze the impact of play- based pedagogies on children’s developmental and/or learning outcomes, distin guishing between naturalistic and intervention studies. 1 Introduction The fourth section critically analyzes the existing literature and identifies research gaps. Finally, the fifth section outlines future research agendas and discusses practical implications. Chapter 21 Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know and How to Move Forward? Alfredo Bautista, Jimmy Yu, Kerry Lee, and Jin Sun Abstract In the Asian continent, many Early Childhood Education (ECE) policies have been influenced by Western theories and pedagogies. An example is the wide spread presence of the notion of play in curriculum policy frameworks, which in part responds to research findings originated in the West. However, given what we know about cross-cultural differences in child development and learning, it is imperative to examine the state of the art on play research conducted with Asian children. This chapter reviews the literature on the impact of play-based pedagogies in Mainland China, Hong Kong, Singapore, and Japan. We describe the types of studies conducted in these jurisdictions and their overall findings, with the aim of outlining future research agendas. We describe the socio-cultural beliefs about ECE in the selected contexts and the visions of play articulated in their official policies. Then, we provide an overview of the empirical studies available, distinguishing between naturalistic and intervention studies. Studies published in English aca demic journals have mainly analyzed the impact of structured and guided forms of play, focusing primarily on socio-emotional outcomes, with minimal research on domains such as scientific thinking, number sense, or creativity, and no research on other areas. We argue that the existing work reflects traditional Asian values and deep-rooted beliefs about ECE, where play is seen as a rather unimportant activity. We conclude that to better justify the inclusion of play in ECE policies across Asia, it would be vital to produce an extensive, rigorous, and locally situated corpus of play impact studies. Keywords Early childhood education · Play · Policy and practice · Child outcomes · Asia 473 474 A. Bautista et al. A. Bautista et al. 2 Western Research on Play and Its Impact on Children Nowadays, official curriculum frameworks around the world (including Asian countries) suggest ECE teachers to implement different types of play-based pedago gies, in a continuum that ranges from structured play (activities led by teachers with Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know… 475 21 educational purposes in mind) to free play (activities led by children and allowing them freedom, choice, and internal agency) (Bautista et al., 2019; Hassinger-Das et al., 2017). The emphasis on play reflects the theories and pedagogies developed by influential Western authors such as Jean Piaget, Lev Vygotsky, Jerome Bruner, Carl Jung, Friedrich Fröbel and Maria Montessori, who extensively wrote about the multiple manifestations of children’s play throughout the various developmental stages and/or educational levels. For example, Piaget (1962) argued that play reflects children’s stages of cognitive development, starting from functional play (allows children to master physical actions, with or without objects), constructive play (children use materials to make or build something), symbolic/fantasy play (chil dren invent pretend scenarios where objects or toys are used as symbols represent ing something else), and finally games (activities with pre-established rules, normally involving competition among players). Numerous taxonomies and classi fications of play types have been proposed by Western scholars (for reviews, see Burghardt, 2011; Johnson et al., 2005). Furthermore, the adoption of play-based pedagogies in Asian ECE settings might reflect the extensive body of Western research documenting the positive impacts of play on children’s developmental and learning. Play impact studies in the West have utilized a variety of research methodologies (quantitative, mix-methods, qualitative) and have adopted a wide range of research designs (e.g., experimental, correla tional, longitudinal, case studies). Moreover, Western scholars have documented the impact of play-based pedagogies on a variety of developmental and learning out comes, including physical, cognitive, academic, socio-emotional, as well as mental health outcomes. For example, Western research has found a direct correlation between playful learning environments and reduced levels of obesity, heart-related problems, and chronic stress (Burdette & Whitaker, 2005). In a recent meta-analysis of 25 studies conducted in schools across Europe, Australia, United States of America (USA), Bedard et al. (2019) found that play-based and physically active classrooms may improve academic achievement and enjoyment outcomes, as com pared to traditional teacher-directed schools. 3 Societal Beliefs About ECE and Curriculum Policy Visions on Play in Selected Asian Contexts 3 Societal Beliefs About ECE and Curriculum Policy Visions on Play in Selected Asian Contexts While Asia is often seen by Western scholars as a homogenous whole, the various Asian countries have specific traditions and socio-cultural characteristics (e.g., val ues, norms, priorities, beliefs), varied conceptions about early childhood and child development, as well as different official discourses on the role of play in ECE (Bautista, Yu, et al., 2021b; Grieshaber, 2016; Gupta, 2014). Given the lack of review studies on play conducted in Asia, this chapter focuses on four specific con texts: Mainland China, Hong Kong, Singapore, and Japan. These jurisdictions were selected due to the availability of (a) ECE policy frameworks written in English or Chinese and (b) published journal articles focusing on the impact of play-based pedagogies in young children. Mainland China has a strong cultural tradition of placing emphasis on academic learning and achievement (Gopinathan & Lee, 2018; Li, 2010). Although this tradi tion is rooted in Confucianism, academic achievement in modern Chinese society is still regarded as a vehicle for social mobility. In his review on Chinese perceptions of early childhood, Luo et al. (2013) argued that this emphasis on the Confucian principle of knowledge (Zhi) has steered Chinese parents beliefs on learning away from avenues that entail high degrees of playfulness and enjoyment. The authors argued that other aspects of Confucian culture, in particular, the notion of Guan (i.e., Chinese term that means training children in the appropriate or expected behaviors) renders much of learning top-down and directed by adults. In this light, the ECE curriculum framework in Mainland China can be seen as somewhat revo lutionary in its emphasis on the role of play in learning (Ministry of Education of the People’s Republic of China [MOE-PRC], 2012). Indeed, the curriculum in China states that children’s learning should be derived from their “play and daily life” and that “we need to treasure the unique value of play” (p.2). This strong stance on the importance of play, in its own right, is tempered in other parts of the framework where play is referred to as a tool for academic learning. For instance, the curriculum suggests that teachers let children construct their play with materials in different shapes to learn shapes and play games like drawing circles to build a foundation for writing. 2 Western Research on Play and Its Impact on Children In a quasi-experimental study con ducted in Norway with children aged 5–7, Fjortoft (2004) found that playing in a natural environment enhanced children’s physical fitness, coordination, balance and agility, as children were able to play and move in landscapes that offered challenge and unpredictability. Another large body of Western literature shows that socio-emotional competen cies are best nurtured through socio-dramatic and pretend play with peers and car ing adults, and other social interactions in small group settings (Yogman et al., 2018). In the USA, Pellegrini et al. (2002) conducted a longitudinal study of chil dren’s playground games, with emphasis on how play affected children’s social competence and adjustment to school. It was found that facility with games pre dicted boys’ social competence, and that play enhanced both boys’ and girls’ adjust ment to the first year of Primary school. Finally, a large body of Western research has documented that cognitive and linguistic development are also optimized through active and exploratory forms of play (Fox et al., 2010; Liu et al., 2017a). Play enhances brain structure and promotes self-regulation and executive function ing (i.e, working memory, inhibition, shifting), which allow young children to A. Bautista et al. A. Bautista et al. 476 pursue goals and ignore distractions (Diamond, 2013). Quinn et al. (2018) con ducted a meta-analysis of the literature focusing on the relationship between sym bolic play and language acquisition. Drawing on 35 studies conducted in Australia, United Kingdom, Finland, and USA, the authors identified a robust association between symbolic play and language development. 3 Societal Beliefs About ECE and Curriculum Policy Visions on Play in Selected Asian Contexts Suggestions such as these reveal that play is, in fact, seen as vehicle to acquire academic knowledge and skills (Li et al., 2016). Hong Kong and Singapore are highly developed and densely populated metropo lises. Both are regional trading hubs with highly developed infrastructure and world leading educational systems. Although both cities have a predominately Chinese Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know… 477 21 population and share a British colonial history, Singapore has a much larger propor tion of non-Chinese in her population (~35%). Regarding parenting practices, Chinese parents in both Hong Kong and Singapore share the traditional Confucian values placed on academic achievement and tend to send their children for private tuition even before the commencement of primary school (Bull et al., 2018; Rao & Lau, 2018). In addition to cultural values, this behavior is also likely driven by par ents’ concerns about their children’s readiness for primary education, which is often characterized as competitive and academic-oriented (Gopinathan & Lee, 2018). Perhaps because of the immediacy of other cultures in Singapore society, the degree to which Singaporean Chinese holds on to traditional Confucian values also tends to be stronger than in Hong Kong. Although not likely an overt consideration in policy making, it is interesting to note the differences in how play is conceptual ized in the curriculum frameworks of the two metropolises. Singapore’s curriculum advocates purposeful play, that is, play-based pedagogies that involve activities pur posefully planned by ECE teachers to achieve intended learning goals (for example, educational games, blocks, puzzles) (Singapore Ministry of Education [MOE], 2013). In contrast, Hong Kong refers to free play in her official curriculum frame work, emphasizing the importance of play in drawing on or cultivating children’s intrinsic motivation, autonomy, creativity, and freedom for exploration and curiosity (Curriculum Development Council [CDC], 2017). p Japan presents a very different case. In contrast to the three Chinese dominated societies examined thus far, the Japanese do not emphasize academic achievement before primary schooling. Rather, they emphasize the notion of mimamoru (i.e., teaching by watching and waiting), grounded in the belief of respecting children and giving children opportunities for taking up responsibility (Hayashi, 2011). Besides the hands-off approach, a key early childhood practice is group-based cur riculum, which is thought to be beneficial for children’s socio-emotional develop ment (Izumi-Taylor, 2013). 3 Societal Beliefs About ECE and Curriculum Policy Visions on Play in Selected Asian Contexts Rather than having direct instruction as its main function, the Japanese believe that kindergartens serve to provide opportunities for children to interact and play with others who are outside of their family circle. Echoing these societal beliefs, Japan’s curriculum framework does not prescribe play for academic learning but instead focuses on the child-directed quality of play (Japan Ministry of Education, Culture, Sports, Science and Technology [MEXT], 2008). The curriculum characterizes play as a voluntary and spontaneous activity enacted by children. Play is seen as a basic form of early childhood learning. Rather than a focus on learning outcomes, the role of the teachers is to prepare an appropri ate environment that corresponds to the children’s play patterns and to facilitate children’s engagement and enjoyment (Fujisawa et al., 2008; Takahashi, 2016). In sum, the four selected jurisdictions have both similarities and differences in their socio-cultural beliefs about early childhood. However, play is a central compo nent of their official ECE curriculum policy frameworks (Bautista, Yu, et al., 2021b; Grieshaber, 2016; Gupta, 2014), although with differences in the specific play approaches that teachers are encouraged to facilitate, ranging from structured (teacher-led) to free (child-led) play (Hassinger-Das et al., 2017). 478 A. Bautista et al. A. Bautista et al. 4 Reviewing the Asian Literature on the Impact of Play on Child Outcomes The key research question addressed in this section is: Drawing on the available empirical research, what do we actually know about the impact of play-based peda gogies on children’s outcomes in Mainland China, Hong Kong, Singapore, and Japan? We conducted a literature search of empirical studies published up to May 2020, using the EBSCO research database. EBSCO is often used in similar review studies, given that it includes a large number of high-quality academic journals. Keywords in the search included the name of each individual Asian jurisdiction (e.g., Singapore, Japan), play, preschool OR kindergarten OR playschool, learning OR development OR outcomes, and child OR children. As this was the first system atic exploration of the topic, we decided to focus exclusively on peer-reviewed jour nal articles written in English. 21.1 4.1 Naturalistic Studies Only five naturalistic studies were identified, one conducted in Hong Kong (Cheung et al., 2015), two in Singapore (Lee & Goh, 2012; Ng & Bull, 2018), and two in Japan (Fujisawa et al., 2008; Takahashi, 2016). They all showed that play-based pedagogies have the potential to positively impact specific aspects of Asian chil dren’s socio-emotional development. Note that the five studies are qualitative and based on small, non-representative samples. English journal publications in Hong Kong and Singapore have focused on investigating the effect of guided or structured forms of play on children, at the expense of free play which has been only investigated in Japan. In Hong Kong, Cheung et al. (2015) conducted a comparative case study in two contrasting pre schools. In the academically focused preschool, learning activities were organized following teachers’ plans, with specified learning objectives; children were permit ted to play in interest corners only after they finished the compulsory learning activ ities. In the play-based preschool, children were usually engaged in small-group activities and encouraged to choose their own activities, for example enjoying their 21 Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know… 21 479 Table 21.1 Descriptive information about the 16 empirical research articles identified in the four Asian jurisdictions Author(s) Year Jurisdiction Study type Research approach Play type(s) Outcome measured Cheung 2018 Hong Kong Intervention Quantitative Free and guided play Creativity Ng & Bull 2018 Singapore Naturalistic Qualitative Outdoor play Socio-emotional skills Fung & Cheng 2017 Hong Kong Intervention Quantitative Pretend play Socio-emotional skills Liu et al. 2017 Hong Kong Intervention Quantitative Eduplay Socio-emotional skills Teo et al. 2017 Singapore Intervention Qualitative Purposeful play Scientific thinking Takahashi 2016 Japan Naturalistic Qualitative Pretend play Socio-emotional skills Li et al. 2016 Mainland China Intervention Quantitative Free and guided play Socio-emotional skills Cheung et al. 2015 Hong Kong Naturalistic Qualitative Multiple play types Socio-emotional skills Hui et al. 2015 Hong Kong Intervention Quantitative Dramatic play Creativity Leung 2015 Hong Kong Intervention Quantitative Eduplay Socio-emotional skills Qu et al. 2015 Singapore Intervention Quantitative Sociodramatic play Socio-emotional skills Lee & Goh 2012 Singapore Naturalistic Qualitative Pretend play Cognitive and socio-emotional skills Leung 2011 Hong Kong Intervention Quantitative Eduplay Socio-emotional skills Wang & Hung 2010 Hong Kong Intervention Quantitative Board games Mathematics Fujisawa et al. 2008 Japan Naturalistic Quantitative Free play Socio-emotional skills Kok et al. 4.1 Naturalistic Studies 2002 Singapore Intervention Quantitative Guided play Socio-emotional skills time in a variety of interest corners freely. Social interaction and collaborative work among children were highly encouraged. A total of 60 4–5-year-old children (30 children from each preschool) were interviewed to understand their agency orienta tion. Cheung et al. (2015) found that children in the academically oriented pre school had more uncertain and less participative orientation than children in the play-based preschool. The authors argued that play-based pedagogies stimulated children’s capacities for agentive and participative social engagement, which in turn enhanced their chances to obtain versatile social skills. In contrast, the teacher- directed environment was more likely to undermine children’s capability in express ing their own ideas and inhibiting the opportunities for children to build interactive relationships with peers and teachers. time in a variety of interest corners freely. Social interaction and collaborative work among children were highly encouraged. A total of 60 4–5-year-old children (30 children from each preschool) were interviewed to understand their agency orienta tion. Cheung et al. (2015) found that children in the academically oriented pre school had more uncertain and less participative orientation than children in the play-based preschool. The authors argued that play-based pedagogies stimulated children’s capacities for agentive and participative social engagement, which in turn enhanced their chances to obtain versatile social skills. In contrast, the teacher- directed environment was more likely to undermine children’s capability in express ing their own ideas and inhibiting the opportunities for children to build interactive relationships with peers and teachers. 480 A. Bautista et al. A. Bautista et al. In Singapore, Lee and Goh (2012) undertook an action research project that examined how pretend play benefited children’s development and helped in their transition to primary school. Pretend play is a form of symbolic play where children use something (e.g., objects, actions ideas) to represent something else, and/or use their creativity to perform the role of imaginary characters (e.g., being superheroes, playing mummies and daddies). Children were observed as engaged in pretend play activities and comfortable to initiate activities, which echoed with newly learned knowledge. The authors concluded that young children’s cognitive and affective outcomes were supported during the pretend play activities, as they were exposed to multiple opportunities to apply the knowledge learned to solve real-life problems. 4.2 Intervention Studies Intervention studies have examined the impact on children of play-based programs and/or identified the factors influencing their effectiveness. These have been more numerous than naturalistic studies, with one study conducted in Mainland China (Li et al., 2016), seven in Hong Kong (Cheung, 2018; Fung & Cheng, 2017; Hui et al., 2015; Leung, 2011, 2015; Liu et al., 2017b; Wang & Hung, 2010), and three in Singapore (Kok et al., 2002; Qu et al., 2015; Teo et al., 2017). We did not identify research of this nature in Japan, which could be interpreted as consistent with their curriculum vision of free or unguided play (MEXT, 2008). Compared to naturalistic studies, this research has been based on more rigorous research designs, including experimental and quasi-experimental designs, with the use of both quantitative and mix-methods analytical techniques. Play interventions have been rather short in terms of duration (e.g., eight weekly sessions), typically guided or facilitated by adults (e.g., ECE teachers, parent volunteers, researchers), and implemented as extra-curricular activities. Similar to naturalistic studies, most interventions have targeted specific out comes related to children’s socio-emotional development, with both educational and/or therapeutic purposes. For example, Liu et al. (2017a, b) showed that Hong Kong children’s social competence could be improved with a parent-guided eduplay intervention. The notion of eduplay (Rao & Li, 2009) is a hybrid between the Western idea of ‘playing to learn’ and the Chinese Confucian emphasis on achiev ing outcomes pre-determined by adults. The program designed by Liu et al. (2017a, b) involved eight 1-h weekly sessions. Children engaged in collaborative group games in a classroom setting, led by trained parent volunteers. Games focused on themes related to social situations such as lining up, gathering, and dispersing. While the children were participating in the games, there were two major roles for the parent volunteers: (1) decoding social cues for children, such as summarizing the positive manners demonstrated by children during the game; (2) reinforcing children’s prosocial behaviors, such as sharing and turns taking, with a star reward ing system and affirmative body language. After 8-weeks of intervention, assessed with The Early School Behavior Rating Scale (ESBRS), children’s enhancement in social competence was significant based on both teacher and parent reports. The effect of the play intervention was sustainable over 5 months and generalizable to both home and ECE settings. 4.1 Naturalistic Studies 21 Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know… 481 21 4.1 Naturalistic Studies Through systematic natural observations in six kindergartens, Ng and Bull (2018) explored the role of teacher-child interactions in outdoor play in supporting chil dren’s social-emotional learning. The authors found that teachers provided most socio-emotional learning opportunities to children in outdoor play compared in the other three major types of learning activities (i.e., lesson time, mealtime/transition time, learning centers). During outdoor play, children were able to freely choose their activities (e.g., climbing equipment, playground play) and teachers were found to support children’s interactions with peers by relating, talking and playing with peers, which facilitated relationship management and social awareness and pro moted children’s self-awareness and positive self-concept. The two studies looking into the impact of free play were conducted in Japan. Fujisawa et al. (2008) investigated the reciprocity of prosocial behaviors among 3- and 4-year-old Japanese preschool children in the free play time. Two classes of 3-year-old children and two classes of 4-year-old children were observed during morning free play time for a school year. Each child was observed for 20 5-min focal observation sessions. The affiliative and prosocial behaviors occurring between the focal child and his/her peers were coded and the frequencies of each of the two types of behaviors were calculated. The results indicated positive correla tions between given and received object offering and helping, as well as between the object offering and helping behaviors in the dyads. This indicated a reciprocity of prosocial behaviors during the free play time in Japanese preschool children. Findings suggest that children’s prosocial behaviors can be developed and sup ported in positive interactions with peers during free play. In an ethnographic study, Takahashi’s (2016) investigated how Japanese young children collectively con structed identities with peers in pretend play. A class with 25 children of 5 years of age in a local preschool in Japan was observed over 4 months with 8 h, 3 days a week. Based on the detailed analyses of children’s conversations and interactions, three characteristic forms of interaction during play were identified, as featuring children’s construction of pretend identifies: (1) Reciprocal immediacy; (2) Maintaining and challenging participation; and (3) Willingness and collaboration. The author argued that play is not only for fun but implies the deliberate process of working out the roles and rules between the playmates. As a result, children co- construct their pretend identities in play situations, which contributes to support their social-emotional development (Takahashi, 2016). 4.2 Intervention Studies The authors further argued that recruiting parent vol unteers as instructors in play-based interventions would enhance parents’ awareness and skills in facilitating children’s play. In this light, parents would likely continue providing children with play opportunities and would be able to better facilitate play activities in the future. The other classroom interventions implemented were also short and involved pretend and socio-dramatic play, which have proven effective to enhance Singaporean children’s theory of mind (Qu et al., 2015) and to reduce Hong Kong children’s disruptive behaviors during peer interactions (Fung & Cheng, 2017). Furthermore, interventions designed with therapeutic purposes have found that 482 A. Bautista et al. A. Bautista et al. guided forms of play contribute to reducing internalizing and externalizing behav ioral problems in Hong Kong (Leung, 2011, 2015), increase time spent on social interactions in extremely shy children in Mainland China (Li et al., 2016), and enhance appropriate communication in children with autism in Singapore (Kok et al., 2002). Beyond socio-emotional development, only three studies have analyzed the impact of play-based interventions on other child outcomes, specifically related to scientific thinking (Teo et al., 2017), creativity and problem-solving (Cheung, 2018), and mathematics (Wang & Hung, 2010). None of the intervention studies conducted in these four Asian contexts have focused on domains such as linguistic, physical, artistic, or spiritual/moral development. In the area of scientific thinking, the qualitative study by Teo et al. (2017) documented how a 90-min purposeful play session (facilitated by the researchers) allowed Singaporean children to expand their intuitive conceptions about floating and sinking. In a quasi-experimental study focusing on creativity and problem-solving, Cheung (2018) found that Hong Kong kindergarten children benefited more from a teacher-guided play approach than from a hands-off approach. In Hong Kong, Wang and Hung (2010) conducted a small-scale quasi-experimental study to examine the effect of teacher-designed boardgames on 5-year-old children’s number sense. Children in the intervention group showed better number sense after 8 weekly gameplays, especially in the domain of addition-subtraction. The authors concluded that play-based pedagogies facilitate curriculum innovation and pedagogical reform, allowing ECE teachers to gain flexibility to cope with the demands of Asian parents. However, note that the small sample size was insufficient for the authors to run inferential analysis. Further studies are needed to confirm the effectiveness of this math play-based intervention. 5 Discussion The low number of play impact studies in these four Asian contexts may be due to multiple factors. First, despite the strong advocacy of play in official curriculum policy guidelines (CDC, 2017; MEXT, 2008; MOE, 2013; MOE-PRC, 2012), play- based pedagogy is still in its infancy in many parts of Asia. In fact, except for Japan, there is a large gap between the officially sanctioned perspectives on play and the observed practices on the ground, as extensively documented in classroom-based studies (Bautista, Yu, et al., 2021b; Grieshaber, 2016; Gupta, 2014). Playtime tends to be low within many ECE settings, and it is often used instrumentally to teach about academic learning areas (Bautista et al., 2019; Lam, 2018). Contextual con straints (e.g., lack of time and space) and cultural ideologies (e.g., lack of support from school leaders, parental pressures for academic learning) are other important factors that contribute to making it difficult for ECE teachers to embrace play-based pedagogies (Bull et al., 2018; Rao & Lau, 2018). In sum, the paucity of studies may be related to the availability of ECE settings where Asian children are consistently exposed to play-based pedagogies. 21 483 Impact of Play-Based Pedagogies in Selected Asian Contexts: What Do We Know… This thin body of literature could be also interpreted as a manifestation of tradi tional Asian values and of the deep-rooted beliefs about teaching and learning, especially within Chinese societies (Gopinathan & Lee, 2018), in which play is often seen as a rather unimportant activity. Indeed, the limited work on the effects of play on domains other than socio-emotional development may be due to the Confucian belief that play is an activity with little benefit for learning, specifically for academically related learning (Luo et al., 2013). Furthermore, consistent with traditional Chinese norms, researchers have clearly favored adult-guided forms of play, also referred to as eduplay (Rao & Li, 2009) or purposeful play (Bautista et al., 2019), characterized by high degree of teacher structure or control, the exis tence of given rules, and children’s lack of freedom to engage in these activities, mainly designed to achieve pre-determined outcomes. Interestingly, the essential ingredients of play (e.g., freedom, autonomy, choice, intrinsic motivation, free par ticipation), as described by Western play theorists (e.g., Van Oers, 2013), are not visible in the studies reviewed in this chapter, except for the studies carried out in Japan. 6 Conclusion and Limitations We conclude that Mainland China, Hong Kong, Singapore and Japan have con ducted little empirical research on the impact of play-based pedagogies on chil dren’s development and learning. Taking peer-reviewed journal articles written in English as a reference, the volume of work is minimal, with only five naturalistic studies (none of them conducted in Mainland China) and 11 interventions (none of them conducted in Japan). Existing studies are small in scale, limited in scope, and often methodologically weak (i.e., short duration, focused on limited outcomes, small sample sizes, lack of control groups, lack of locally developed measures, lim ited generalizability). Findings suggest that little research funding has been allocated to investigate the impact of play-based pedagogies on children in these four Asian jurisdictions. As a result of globalization in ECE (Gupta, 2018; Yang & Li, 2019), Western discourses pertaining to play seem to have been assumed as universally valid in these jurisdic tions, where play-based pedagogies are recommended to teachers within ECE poli cies with little empirical evidence about their impact on local children (Bautista, Yu, et al., 2021b; Grieshaber, 2016; Gupta, 2014). However, we agree with J. Li (2010) in that “long-held Western assumptions about processes, efficacy, and effectiveness of learning cannot be readily applied to the study of learners from non-Western cultures” […] because these assumptions “were developed by Western researchers to study Western people based on Western cultural norms and values” (p. 42). In other words, what is known from Western research about how play impacts on Western children may or may not be applicable to children in other parts of the world, including Asia, as cultural contexts lead to significant differences in develop mental and learning pathways (UNESCO, 2010). A given play-based pedagogy that A. Bautista et al. 484 A. Bautista et al. is effective (and culturally appropriate) in the West may or may not be effective (or culturally appropriate) in the East (Bautista, Bull, et al., 2021a; Gupta, 2014). One obvious limitation of this review is that we only included articles published in English. Nevertheless, compared to the vast volume of Western work in this area, it seems clear that there is a need for a more solid corpus of play-based research in these four Asian societies; research that takes into consideration their socio-cultural characteristics and the developmental pathways of local young children (King & Bernardo, 2016; Li, 2010). 6 Conclusion and Limitations We propose future lines of research and implications in the following section. Acknowledgements This study was supported by the Central Reserve Fund at The Education University of Hong Kong, as part of the project “A multi-disciplinary research program in research on child development” (04A05). The views expressed in this paper are the authors’ and do not necessarily represent the views of their respective institutions. References Bautista, A., Habib, M., Eng, A., & Bull, R. (2019). Purposeful play during learning center time: From curriculum to practice. Journal of Curriculum Studies, 51(5), 715–736. https://doi.org/1 0.1080/00220272.2019.1611928 Bautista, A., Bull, R., Ng, E. L., & Lee, K. (2021a). “That’s just impossible in my kindergarten.” Advocating for ‘glocal’ early childhood curriculum frameworks. Policy Futures in Education, 19(2), 155–174. https://doi.org/10.1177/1478210320956500 p g Bautista, A., Yu, J., Lee, K., & Sun, J. (2021b). Play in Asian preschools? Mapping a landscape of hindering factors. Contemporary Issues in Early Childhood, 22(4), 312–327. https://doi. org/10.1177/14639491211058035 g Bedard, C., St John, L., Bremer, E., Graham, J. D., & Cairney, J. (2019). A systematic review and meta-analysis on the effects of physically active classrooms on educational and enjoyment outcomes in school age children. PLoS One, 14(6), e0218633. https://doi.org/10.1371/journal. pone.0218633 Bull, R., & Bautista, A. (2018). A careful balancing act: Evolving and harmonizing a hybrid sys tem of ECEC in Singapore. In S. L. Kagan (Ed.), The early advantage: Early childhood sys tems that Lead by example (pp. 155–181). Teachers College Press. Bull, R., Bautista, A., Salleh, H., & Karuppiah, N. (2018). A case study of the Singapore early childhood education and care system. Evolving a harmonized hybrid system of ECEC: A care ful balancing act. Teachers College Press. Report commissioned by USA National Center on education and the economy (NCEE). Retrieved from: ncee.org/wp-content/uploads/2019/03/ EA-Singapore-Case-Study-022819.pdf. Burdette, H., & Whitaker, R. (2005). Resurrecting free play in young children: Looking beyond fitness and fatness to attention, affiliation, and affect. Archives of Pediatrics & Adolescent Medicine, 159, 46–50. Burghardt, G. M. (2011). Defining and recognizing play. In A. D. Pellegrini (Ed.), Oxford hand book of the development of play (pp. 9–18). Oxford University Press. Cheung, R. (2018). Play-based creativity-fostering practices: The effects of different pedagogi cal approaches on the development of children’s creative thinking behaviours in a Chinese preschool classroom. Pedagogy, Culture & Society, 26(4), 511–527. https://doi.org/10.108 0/14681366.2018.1424725 Cheung, D., Reunamo, J., Cooper, P., Liu, K., & Vong, K.-i. P. (2015). Children’s agen tive orientations in play-based and academically focused preschools in Hong Kong. Early Child Development and Care, 185(11–12), 1828–1844. https://doi.org/10.1080/0300443 0.2015.1028400 Curriculum Development Council (CDC). (2017). Kindergarten education curriculum guide: Joyful learning through play, balanced development all the way. https://www.edb.gov.hk/attach ment/en/curriculum-development/major-level-of-edu/preprimary/ENG_KGECG_2017.pdf ment/en/curriculum-development/major-level-of-edu/preprimary/ENG_KGECG_2017.pdf Diamond, A. (2013). Executive functions. Annual Review of Psychology, 64, 135–168. https://doi org/10.1146/annurev-psycho-113011-143750 Fjortoft, I. (2004). 7 Future Research and Implications To better justify the inclusion of play and play-based pedagogies within Asian ECE curriculum frameworks, we claim it would be vital to conduct more rigorous and ambitious impact studies. Long-term longitudinal projects, which track children educated in various types of ECE settings (from academically oriented to play- based), are needed to understand the extent to which exposure to play in ECE makes a difference in the life of children (Cheung et al., 2015; Fung & Cheng, 2017). Consistent with the vision of holistic and balanced development (e.g., CDC, 2017; MOE, 2013), a wide range of outcomes should be investigated in these studies (e.g., physical, socio-emotional, cognitive, academic, mental health). Large-scale inter vention studies, including randomized controlled trials, should be also undertaken to examine the benefits of specific play pedagogies within Asian ECE settings (Bull & Bautista, 2018; Cheung, 2018). In particular, it would be vital to examine the impact of different types of play in the continuum from structured (teacher-led) to free (child-led) play. As argued by Fung and Cheng (2017), studies on gender differ ences in response to diverse play approaches would be also desirable. Following the example of Western scholars, a wide range of research methodologies (quantitative, mix-methods, qualitative) and research designs (e.g., correlational, longitudinal, case studies) should be employed. Developing this future research agenda would be vital not only to better justify the inclusion of play in curriculum frameworks, but also to influence (and eventu ally change) societal mindsets about the importance of play among Asian parents, who often prioritize academic work, discipline, and effort over other forms of learn ing (Lam, 2018; Rao & Li, 2009). An extensive, rigorous, and locally situated cor pus of play impact studies would allow them to choose the best ECE for their children, within the frame of their respective cultural contexts (Bull et al., 2018; Rao & Lau, 2018; Yang & Li, 2019). Acknowledgements This study was supported by the Central Reserve Fund at The Education University of Hong Kong, as part of the project “A multi-disciplinary research program in research on child development” (04A05). 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Pediatrics, 142(3), e20182058. https://doi.org/10.1542/peds.2018-2058 Yogman, M., Garner, A., Hutchinson, J., Hirsh-Pasek, K., & Golinkoff, R. M. (2018). The power of play: A pediatric role in enhancing development in young children. Pediatrics, 142(3), e20182058. https://doi.org/10.1542/peds.2018-2058 Alfredo Bautista is Associate Professor and Associate Head of the Department of Early Childhood Education (ECE) at The Education University of Hong Kong (EdUHK). He is also Co-Director of the Centre for Educational and Developmental Sciences (CEDS). Alfredo is interested in curricu lum, pedagogy, instructional practices, and teacher learning and professional development, with emphasis on the field of ECE. His work has been published in prestigious academic journals. Alfredo serves as Editor-in-Chief for the Journal for the Study of Education and Development, and Associate Editor for other academic journals. Address: Department of Early Childhood Education. B3–2/F-34 \| 10 Lo Ping Road, Tai Po, New Territories. Hong Kong SAR (China). email: abau tista@eduhk.hk Jimmy Yu is a Research Assistant at CEDS at EdUHK. He works for “The PACE Project: A multi- disciplinary research program in research on child development”, which investigates preschoolers’ multi-disciplines developmental trajectories in Hong Kong. Jimmy graduated in Psychology from Shue Yan University. He is interested in investigating effective and joyful learning strategies for children through evidence-based practices. He is also interested in the neuroscience of morality, how moral sense emerges in young children, and the nature of their ethical decisions. Address: Department of Early Childhood Education. B3–2/F-34 \| 10 Lo Ping Road, Tai Po, New Territories. Hong Kong SAR (China). Kerry Lee is Professor, Head of the ECE Department, and Director of the CEDS at EdUHK. Professor Lee is trained as a cognitive developmental psychologist. His work focuses on mathematical achievement, working memory, and the development of executive functioning. He serves on the editorial or review boards of several academic journals. Address: Department of Early Childhood Education. B2–1/F-36 \| 10 Lo Ping Road, Tai Po, New Territories. Hong Kong SAR (China). Jin Sun is Assistant Professor and Associate Head of ECE Department at EdUHK, as well as Co-Director for CEDS. References https://doi.org/10.1007/s13158-018-0225-9 Organisation for Economic Co-operation and Development (OECD). (2004). Starting strong: Curricula and pedagogies in early childhood education and care: Five curriculum outlines. OECD Publishing. Retrieved from http://www.oecd.org/edu/school/31672150.pdf Pellegrini, A., Kato, K., Blatchford, P., & Baines, E. (2002). A short-term longitudinal study of children’s playground games across the first year of school: Implications for social competence and adjustment to school. 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(2009). “Eduplay”: Beliefs and practices related to play and learning in Chinese kindergartens. In I. Pramling-Samuelsson & M. Fleer (Eds.), Play and learning in early child hood settings (Vol. 1, pp. 97–116). Springer. https://doi.org/10.1007/978-1-4020-8498-0_5 Singapore Ministry of Education (MOE). (2013). Nurturing early learners: A curriculum for kin dergartens in Singapore. Educators’ guide: Overview. Ministry of Education. Retrieved from https://www.moe.gov.sg/docs/default-source/document/education/preschool/files/nel-edu- guide-overview.pdf Takahashi, M. (2016). Consumers at play: Negotiations of identity in a Japanese preschool. Young Consumers, 17(1), 94–106. https://doi.org/10.1108/YC-08-2015-00549 Teo, T.-W., Yan, Y.-K., & Ong, M. (2017). An investigation of Singapore preschool children’s emerging concepts of floating and sinking. Pedagogies, 12(4), 325–339. https://doi.org/10.108 0/1554480X.2017.1374186 The LEGO Foundation & UNICEF. (2018). Learning through play. Strengthening learning through play in early childhood education programmes. Education section, Programme divi sion. United Nations Children’s Fund (UNICEF). United Nations Educational Scientific and Cultural Organization (UNESCO). (2010). Culture and development. Evolution and prospects. UNESCO. Van Oers, B. (2013). Is it play? Towards a reconceptualisation of role play from an activity theory perspective. European Early Childhood Education Research Journal, 21(2), 185–198. Wang, Z., & Hung, L. (2010). Kindergarten children’s number sense development through board games. International Journal of Learning, 17(8), 19–31. https://doi.org/10.18848/1447-9494/ CGP/v17i08/47181 488 A. Bautista et al. A. Bautista et al. Yang, W., & Li, H. (2019). References Her research interests include the assessment of early learning and devel opment, early self-regulation and math development, and Chinese socialization. She is particularly interested in early development and education of socially and economically disadvantaged chil dren. Dr. Sun has undertaken consultancies for UNICEF, UNESCO, Education for All, and the Plan. Address: Department of Early Childhood Education. B3–1/F-16 \| 10 Lo Ping Road, Tai Po, New Territories. Hong Kong SAR (China). 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 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 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 statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
https://openalex.org/W1996681236	https://hal.archives-ouvertes.fr/hal-01137055/document	English	null	Intuitive Visualization and Analysis of Multi-Omics Data and Application to Escherichia coli Carbon Metabolism	PloS one	2,011	cc-by	8,147	Introduction forward the regulatory aspect conjointly to omic-data display. Finally, the representations of biomolecular networks that are automatically generated by current software are often far from both the academic conventions and biological perception of the net- works, thereby making difficult the intuitive interpretation of data. The graphical visualization and analysis of multi-omics data is a challenge in systems biology [1,2]. The representation of true biological networks includes several layers of complexity due to the embedding of multiple biological components and processes – e.g gene expression, protein biosynthesis, regulatory processes, etc -. Given that each layer includes thousand of components even in the simplest cell, there are strong needs for visualization tools that ease the intuitive interpretation of multi-omics data. In this regard, such a tool should explicitly represent all the molecular components in the studied phenomenon (data representation), as well as all the interactions between these components (data understanding). The objective of this work was to propose a cost-less and straightforward strategy to represent both complex biomolecular networks and multi-omics data in the same graphical representa- tion. A simple graphical formalism was designed to represent all network components (structural and functional components). These components were compiled using the open source software Cytoscape [8]. MODAM, a custom-made Cytoscape plugin, was developed to optimize the mapping of multi-omics data and their interpretation. This approach was applied to the central metabolic network of the bacterium Escherichia coli, as a typical example of cellular metabolism and its regulation, with hundreds of metabolic or regulatory interactions. The resulting network encompasses 774 components and 1232 interactions that are represented accordingly to biochemistry text-book drawing conventions. The mapping of multi-omics data from Ishii et al. [9] offered a valuable example of the approach, as discussed in the final part of this publication. Numerous solutions have been recently developed for the re- presentation of complex networks as well as the analysis of multi- omics datasets [3]. Some of the most complex tool packages (‘‘Cyclone’’ [4], ‘‘the Gaggle’’ [5], ‘‘Prometra’’ [6]) succeed to present together data from multiple dimensions through the association of several software. These tools are powerful and versatile but require significant computing efforts, which can limit their use by biologists. Some commercial tools also offer a combination of data and net- work visualization (i.e, ‘‘Genespring’’: http://www.genespring.com; ‘‘Ingenuity Pathways Analysis’’: http://www.ingenuity.com/), but they are quite expensive and lack the flexibility of open-source software. Brice Enjalbert1,2,3, Fabien Jourdan4, Jean-Charles Portais1,2,3* UPS, INP, Toulouse, France, 2 INRA, UMR792 Inge´nierie des Syste`mes Biologiques et des Proce´de´s, Toulouse, France, 3 CNRS, UMR5504 089 Xe´nobiotiques, Toulouse, France 1 Universite´ de Toulouse, INSA, UPS, INP, Toulouse, France, 2 INRA, UMR792 Inge´nierie des Syste`mes Biologiques et des Proce´de´s, Toulouse, France, 4 INRA, UMR 1089 Xe´nobiotiques, Toulouse, France 1 Universite´ de Toulouse, INSA, UPS, INP, Toulouse, France, 2 INRA, UMR792 Inge´nierie des Syste`mes Biologiques et des Proce´de´s, Toulouse, France, 3 CNRS, UMR5504, Toulouse, France, 4 INRA, UMR 1089 Xe´nobiotiques, Toulouse, France Abstract Competing Interests: The authors have declared that no competing interests exist supported by the French "Agence Nationale de la Recherche" (ANR, Project nu ANR-06-BYOS-0003-03) from which BE received a post- funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by the French "Agence Nationale de la Recherche" (ANR, Project nu ANR-06-BYOS-0003-03) from doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Competing Interests: The authors have declared that no competing interests exist. * E-mail: jean-charles.portais@insa-toulouse.fr Introduction Some tools have been developed to represent either metabolic or regulatory networks (e.g., Pathway Tools [7]), but to our knowledge, no freely-available solution has been developed to bring Intuitive Visualization and Analysis of Multi-Omics Data and Application to Escherichia coli Carbon Metabolism Brice Enjalbert1,2,3, Fabien Jourdan4, Jean-Charles Portais1,2,3* PLoS ONE \| www.plosone.org Citation: Enjalbert B, Jourdan F, Portais J-C (2011) Intuitive Visualization and Analysis of Multi-Omics Data and Application to Escherichia coli Carbon Metabolism. PLoS ONE 6(6): e21318. doi:10.1371/journal.pone.0021318 Abstract Combinations of ‘omics’ investigations (i.e, transcriptomic, proteomic, metabolomic and/or fluxomic) are increasingly applied to get comprehensive understanding of biological systems. Because the latter are organized as complex networks of molecular and functional interactions, the intuitive interpretation of multi-omics datasets is difficult. Here we describe a simple strategy to visualize and analyze multi-omics data. Graphical representations of complex biological networks can be generated using Cytoscape where all molecular and functional components could be explicitly represented using a set of dedicated symbols. This representation can be used i) to compile all biologically-relevant information regarding the network through web link association, and ii) to map the network components with multi-omics data. A Cytoscape plugin was developed to increase the possibilities of both multi-omic data representation and interpretation. This plugin allowed different adjustable colour scales to be applied to the various omics data and performed the automatic extraction and visualization of the most significant changes in the datasets. For illustration purpose, the approach was applied to the central carbon metabolism of Escherichia coli. The obtained network contained 774 components and 1232 interactions, highlighting the complexity of bacterial multi-level regulations. The structured representation of this network represents a valuable resource for systemic studies of E. coli, as illustrated from the application to multi-omics data. Some current issues in network representation are discussed on the basis of this work. Citation: Enjalbert B, Jourdan F, Portais J-C (2011) Intuitive Visualization and Analysis of Multi-Omics Data and Application to Escherichia coli Carbon Metabolism. PLoS ONE 6(6): e21318. doi:10.1371/journal.pone.0021318 Editor: Nicholas James Provart, University of Toronto, Canada Editor: Nicholas James Provart, University of Toronto, Canada Received December 22, 2010; Accepted May 27, 2011; Published June 22, 2011 Copyright: 2011 Enjalbert 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 the French "Agence Nationale de la Recherche" (ANR, Project nu ANR-06-BYOS-0003-03) from which BE received a post- doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Visualization of Multi-Omics Data intuitive interpretation of multi-omics datasets. A graphical for- malism (figure 1) was introduced to represent explicitly any com- ponent of the system (RNA, proteins, activities, fluxes, and metabolites) as well as any kind of structural and regulatory interaction between two components (metabolic reactions, tran- scriptional and translational regulations, control of enzymes by metabolic effectors or by phosphorylation, and hierarchical rela- tionships – genes to proteins, proteins to activities, and activities to reactions). This formalism can be applied to generate maps repre- senting the structural and regulatory knowledge for all types of biomolecular networks. Beside its universality, the presented for- malism provides a graphical representation compatible with an intuitive understanding of the network structure. subjected to intense regulation. The central carbon metabolism and its regulation have been extensively studied and a large wealth of information is available to generate a highly detailed network describing all known metabolic and regulatory interactions. The first step is the network delineation, which is determined by the biologic purpose. For this work, the network has to be large enough to illustrate the strategy and consistent enough to depict E. coli central carbon metabolism. The network was consequently delimited to include all the central carbon metabolic pathways: glycolysis/gluconeogenesis, pentose phosphate, TCA, glyoxylate, Entner-Doudoroff, methylglyoxal and acetate (figure 2). The second step was to collect and gather all the molecular and functional information related to this network and its regulation from relevant databases: the information was compiled for meta- bolic pathways (KEGG [10], Ecocyc [11]), biochemical reactions (Brenda [12]), and their regulations (Ecocyc [11], Colibri [13], RegulonDB [14]) in addition to literature data (e.g. NCBI [15]). Inconsistencies in the so-established network were curated using existing literature (NCBI) and personal expertise. The final network (figure 2 and figure S1) contained 55 metabolic reactions, 63 metabolites, and required 77 enzymatic activities generated from 93 different polypeptides and as many mRNAs. A total of 41 small molecules were identified as effectors of enzymatic activities and were responsible for 43 activations and 83 inhibitions. A total of Application to the assembly of E. coli central carbon metabolic network A color code can be applied to each node (symbol) in the network to visualize experimental data (gene expression for the squares, protein abundance for the rounded squares, specific activity for the hexagons, metabolite concentrations for the diamonds and flux values for the circle). Interactions between the components are indicated with lines (edges). Four main kinds of interactions were considered and were represented using lines with specific colors: biochemical conversions (grey lines), transcriptional and translational regulations (blue lines), control of enzymatic activities by metabolic effectors or by phosphorylation (green lines), hierarchical relationships – i.e. RNAs to proteins, proteins to activities, activities to reactions - (pink lines). In the given example, a metabolite X is converted in Y through the reaction ECx.x.x.x. The reaction requires a molecule of H2O and produces a molecule of CO2. The metabolite W is a negative effector of this reaction. The reaction depends on the enzymatic activity ‘‘Actv’’ which is a property of the protein ‘‘Actv Prot’’. This protein is encoded by the gene ‘‘Actv gene’’ whose transcription is induced by the activity ‘‘Trxu Factor’’, itself l i f h i d ‘‘T u’’ T l i f ‘‘A P ’’ i ll d b h l i f ‘‘T lu F ’’ Figure 1. Dedicated formalism. All molecular or functional components of the metabolic and regulatory networks are explicitly represented using specific symbols. Each RNA (square) encodes a polypeptide (rounded square). Polypeptides or polypeptide complexes generate functional entities – i.e., enzymes or regulators – (hexagons). Enzymes catalyze reactions (circles), which allow the inter-conversion of metabolites (diamonds). A color code can be applied to each node (symbol) in the network to visualize experimental data (gene expression for the squares, protein abundance for the rounded squares, specific activity for the hexagons, metabolite concentrations for the diamonds and flux values for the circle). Interactions between the components are indicated with lines (edges). Four main kinds of interactions were considered and were represented using lines with specific colors: biochemical conversions (grey lines), transcriptional and translational regulations (blue lines), control of enzymatic activities by metabolic effectors or by phosphorylation (green lines), hierarchical relationships – i.e. RNAs to proteins, proteins to activities, activities to reactions - (pink lines). In the given example, a metabolite X is converted in Y through the reaction ECx.x.x.x. The reaction requires a molecule of H2O and produces a molecule of CO2. Dedicated formalism The aim of this work was to develop a strategy for the repre- sentation of complex biomolecular networks that facilitates the PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 1 Visualization of Multi-Omics Data Application to the assembly of E. coli central carbon metabolic network For illustration, the graphical formalism was applied to build up a map of Escherichia coli central carbon metabolism and its regulations. Cellular metabolism represents a valuable example of a complex and tightly regulated biomolecular network. The central carbon metabolism is composed of a set of highly inter- connected reactions that provide a variety of molecules, energy and redox power to the cell to sustain survival, growth, and adaptation. It carries some of the most basic processes of life and is Figure 1. Dedicated formalism. All molecular or functional components of the metabolic and regulatory networks are explicitly represented using specific symbols. Each RNA (square) encodes a polypeptide (rounded square). Polypeptides or polypeptide complexes generate functional entities – i.e., enzymes or regulators – (hexagons). Enzymes catalyze reactions (circles), which allow the inter-conversion of metabolites (diamonds). A color code can be applied to each node (symbol) in the network to visualize experimental data (gene expression for the squares, protein abundance for the rounded squares, specific activity for the hexagons, metabolite concentrations for the diamonds and flux values for the circle). Interactions between the components are indicated with lines (edges). Four main kinds of interactions were considered and were represented using lines with specific colors: biochemical conversions (grey lines), transcriptional and translational regulations (blue lines), control of enzymatic activities by metabolic effectors or by phosphorylation (green lines), hierarchical relationships – i.e. RNAs to proteins, proteins to activities, activities to reactions - (pink lines). In the given example, a metabolite X is converted in Y through the reaction ECx.x.x.x. The reaction requires a molecule of H2O and produces a molecule of CO2. The metabolite W is a negative effector of this reaction. The reaction depends on the enzymatic activity ‘‘Actv’’ which is a property of the protein ‘‘Actv Prot’’. This protein is encoded by the gene ‘‘Actv gene’’ whose transcription is induced by the activity ‘‘Trxu Factor’’, itself resulting from the protein and gene ‘‘Trxu’’. Translation of ‘‘Actv Prot’’ is controlled by the translation factor ‘‘Trlu Fact’’. doi:10.1371/journal.pone.0021318.g001 Figure 1. Dedicated formalism. All molecular or functional components of the metabolic and regulatory networks are explicitly represented using specific symbols. Each RNA (square) encodes a polypeptide (rounded square). Polypeptides or polypeptide complexes generate functional entities – i.e., enzymes or regulators – (hexagons). Enzymes catalyze reactions (circles), which allow the inter-conversion of metabolites (diamonds). Application to the assembly of E. coli central carbon metabolic network The metabolite W is a negative effector of this reaction. The reaction depends on the enzymatic activity ‘‘Actv’’ which is a property of the protein ‘‘Actv Prot’’. This protein is encoded by the gene ‘‘Actv gene’’ whose transcription is induced by the activity ‘‘Trxu Factor’’, itself resulting from the protein and gene ‘‘Trxu’’. Translation of ‘‘Actv Prot’’ is controlled by the translation factor ‘‘Trlu Fact’’. doi:10.1371/journal.pone.0021318.g001 June 2011 \| Volume 6 \| Issue 6 \| e21318 PLoS ONE \| www.plosone.org 2 Visualization of Multi-Omics Data Figure 2. E. coli central carbon metabolism pathways and its regulations. The central part of the figure represents the central carbon metabolism (yellow background), with glucose entry (central topmost part) and gluconate entry (right topmost part), methylglyoxal pathway (leftmost central part), glycolysis/gluconeogenesis (left central part), Entner-Doudoroff pathway (rightmost central part), pentose phosphate pathway (right top part), TCA cycle with glyoxylate shunt (bottommost part), acetate metabolism (right bottom part) and transhydrogenase reactions (right PLoS ONE \| www.plosone.org 3 June 2011 \| Volume 6 \| Issue 6 \| e21318 Figure 2. E. coli central carbon metabolism pathways and its regulations. The central part of the figure represents the central carbon metabolism (yellow background), with glucose entry (central topmost part) and gluconate entry (right topmost part), methylglyoxal pathway (leftmost central part), glycolysis/gluconeogenesis (left central part), Entner-Doudoroff pathway (rightmost central part), pentose phosphate pathway (right top part), TCA cycle with glyoxylate shunt (bottommost part), acetate metabolism (right bottom part) and transhydrogenase reactions (right PLoS ONE \| www.plosone.org 3 Visualization of Multi-Omics Data central/bottom part). The first outskirt (green background) corresponds to the enzymatic activities and their metabolic controls. The second outskirt (pink background) represents the gene and protein encoding the activities. The third outskirt exhibits the direct and indirect transcriptional and traductional regulations (blue background). See the text for additional descriptions. doi:10.1371/journal.pone.0021318.g002 411 transcriptional controls were also identified. Translational controls exerted by small non-coding RNAs were also introduced where needed (e.g. Csr system). As most of the transcriptional factors are themselves under transcriptional control, all the in- direct degrees of transcriptional regulations were included. In addition to graphical representation and compilation of biological information, Cytoscape offers also the possibility to map the metabolic/regulatory network with multi-omics data. Since all network components – i.e. RNAs, proteins, metabolites, fluxes -, are explicitly represented, the various omics data – i.e. Cytoscape y All the relevant biological knowledge was compiled as an inter- active graph object using the open source software Cytoscape [8]. Combined with the graphical formalism, this network-dedicated software was found to be a convenient and handy platform to bypass the complexity of representing multi-level regulated networks. Besides facilitating the compilation of the data in the form of a graph, it offers numerous additional benefits. A first useful functionality of Cytoscape is the possibility to link any edge or node of a graph to a specific webpage. This functionality was used to link each component of the interaction network to cor- responding information web pages in relevant databases (e.g., Ecocyc, Brenda, Pubmed, etc.). In most cases, this allows getting molecule structures, reaction details, enzymatic effectors and their targets, gene and protein properties, or gene regulation networks (figure 3). Therefore, this function is extremely useful to get access to detailed information about the displayed network and its components in an interactive manner, and thereby to speed up data interpretation. Application to the assembly of E. coli central carbon metabolic network transcrip- tomics, proteomics, metabolomics, and fluxomics – can be visualized in parallel on the same graph. A unique color scale can be applied to all nodes to plot the experimental values for all types of data. To validate this functionality, complete sets of multi- omics data extracted from the work of Ishii et al. [9] were plotted on the graphical display. This is illustrated in figure 4 for the comparison of two datasets corresponding to E. coli cells grown at m = 0.7 h21 and m = 0.2 h21, respectively. The display of the multi-omics data on the network showed the activation (in red) of the PTS gene expression at both the transcriptional and trans- lational levels. This activation is likely to be controlled by the Mlc/ dgsA transcriptional factor. Another transport system, i.e. the mannose PTS operon, is also under the control of Mlc/DgsA. However, this operon is down-regulated (green), which could be explained by the influence of the NagC transcriptional factor. In spite of the induction of the PTS transport system, the glycolytic flux seems to be stable (yellow). This apparent lack of effect is due to the fact that flux data were expressed relative to the rate of glucose uptake. Indeed, the absolute rate of glycolysis in the fast- growing cells was higher than in the slow-growing cells. In ad- dition, the display of the relative flux data showed a significant redirection of the carbon flux towards the pentose phosphate pathway (PPP) when the growth rate is increased. The genes encoding the PPP enzymes were mostly upregulated at the transcriptional level (zwf, gnd, rpiA, rpe, tktA, talB), but not at the protein level. The correlation of transcriptomic and proteomic data showed a Pearson score of 0.04 for PPP components, compared to 0.68 for glycolysis (using the data for the four growth rates described in Ishii et al. [9]; data not shown). It is beyond the scope of this paper to re-interpret the authors’ results but the application of the proposed formalism to this particular case does point out either inconsistencies in the data or the occurrence of an unidentified translational mechanism that controls PPP. It does however illustrate the usefulness of the introduced formalism and representation to assess the overall coherence of complex datasets and, thereby, to help formulate working hypothesis for future investigations. 411 transcriptional controls were also identified. Application to the assembly of E. coli central carbon metabolic network Translational controls exerted by small non-coding RNAs were also introduced where needed (e.g. Csr system). As most of the transcriptional factors are themselves under transcriptional control, all the in- direct degrees of transcriptional regulations were included. The third step consisted in organizing the network layout to facilitate the intuitive reading of the biological information despite the considerably high number of components. The aim is to opti- mize the clarity of the representation (e.g. reduction of edge lengths, etc) while respecting the academic conventions for intuitiveness. This is not feasible with the automatic tools for network repre- sentation that are currently available (figure S2). Because a meta- bolic-centric representation was considered, the graphical layout of E. coli central metabolism was designed so as to make clear the structure of the metabolic network according to usual conventions (such as a vertical glycolytic pathway from glucose at the top to a circular TCA at the bottom). Consequently, the graphical layout (figure 2) was organized as a core of metabolic processes surrounded by 3 successive layers representing respectively metabolic control (inner layer), genome expression (medium layer), and transcriptional control (outer layer, or ‘outskirt’). Some transcriptional factors (for example DgsA/Mlc, DcuR, FlhCD) were highly specific to a pathway meanwhile other factors (e.g. CRP, ArcA, FNR, IHF, FruR, Fis and Sigma S) were extremely pleiotropic. Likewise, ATP, ADP, AMP, phosphate and coenzyme A have numerous implica- tions in enzymatic activity control. For these pleiotropic regulators, a ‘‘passageway’’ representation was introduced to avoid too many crossing lines over the central part of the graph (external blue lines, green lines surrounding the central part). The resulting interaction map provides a unique graphical interface to access the knowledge accumulated about E. coli central carbon metabolism and a valuable illustration of the complexity of its regulations. It has to be noted that, using the same strategy, the same net- work could be represented in a different manner depending on the biological question. For illustration, it could be organised around pleiotropic transcriptional regulators if the question was mainly related to global regulation. PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 Multi-Omic Data Miner (MODAM): a Cytoscape plugin to facilitate multilayer data interpretation Cytoscape offers the possibility to represent different entities through dedicated symbols and to apply a colour scale according to numerical values. The latter functionality is useful for the visualization of one particular type of omic data but is limited for multi-omics data representation. In particular, a graphical pro- perty (i.e. colour) can be associated to only one kind of attribute (e.g. transcriptomic values). To apply the colour scale to all omics data, all biological components must be declared with the same and unique attribute. Such generic design does not allow pro- cessing separately the different types of omics data to account for differences in data format (e.g. ratios, absolute values, etc) or differences in the amplitudes of changes between, which can be highly different from one type of data to another. The nature of changes can be different too. For instance, the flux through a PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 June 2011 \| Volume 6 \| Issue 6 \| e21318 PLoS ONE \| www.plosone.org 4 Visualization of Multi-Omics Data Figure 3. Utilization as a web-platform for biological information. Cytoscape allows linking any component of the network (node or edge) to a webpage. This functionality was used to link all the nodes and edges of the graph to relevant information in the databases from which the biological network was generated. For example, one can select the fumarate reductase activity (highlighted in green in the cytoscape main visualization window) and click on the associated link (‘‘Source’’) to access the corresponding information page in Ecocyc (bottom window). doi:10.1371/journal.pone.0021318.g003 PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 To do so, MODAM includes extra features like the automatic extraction of three sub-networks highlighting the most significant data. A first sub-network contains all the biological entities that are down regulated (Figure 5B). Since the components extracted by such process are not necessarily directly connected one to each other, the sub-network extraction was extended to include all their direct neighbours (i.e. components they are directly connected to). A and protein expression, metabolite accumulation) or edge width (fluxes) to the fold change. This display mode (refered to as ‘‘impact mode’’) is well-suited to embrace large scale network information. Both modes (‘‘impact mode’’ and ‘‘normal mode’’; figures S3 and S4) could be easily switched using the GUI interface of MODAM. The complete interaction network contains hundreds of com- ponents among which many of them were not monitored by omics data or for which changes were not significant. In the biological interpretation of the results, these parts of the network can be discarded in order to focus on the core of the modifications. To do so, MODAM includes extra features like the automatic extraction of three sub-networks highlighting the most significant data. A first sub-network contains all the biological entities that are down regulated (Figure 5B). Since the components extracted by such process are not necessarily directly connected one to each other, the sub-network extraction was extended to include all their direct neighbours (i.e. components they are directly connected to). A Figure 5. MODAM plug-in interface. The main MODAM interface is the panel below the graph representations (A). For each omics data it is possible to change the coloring threshold expressed in percentage of the significant elements. The user can also select, via the radio buttons, which omics data will be displayed and taken into account for the subnetwork extraction. Note that any modification is directly applied to the view in order to provide an interactive feedback to users. Four representations are available:,the subnetwork of down-regulated elements (B), the sub-network of up-regulated ones (C), the joint up-and down regulated elements subnetwork (D), and the global representation (E). doi:10.1371/journal.pone.0021318.g005 Figure 5. MODAM plug-in interface. The main MODAM interface is the panel below the graph representations (A). For each omics data it is possible to change the coloring threshold expressed in percentage of the significant elements. Figure 3. Utilization as a web-platform for biological information. Cytoscape allows linking any component of the network (node or edge) to a webpage. This functionality was used to link all the nodes and edges of the graph to relevant information in the databases from which the biological network was generated. For example, one can select the fumarate reductase activity (highlighted in green in the cytoscape main visualization window) and click on the associated link (‘‘Source’’) to access the corresponding information page in Ecocyc (bottom window). doi:10.1371/journal.pone.0021318.g003 GUI (figure 5A). A strong benefit of MODAM is to allow multiple independent colour scales to be applied in parallel. The adjustment of the colour scale using independent cursors for each omic set is a straightforward and convenient operation. These cursors allow mapping the data according to their relative distributions and not only according to arbitrary thresholds [16]. Each individual clour scale ranges from green (ratios below one) to yellow (similar values) and red (ratios above one). Negative flux ratios were represented with a dedicated blue colour scale (see example of the PPP fluxes in figures S3 and S4) We also offer through MODAM the possibility to highlight the strongest variations by matching the node size (gene reversible reaction can be orientated in the direction opposite to that of a reference condition, The reverse direction can be expressed as a negative value, and has to be explicitly visualized on the graphical representation using dedicated attributes. The appli- cation of a unique colour scale to all types of data can hamper the visibility or nature of changes associated with some types of omics data compared to other types. Here, we propose MODAM (stands for Multi-Omic Data Miner), a new plugin to overcome this problem and to extend Cytoscape functionalities toward data mining in multi-omics datasets. These functionalities are accessible through a user-friendly June 2011 \| Volume 6 \| Issue 6 \| e21318 5 Visualization of Multi-Omics Data PLoS ONE \| www.plosone.org 6 June 2011 \| Volume 6 \| Issu PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 6 Visualization of Multi-Omics Data Visualization of Multi-Omics Data Figure 4. Detail of the network mapped with multi-omics data. The network was used to visualize a set of multi-omics data – including transcriptomic, proteomic, metabolomic and fluxomic data - from Ishii et al. [9]. The figure shows a detail of the whole network, which is displayed in miniature in the upper right corner with a highlight of the expanded region (using the ‘‘impact mode’’ while the central figure is displayed in ‘‘normal mode’’ ; see text for details). The displayed data correspond to the comparison of E. coli MG1655 K12 grown at a growth rate of 0.7 h21 compared to the same strain grown at 0.2 h21. The color scale tends from green for a greater value of the low growth rate to red for a greater value in the rapidly growing cells, through yellow for equivalent values. Grey shapes are nodes with no associated values. Full size of the figure in ‘‘normal mode’’ is available on figure S1. doi:10.1371/journal.pone.0021318.g004 second network can be obtained by extracting only up-regulated components and their neighbours (Figure 5C). Finally a third network containing both up- and down- regulated elements is proposed (Figure 5D). To illustrate, MODAM was used to extract the main changes between an E. coli strain (Dzwf) deleted for the gene zwf encoding glucose-6-phosphate dehydrogenase (G6PDH) and its isogenic wild type strain [9]. The G6PDH is the first committed step of the PPP and its absence in the Dzwf strain blocks the flux of carbon through the oxidative part of this pathway, resulting in significant metabolic rearrangements. The sub-network of transcriptional factors involved in the metabolic adaptation to zwf knock-out could be automatically extracted from the selection (Figure 5) of the most (up- and down-) regulated components. This sub-network (Figure 6) nicely highlights the role of the transcriptional factors CRP, FNR, ARCA and IHF in the resulting differential expression of central carbon metabolic genes. In particular, a pool of four less expressed genes (sdhB, sucB, sucC, and protein expression, metabolite accumulation) or edge width (fluxes) to the fold change. This display mode (refered to as ‘‘impact mode’’) is well-suited to embrace large scale network information. Both modes (‘‘impact mode’’ and ‘‘normal mode’’; figures S3 and S4) could be easily switched using the GUI interface of MODAM. The complete interaction network contains hundreds of com- ponents among which many of them were not monitored by omics data or for which changes were not significant. In the biological interpretation of the results, these parts of the network can be discarded in order to focus on the core of the modifications. The user can also select, via the radio buttons, which omics data will be displayed and taken into account for the subnetwork extraction. Note that any modification is directly applied to the view in order to provide an interactive feedback to users. Four representations are available:,the subnetwork of down-regulated elements (B), the sub-network of up-regulated ones (C), the joint up-and down regulated elements subnetwork (D), and the global representation (E). doi:10.1371/journal.pone.0021318.g005 PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 7 Visualization of Multi-Omics Data Figure 6. Major transcriptional factors implicated in a Dzwf metabolic reshuffling using MODAM. The ‘‘select experiment’’ option MODAM interface was used to pick the Dzwf data provided by Ishii et al. [9]. Each cursor (gene, protein, reaction and metabolite) were set on 1 highlighted differential data. The ‘‘extract subnetworks’’ option was then activated to select only the differentially expressed nodes and the neighbours (as in 5d). From the ‘‘up and down regulated’’ network, nodes were ranked according to their number of connections (degree). A the nine most connected elements, four are transcription factors. The selection of these transcription factors and their neighbours was reiterated iterative process resulted in the presented subnetwork. The drawing was obtained using a force directed algorithm, manually adjusted to genes that are regulated by the same sets of transcription factors (e.g. the four genes in the centre are regulated by the four transcription fa d i 10 1371/j l 0021318 006 Figure 6. Major transcriptional factors implicated in a Dzwf metabolic reshuffling using MODAM. The ‘‘select experiment’’ option of MODAM interface was used to pick the Dzwf data provided by Ishii et al. [9]. Each cursor (gene, protein, reaction and metabolite) were set on 10 highlighted differential data. The ‘‘extract subnetworks’’ option was then activated to select only the differentially expressed nodes and their neighbours (as in 5d). From the ‘‘up and down regulated’’ network, nodes were ranked according to their number of connections (degree). Am the nine most connected elements, four are transcription factors. The selection of these transcription factors and their neighbours was reiterated. iterative process resulted in the presented subnetwork. The drawing was obtained using a force directed algorithm, manually adjusted to gr genes that are regulated by the same sets of transcription factors (e.g. the four genes in the centre are regulated by the four transcription fact doi:10.1371/journal.pone.0021318.g006 Figure 6. Major transcriptional factors implicated in a Dzwf metabolic reshuffling using MODAM. The ‘‘select experiment’’ option of the MODAM interface was used to pick the Dzwf data provided by Ishii et al. [9]. Each cursor (gene, protein, reaction and metabolite) were set on 10% of highlighted differential data. The ‘‘extract subnetworks’’ option was then activated to select only the differentially expressed nodes and their first neighbours (as in 5d). From the ‘‘up and down regulated’’ network, nodes were ranked according to their number of connections (degree). Among the nine most connected elements, four are transcription factors. The selection of these transcription factors and their neighbours was reiterated. This iterative process resulted in the presented subnetwork. The drawing was obtained using a force directed algorithm, manually adjusted to group genes that are regulated by the same sets of transcription factors (e.g. the four genes in the centre are regulated by the four transcription factors). doi:10.1371/journal.pone.0021318.g006 from activities to fluxes. It was possible to represent the different types of interaction and therefore, to conciliate regulatory and structural networks on the same figure, and to map this network with multi-omics data. The visualization strategy could be divided in three steps: (i) network delineation and data gathering; (ii) com- pilation, justification and organization, using Cytoscape; (iii) data mapping and interpretation using the custom-made plugin MODAM. The graphical representation of complex biological networks and multi-omics data is currently a challenge as many problems are known to be computationally difficult [17,18,19,20]. The main difficulty lies in the design of graphical layouts that contain the complete information but facilitate data visualization and inter- pretation. For intuitiveness, there is likely no generic layout that can be considered since both network limits and representation depend on the biological question to be addressed. In this work, the purpose was to illustrate the potential of the visualization strategy by tackling the complexity of E. coli central carbon meta- bolism and its many regulations. Hence, metabolic processes were represented at the center of the figure and usual metabolic conventions were applied to facilitate intuitive reading. For other biological focuses (e.g. a genetic-centric purpose), different network sucD), all implicated in succinate metabolism, is regulated by the four transcription factors. This kind of conclusion could not be directly raised from the initial network representation since the considered components and their interactions are widely distrib- uted over the graph. from activities to fluxes. It was possible to represent the different types of interaction and therefore, to conciliate regulatory and structural networks on the same figure, and to map this network with multi-omics data. The visualization strategy could be divided in three steps: (i) network delineation and data gathering; (ii) com- pilation, justification and organization, using Cytoscape; (iii) data mapping and interpretation using the custom-made plugin MODAM. Last but not least, the E. coli central carbon metabolism network presented in this work has been set up as default on the launch of MODAM, as well as the best Cytoscape parameters and visual properties to display the whole network. This will provide any user with fast access to the knowledge compiled in this work. Users can also create their own networks following the conventions given in the manual of the plugin and, this way, can apply MODAM to their own networks. The graphical representation of complex biological networks and multi-omics data is currently a challenge as many problems are known to be computationally difficult [17,18,19,20]. The main difficulty lies in the design of graphical layouts that contain the complete information but facilitate data visualization and inter- pretation. For intuitiveness, there is likely no generic layout that can be considered since both network limits and representation depend on the biological question to be addressed. In this work, the purpose was to illustrate the potential of the visualization strategy by tackling the complexity of E. coli central carbon meta- bolism and its many regulations. Hence, metabolic processes were represented at the center of the figure and usual metabolic conventions were applied to facilitate intuitive reading. For other biological focuses (e.g. a genetic-centric purpose), different network PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 Supporting Information (EPS) Figure S3 Mapping of multi-omics data (zwf) using the ‘‘Normal mode.’’ Plot of a full set of omics data extracted from Ishii et al (2007), and corresponding to the comparison of the growth of E. coli zwf mutant (encoding the first reaction of the pentose phosphate pathway) versus its isogenic wild type control. Data are displayed as ratios relative to the wild type. The color scale tends from green for a greater value at the low growth rate to red for a greater value in the rapid growing cells, through yellow for equivalent values. Negative fluxes are represented by a blue scale (deeper blue for stronger negative values). Grey shapes are nodes with no associated values. (EPS) The main limitation was the representation and exploration of multi-omic datasets, which was overcome by developing the MODAM plugin. As shown from the examples provided in this work, MODAM greatly facilitates data mapping (intuitive graphical user interface and two display modes), allows multi- omic data representation (application of independent scales) and improves interpretation (automatic extraction of most significant information). MODAM could highlight the transcriptional factors involved in the metabolic reorganizations caused by the zwf deletion, which is of special interest for further comprehensive understanding of metabolic robustness. It allowed also the detection of discrepancies between transcriptomics and proteomics data in E. coli growing at different growth rates, rising the need for further investigations to determine whether this was due to technical issues or to the occurrence of post-transcriptional controls. Figure S4 Mapping of multi-omics data (zwf) using the ‘‘Impact mode.’’ Plot of a full set of omics data extracted from Ishii et al (2007), and corresponding to the comparison of the growth of E. coli zwf mutant (encoding the first reaction of the pentose phosphate pathway) versus its isogenic wild type control. Data are displayed as ratios relative to the wild type. The color scale tends from green for a greater value at the low growth rate to red for a greater value in the rapid growing cells, through yellow for equivalent values. Negative fluxes are represented by a blue scale (deeper blue for stronger negative values). Grey shapes are nodes with no associated values. Sizes of the gene, protein and metabolite nodes are adjusted depending on the fold change. (EPS) The application to the central carbon metabolism of E. Discussion This work presents a simple, handy and inexpensive strategy to facilitate the visualization and analysis of complex multi-omics datasets (companion web-page at https://sites.google.com/site/ modamplugin/). The central concept is to decompose the struc- tural and functional components from gene expression to translation, June 2011 \| Volume 6 \| Issue 6 \| e21318 8 Visualization of Multi-Omics Data languages [21;22], this initiative can be amplified and extended from a sub-network scale to a whole-cell scale. layouts and representation conventions could be adopted for the same biomolecular network. Whatever the focus, a generic layout issue is the density of nodes and interactions to represent, which increases with the network size. The first rule is to minimize the distance between interacting nodes. However, some nodes – e.g. pleiotropic regulators in the E. coli network - have multiple inter- actions that spread over multiple sub-parts of the network. For such highly connected nodes, the ‘proximity rule’ was by-passed by creating ‘‘passageways’’ circling the zone of interaction. This passageway solution was very efficient to clarify the representation without loss of information or in data visualization capability. In addition to the visualization aspects, the formalism introduced here will be of interest to translate classical drawing conventions into computation. The graph structure facilitates the application of automatic mining methods (e.g. automatic search of highly con- nected genes that can correspond to important regulators) and can be exported to other bioinformatics software. layouts and representation conventions could be adopted for the same biomolecular network. Whatever the focus, a generic layout issue is the density of nodes and interactions to represent, which increases with the network size. The first rule is to minimize the distance between interacting nodes. However, some nodes – e.g. pleiotropic regulators in the E. coli network - have multiple inter- actions that spread over multiple sub-parts of the network. For such highly connected nodes, the ‘proximity rule’ was by-passed by creating ‘‘passageways’’ circling the zone of interaction. This passageway solution was very efficient to clarify the representation without loss of information or in data visualization capability. In addition to the visualization aspects, the formalism introduced here will be of interest to translate classical drawing conventions into computation. The graph structure facilitates the application of automatic mining methods (e.g. automatic search of highly con- nected genes that can correspond to important regulators) and can be exported to other bioinformatics software. Acknowledgments The continuous support from the Re´gion Midi-Pyre´ne´es, from the Ministe`re de l’Education Nationale et de la Recherche, from the European Regional Development Fund, from the Institut National de la Recherche Agronomique, and from the SICOVAL is gratefully acknowledged. We thank S. Sokol for fruitful comments. 1. Noble D (2006) The Music of Life: Biology beyond the genome. Oxford University Press. ISBN 978-0199295739. 21 p. 2. Frankel F, Reid R (2008) Big data: Distilling meaning from data. Nature 455: 30. Supporting Information coli proved to be a valuable illustration of the potential of the proposed visualization strategy, of its value for data assessment and mining, and for the formulation of new working hypothesis. The com- pilation work performed here can be freely exploited and trans- posed to different format/applications. In spite of the biological system complexity, the strategy does not require strong bioinfor- matics background and is accessible and user-friendly to users interested in omics data visualization and compilation of biological knowledge. Finally, the richness of the information displayed on a single figure as presented in this work is a first achievement and demonstrates the possibility of the approach. Taken in conjunction with current similar efforts, like the standardization of visual 3. Gehlenborg N, O’Donoghue SI, Baliga NS, Goesmann A, Hibbs MA, et al. (2010) Visualization of omics data for systems biology. Nat Methods 7: S56–68. 4. Le Fe`vre F, Smidtas S, Scha¨chter V (2007) Cyclone: java-based querying and computing with Pathway/Genome databases. Bioinformatics 23: 1299–300. Author Contributions Conceived and designed the experiments: BE J-CP. Performed the experiments: BE. Analyzed the data: BE. Contributed reagents/materi- als/analysis tools: FJ. Wrote the paper: BE FJ J-CP. Conceived and designed the experiments: BE J-CP. Performed the experiments: BE. Analyzed the data: BE. Contributed reagents/materi- als/analysis tools: FJ. Wrote the paper: BE FJ J-CP. Supporting Information Figure S1 Mapping of multi-omics data (fast growth versus slow growth) using the ‘‘normal mode’’. Plot of a full set of omics data extracted from Ishii et al. (2007), and corresponding to the comparison of the growth of E. coli at two growth rates (0.7 and 0.2 h21). Data are displayed as ratios relative to the wild type. The color scale tends from green for a greater value at the low growth rate to red for a greater value in the rapid growing cells, through yellow for equivalent values. Grey shapes are nodes with no associated values. (EPS) Figure S2 Automatic layout of the network using cytoscape. Manual layout (a), cytoscape circular layout (b), hierarchical layout (c), sugiyama layout (d), spring embedded (e) and organic (f). (EPS) Cytoscape proved to be a highly versatile and flexible graphical platform, and met expectations as regard to the diversity of bio- logical entities and interactions to represent. This versatility may be useful to users willing to extend the formalism introduced in this work. In terms of time, it took a couple of months for a single person to establish the E. coli network presented here. The exten- sion to a different organism (the bacterium Clostridium acetobutylicum) required only two weeks (data not shown). For such less studied organisms, the graphical formalism could be adapted (using specific colors, size, etc) to account for uncertain information or to include data from close organisms. The proposed strategy is not limited to metabolism but can be applied to other cellular pro- cesses, e.g. signaling pathways, cellular cycle, stress responses, etc. Figure S3 Mapping of multi-omics data (zwf) using the ‘‘Normal mode.’’ Plot of a full set of omics data extracted from Ishii et al (2007), and corresponding to the comparison of the growth of E. coli zwf mutant (encoding the first reaction of the pentose phosphate pathway) versus its isogenic wild type control. Data are displayed as ratios relative to the wild type. The color scale tends from green for a greater value at the low growth rate to red for a greater value in the rapid growing cells, through yellow for equivalent values. Negative fluxes are represented by a blue scale (deeper blue for stronger negative values). Grey shapes are nodes with no associated values. References PLoS ONE \| www.plosone.org June 2011 \| Volume 6 \| Issue 6 \| e21318 9 Visualization of Multi-Omics Data Visualization of Multi-Omics Data 5. 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(2007) Multiple high- throughput analyses monitor the response of E. coli to perturbations. Science 316: 593–7. 19. Gehlenborg N, O’Donoghue SI, Baliga N, Goesmann A, Hibbs MA, et al. (2010) Visualization of omics data for systems biology. Nat Methods 7: S56–S68. 10. Aoki KF, Kanehisa M (2005) Using the KEGG database resource. Curr Protoc Bioinformatics 1: 1–12. 20. Liebers A (2001) Planarizing Graphs – A Survey and Annotated Bibliography. Journal of Graph Algorithms and Applications 5: 1–74. 11. Keseler IM, Collado-Vides J, Gama-Castro S, Ingraham J, Paley S, et al. (2005) EcoCyc: a comprehensive database resource for Escherichia coli. Nucleic Acids Res 33: 334–7. 11. Visualization of Multi-Omics Data Keseler IM, Collado-Vides J, Gama-Castro S, Ingraham J, Paley S, et al. (2005) EcoCyc: a comprehensive database resource for Escherichia coli. Nucleic Acids 21. Le Nove`re N, Hucka M, Mi H, Moodie S, Schreiber F, et al. (2009) The Systems Biology Graphical Notation. 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W2991485289.txt	null	en		Tuning Superhydrophobic Materials with Negative Surface Energy Domains	Research	2,019	cc-by	4,583	AAAS Research Volume 2019, Article ID 1391804, 7 pages https://doi.org/10.34133/2019/1391804 Research Article Tuning Superhydrophobic Materials with Negative Surface Energy Domains Zhongzhen Wu,1 Liangliang Liu,2 Shunning Li,1 Shunping Ji,1 Pinghu Chen ,1 Suihan Cui,1 Zhengyong Ma,1 Yuchang Weng,1 Qian Huang,1 Zhongcan Wu,1 Hao Wu,1 Yuan Lin ,1 Ricky K. Y. Fu,2 Hai Lin,1 Xiubo Tian,1 Paul K. Chu ,2 and Feng Pan 1 1 2 School of Advanced Materials, Peking University Shenzhen Graduate School, Shenzhen 518055, China Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China Correspondence should be addressed to Feng Pan; panfeng@pkusz.edu.cn Received 26 September 2019; Accepted 7 November 2019; Published 30 November 2019 Copyright © 2019 Zhongzhen Wu et al. Exclusive Licensee Science and Technology Review Publishing House. Distributed under a Creative Commons Attribution License (CC BY 4.0). Hydrophobic/superhydrophobic materials with intrinsic water repellence are highly desirable in engineering ﬁelds including antiicing in aerocrafts, antidrag and anticorrosion in ships, and antifog and self-cleaning in optical lenses, screen, mirrors, and windows. However, superhydrophobic material should have small surface energy (SE) and a micro/nanosurface structure which can reduce solid-liquid contact signiﬁcantly. The low SE is generally found in organic materials with inferior mechanical properties that is undesirable in engineering. Intriguingly, previous theoretical calculations have predicted a negative SE for θ-alumina (θ-Al2O3), which inspires us to use it as a superhydrophobic material. Here, we report the experimental evidence of the small/negative SE of θ-Al2O3 and a θ-Al2O3-based superhydrophobic coating prepared by one-step scalable plasma arcing oxidation. The superhydrophobic coating has complete ceramic and desired micro/nanostructure and therefore exhibits excellent aging resistance, wear resistance, corrosion resistance, high-temperature tolerance, and burning resistance. Owing to the rarity of the small/negative SE in inorganic materials, the concept to reduce SE by θ-Al2O3 may foster a blowout to develop robust superhydrophobicity by complete inorganic materials. 1. Introduction From the view of thermodynamics, the SE of solid materials in a single-component cannot be negative; otherwise, they will sublimate. However, in a multicomponent system, the SE depends strongly on the chemical environment as follows: γ= 1 Esurf −〠N i μi , A ð1Þ where Esurf is the total energy of the system for the surfaces; A is the surface area; N i and μi are the number and the chemical potential of constituent element i. When no external element exists, the surface energy will correspond to the free energy gain in creating the dangling bonds. If external elements are incorporated into the system, surface reconstruction occurs by for example passivation, adsorption, or hydration, and the newly formed chemical bonds will compensate the energy of the dangling bonds, thus lowering the surface energy to a value that possibly goes below zero after deducting the heat of the adsorbate. Typically, in 2004, Lodziana et al. discovered that the surfaces of θ-Al2O3 have much lower SE after hydration, especially the (10-2) plane which has a negative SE by DFT calculation [1]. The negative surface energy of θ-Al2O3 is induced by the dissociation of water molecules over the (10-2) surfaces, as shown in Figure 1(a). When a water molecule is placed on stoichiometric θ-Al2O3 (10-2) surface, with its O atom at a distance allowing the formation of chemical bonds to one of the exposed Al atoms, a dissociation reaction into OH- and H- fragments would be energetically favored. The OH- fragment remains on top of the Al atom while the H- fragment is transferred onto the surface of a nearby O atom. Since no excess charge is introduced in this process, all the Al and O atoms in the surface layer of θ-Al2O3 could be fully covered by OH- and H-, respectively. By this, the difference in chemical environment between the surface and the bulk atoms would be largely eliminated, relieving the internal 2 Research 𝜃-Al2O3 (102) H O H O Al O H O H O Al O Hydroxylated surface (a) Mixed a-Al2O3 and 𝜃-Al2O3 a-Al2O3 500 Voltage (V) 400 Stage I Stage I Stage II Stage III 300 200 100 0 0 Stage II 50 100 150 200 250 300 Time (s) Mixed a-Al2O3 and 𝜃-Al2O3 Electrolyte 𝜃-Al2O3 Coral-like layer Tube-like layer Stage III Etching Compact layer Al substrate (b) Figure 1: (a) Schematic illustration of the hydration process. Water molecules will dissociate into OH- and H- fragments, releasing energy that can compensate the energy penalty caused by the cleavage of Al-O bonds. The overall surface energy can become negative owing to this compensation eﬀect. (b) Electrochemical mechanism of the coating growth. Three stages are shown: (I) Al dissolution and alumina formation, (II) Al2O3 erosion, and (III) formation of the coral-like layer. stress built up during the creation of these surfaces. The saturation of dangling bonds with hydroxyl species will compensate the energy consumption for the cleavage of Al-O bonds, leading to substantially low or even negative surface energy. The high stability of the hydroxylated surfaces would eventually shape the preference for a high-surface-area micro/nanostructures as the thermodynamic ground state, which guarantees their resistance to structural collapse. The small or negative SE of the hydroxylated θ-Al2O3 [1] suggests a new modiﬁcation layer to obtain superhydrophobicity, which is inorganic and has good mechanical properties. However, θ-Al2O3 is a metastable polymorph obtained in the heating process to synthesize α-Al2O3 and it cannot preserve to room temperature unless it exists as nanograins and coexists with other alumina phase. Although previous work reveals the hydrophobicity of Al2O3 by plasma electrolytic oxidation (PEO) [2], it is far from the regular structure and θ-Al2O3. Here, we employ the arcing discharge plasma chemistry in PEO and surface structure control in anodic oxidation [3] to develop a one-step and scalable method to prepare a multiphase (θ, amorphous, etc.) intergrowth composite Al2O3 coating with the coral-like micro/nanostructure which has a surface layer composed of nanosize θ-Al2O3 grains and micro/nanopores. The observation of superhydrophobicity is reported on this coating surface, which oﬀers excellent performance under harsh conditions including high temperature, open ﬂame, corrosion, wearing, and aging. A high temperature of about 2,000°C produced by instantaneous arcing in PEO can provide enough energy to form θAl2O3, while the shock cooling in the water-based electrolyte cools θ-Al2O3 to room temperature and creates conditions for hydration [4]. Inspired by the controllable micro/nanostructures by pH in anodic oxidation [5], a weakly acidic electrolyte is adopted to produce the nanoporous structure. At the same time, NaF is introduced to the electrolyte to produce HF slowly as the microarc discharge energy is increased in the plasma discharge. HF etches the surface of the multiphase alumina coating [6] producing a thin surface layer with mainly θ-Al2O3. Research 2. Method 2.1. Sample Preparation. The commercial LY12 Al alloy was cut into rectangular pieces with dimensions of 50 mm × 25 mm × 1 mm. The samples were degreased in acetone and dried in warm air. A 20 kW pulsed bipolar power supply and constant current mode were used in the PEO experiment, and the electrolyte was composed of sodium hexametaphosphate ((NaPO3)6, 20 g/L), ethylenediamine tetraacetic acid disodium salt (Na2EDTA, 5 g/L), sodium ﬂuoride (NaF, 3 g/L), and ammonium iron(III) oxalate hydrate (C6H12FeN3O12·3H2O, 4.5 g/L). The experiments were performed at a constant current density (6 A dm-2) at 200 Hz for 5 min with a duty cycle of 20% using a 20 kW AC power supply. The temperature of the electrolyte was controlled to be below 40°C using an external water cooling system. After PEO, the samples were washed with distilled water and vacuum-dried at 120°C for 24 hours before the contact angle measurements. 2.2. Sample Characterization. A ﬁeld emission scanning electron microscope (FESEM, Carl Zeiss, SUPRA® 55) was employed to characterize the surface and cross-sectional morphologies of the PEO coatings and the elemental distribution and composition were determined by energydispersive X-ray spectroscopy (EDS). The phase composition was determined by X-ray diﬀraction (XRD, Bruker, D8 Advance) with a Cu target (λ = 0:15418 nm) and the data were collected at 2θ = 10° ~80° at a scanning rate of 2θ = 2° / min. X-ray photoelectron spectroscopy (XPS, Thermo Fisher, ESCALAB 250X) was utilized to determine the composition of the coatings, and the spectra were referenced to the C1s peak (284.8 eV). The atomic structure of the coating was examined by high-resolution ﬁeld emission transmission electron microscope (TEM: JEM-3200FS, Japan). 2.3. Performance Measurements 2.3.1. Contact Angle Measurements. The water CAs were measured on a commercial contact angle meter (PT-705B, Dong Guan Precise Test Equipment Co., Ltd., China). The volume of water droplets was 2 μL, and three diﬀerent spots were measured on each sample to obtain the average. 2.3.2. Aging Testing. The samples were exposed to air for 360 days, and the contact angle was measured every 30 days. The CAs shown here are the average of 5 measurements. 2.3.3. Wear Testing. The friction and wear tests were conducted on a reciprocating machine (MFT-5000, Rtec Instruments, USA) with the nonwoven fabric as the counterpart. The load was 10 N, friction frequency was 0.5 Hz, and wear distance was 15 mm. The CAs and surface morphology were determined after 2,000 cycles at ambient temperature (relative humidity is about 30%). 2.3.4. Corrosion Testing. The polarization curves were acquired on an electrochemical workstation (1470E, Solartron Metrology) between -2.0 and 0 V versus the saturated calomel electrode (SCE) at a scanning rate of 10 mV/s at 25°C in the NaCl (3.5 wt%) solutions. The corrosion potential 3 (Ecorr) and corrosion current density (icorr) were analyzed by the Tafel extrapolation method. The salt spraying test was performed to evaluate the CAs on the coatings and dependence on corrosion time. 2.3.5. Firing Testing. Three types of materials including polyimide (PI), polytetraﬂuoroethylene (PTFE), and the superhydrophobic coating were examined in an alcohol lamp ﬂame to evaluate the ﬂame resistance and the hydrophobicity was evaluated. 3. Results and Discussion The U-I curves of the discharge and schematic of the coating mechanism are shown in Figure 1(b). In the constant current mode, the oxide layer thickens with discharge time and the impedance increases. As a result, the discharge voltage should increase monotonically until the end of the discharge. However, in the weakly acidic electrolyte, the discharge voltage increases quickly at ﬁrst but decreases slowly when the voltage is over 400 V, revealing three stages in the coating process. At the beginning of PEO, alumina grows on the Al alloy initially and a larger discharge intensity and arcing temperature increase the coating thickness. Transmission electron microscopy (TEM) suggests that the multiphase alumina including the θ phase begins to form (Figure S1) [4]. As the coating becomes thicker and the impedance increases, the total arc number decreases, while the intensity of each arc increases [5]. The higher arc energy improves the corrosion of the coating in the weakly acidic electrolyte resulting in decreased discharge voltage. Therefore, both growth and corrosion (etching) take place and the nanotubular structure starts to from in the coating in the second stage. Although the impedance of the coating decreases slightly because of the porous structure, the thickness of the coating still increases with time. The arc energy continues to increase inducing the reaction between NaF and the acidic electrolyte and subsequent production of HF. Owing to the reduced arc number, the amount of HF is small and so only amorphous alumina, not crystalline, is etched [7]. In this way, crystalline alumina (mainly θ-Al2O3) domain which has high corrosion resistance is formed in the coating surface. The pH and the NaF concentrations are optimized and the CAs are shown in Figure S2. Figure 2 and Figure S3 show the materials and structure of the coatings. The coating surface exhibits an irregular coralline-like interconnected skeleton forming a cellular structure with a size of 300 to 400 nm. In each skeleton, small nanoholes with a size of 10-50 nm are present. This multilevel micro/nanostructure (Figure S3(A)) shows the corrosion eﬀects of the weakly acidic electrolyte. The crosssectional images show three typical layers between the surface and substrate with a total thickness of 4-5 μm. The top layer has an irregular coralline-like structure, and the middle layer has a nanotubular structure with a diameter of about 200 nm and 10-50 nm nanoholes. The bottom layer is a dense layer with a thickness of about 300 nm (Figure S3(B)). The coating is mainly composed of Al and O and a trace amount of C which arises from 4 Research A1 B3 B2 B1 Zone I Zone II 1 𝜇m 50 nm 5 nm A2 2 nm (b) C1 1st cycle 298K 1st cycle 673K A3 2nd cycle 298K C2 C4 3rd cycle 298K 3rd cycle 673K 4000 3500 3000 2500 2000 1500 1000 500 C3 (a) 2nd cycle 673K Wavenumber (cm‒1) (c) (d) Figure 2: Morphology and structure of the prepared coating. (a) Surface and the cross-sectional images by SEM showing three typical layers form the surface to substrate: irregular coralline-like layer, nanotubular layer, and dense layer. (b) TEM images of the powder scraped from the coating: a 2-5 nm crystalline layer (mainly θ-Al2O3) is observed on the surface of the coating. (c) High-resolution images of the θ-Al2O3 grains and electron diﬀraction data: θ-Al2O3 (111) and (10-2) nanograins are embedded in the amorphous alumina matrix. (d) IR spectrum at room temperature and 673 K: the hydration/dehydration repetition shows a repeatable small SE and superhydrophobicity. surface adsorption of hydrocarbon compounds from air. No obvious peaks are observed by XRD, thus suggesting an essentially amorphous structure (Figure S4). The results show that an alumina coating with a multilevel micro/nanostructure is formed. TEM performed on the coating materials scraped oﬀ from the sample shows that the coating contains not only amorphous but also crystalline alumina distributed uniformly in the amorphous alumina matrix as nanograins with a size of 2-5 nm. The d-spacings of the alumina grains determined by electron diﬀraction (Figure 2(c)) are 0.255 nm and 0.354 nm [8], corresponding to the θ-Al2O3 (111) and [102], respectively, which are the planes with small or negative SE after hydration [1, 9]. A 2-5 nm crystalline layer is observed from the surface of the alumina particles as shown in Figure 2(b). It is composed of θ-Al2O3 and expected to play a critical role in the surface hydrophobicity. The coating materials are scraped from the substrate and pressed them into a bulk disc for testing (Figure S5). The disk shows a contact angle of about 120° conﬁrming a low SE surface. The surface hydrophobicity of the coatings depends on hydration/dehydration of θ-Al2O3 which plays an important role to produce smaller or negative SE. The dehydration temperature is about 623 K as measured by infrared spectroscopy (IR) spectrum in Figure S6. The hydrophobicity of the hydration/dehydration coating is performed, as shown in Figure 2(d). At the room temperature, the IR spectrum taken from the powder scraped from the coating shows an obvious vibration peak at 3489 cm-1 which is the typical vibration of OH-, suggesting that the θ-Al2O3 is hydroxylated. When the coating is heated to 673 K, IR shows that the vibration peak of OH- disappears indicating dehydration. At the same time, the coating becomes hydrophilic due to increased surface energy from dehydration [10]. However, the dehydrated θ-Al2O3 can regain superhydrophobicity after hydration again. The IR spectrum of the dehydrated coating after hydration for 24 h in air shows the OHpeak again implying recovery of the hydrophobicity. This phenomenon repeats itself with heating thereby providing evidence that hydration plays a critical role in the small or negative SE and superhydrophobicity of θ-Al2O3. The wetting experiment on the coating shows that the static contact angle is 152° and the sliding angle is no more than 3°. The droplet cannot spread but instead bounces as it contacts the coating surface as observed by high-speed photography (Figure 3(a), Figure S7, and Movie S1). In comparison, a hydrophilic surface wets readily and the droplet does not bounce. To assess the robustness of superhydrophobicity, aging, wear, high-temperature, and burning experiments are performed. After ambient exposure for one year, no obvious decrease in the contact angle occurs as shown in Figure 3(b). The high-temperature experiment is conducted by adding a heater to the contact angle instrument (Figure S8 and Movie S2). The sample is placed on the heating stage and heated to 300°C. At this high temperature, the droplet spreads quickly upon contact with the hydrophilic material surface. A cone forms from the needle to the sample surface and a large amount of bubbles are produced until the droplet evaporates. However, when Research 5 4.6 ms 0 ms 2.3 ms 4.0 ms 17.7 ms 25.6 ms 0 ms 1.3 ms (a) (c) 0.01 Current density (A cm‒2) 160 Contact angles (degree) 27.1 ms 5.8 ms 150 140 130 CA: 149° CA: 152° 120 110 1E‒4 Substrate 1E‒6 1E‒8 Hydrophilic 1E‒10 Hydrophobic 1E‒12 100 0 60 120 180 240 Times (d) (b) 300 360 ‒2.0 ‒1.6 ‒1.2 ‒0.8 Potential (V, vs. SCE) (d) ‒0.4 Figure 3: (a) Droplet bouncing test: the droplet cannot spread but instead bounces as it contacts the coating surface, (b) aging test showing the long-time performance of the coating, (c) high-temperature test at 300°C, and (d) corrosion test showing the excellent corrosion resistance. the droplet makes initial contact with the prepared coating surface as shown in Figure 3(c), it bounces immediately and does not stay on the surface suggesting the occurrence Leidenfrost Phenomenon which means that an insulating vapor layer on the coating surface is produced and keeps the liquid from boiling rapidly [11]. The diﬀerent phenomenon between the hydrophilic surface and prepared coating reveals that the superhydrophobicity decreases the temperature threshold of Leidenfrost Phenomenon to <300°C. Compared to the organic materials, these types of inorganic materials have signiﬁcant advantages concerning ﬁre resistance. In order to check if F contained organics during synthesis retain on the coating surface, the burning experiments are performed on the coating and compared with polyimide (PI) and polytetraﬂuoroethylene (PTFE), as shown in Figure S9 and Movie S3. The PI sample ignites after 3 s on the outerﬂame of the alcohol lamp. Although PTFE is an important refractory material, it does not ignite but melts completely after 12 s. In comparison, the coating keeps the excellent superhydrophobicity after 18 s ﬁring. Besides, the burning test makes sure further that no F contained organics retain on the coating surface because of their small ignition temperature. The coating has good wear resistance, and the contact angle remains over 130° after 2,000 cycles of the friction test using nonwoven fabric at a load of 10 N, as shown in Figure S10. The good wear resistance is mainly attributed to the mechanical performance of alumina but the loose and nanoscale coral-like structure can collapse under a erally have good corrosion resistance because of water repellence. The corrosion resistance of the coating is assessed electrochemically and by salt spraying. Generally, it is diﬃcult to obtain electrochemical data because an air ﬁlm forms between the coating and corrosive solution [12]. In order to destroy the air ﬁlm, the coating is immersed into a corrosive solution for 24 h before the electrochemical test as shown in Figure 3(d). The corrosion current decreases signiﬁcantly and is 4 and 2 orders of magnitude smaller than those of the substrate and hydrophilic coatings, respectively, thereby showing excellent corrosion resistance. Salt spraying reveals the similar corrosion resistance, and the contact angle is still above 140° after 250 h and 370 h as shown in Figure S11. It is noted that the contact angle decreases mainly during initial corrosion possibly due to destruction of the air ﬁlm and is nearly constant afterwards. 4. Conclusions Surface adsorption and hydroxylated surface states can decrease the surface energy of inorganic materials, as demonstrated by previous studies on rare-earth oxides [13, 14]. On the smooth surface, the contact angle of water droplets can be larger than 90° exhibiting hydrophobicity [10]. According to Young’s theory, the contact angle has a theoretical limit and no larger than 119°on a smooth surface morphology (lowest SE = 6:7 mJ/m2 ). Therefore, the low SE materials (generally organic compounds containing F and Si) with contact angles between 90° and 119° on a smooth surface morphology can be categorized as ﬁrst-generation hydrophobic materials as shown in Figure 4. To develop superhydrophobic materials (CA > 150° ) with self-cleaning eﬀects, periodic micro/nanostructures inspired by natural materials such as lotus leaves have been proposed [15–17]. This kind of superhydrophobic surface with the alternating composition of low SE materials and air, which can be considered to have zero SE, is classiﬁed as second-generation hydrophobic materials (G2). However, organic materials tend to have inferior mechanical properties and aging resistance, thus plaguing wider engineering applications [18, 19]. Only a few kinds of inorganic materials, such as ZnO and TiO2, are reported to have superhydrophobicity 6 Research G1 G2 Domains with negative surface energy G3 Domains with positive surface energy Figure 4: Schematic showing the three types of hydrophobic materials: G1: Si and F containing organic materials have a smooth surface and low surface energy and the contact angle is between 90° and 119°; G2: periodic micro/nanostructures with alternate Si, and F containing organic materials and air form a super hydrophobic surface and the contact angle is above 150°; G3: periodic micro/nanostructure containing alternating air and inorganic materials with the average surface energy reduced by negative surface energy materials provide superhydrophobicity along with high mechanical strength. after interface reconstruction by hydroxyl group adsorption. Until now, no synthetic surface reported can possess both high mechanical strength and superhydrophobicity, except the multiphase Al2O3 composed of θ-Al2O3 fabricated in this work. Tuning the multiphase Al2O3, which decrease the SE of a strong material by another strong material with small or negative SE, together with the alternating bulk materials and air, prompts us to term this the third-generation hydrophobic material (G3). It is anticipated that this type of materials tuned with negative SE domains can be explored to serve as superhydrophobic coatings capable of meeting engineering needs under harsh conditions in areas like aeronautics, astronautics, ships, and missiles. Conflicts of Interest The authors declared no competing interests. Authors’ Contributions Z.W., L.L., and S.J. proposed the research; S.J., Y.W., P.C., and Z.M. designed the coating depositions; Q.H., ZC.W., and H.W. did the coating characterizations and properties testing; Z.W., S.L., S.C., Y.L., H.L., and F.P. analyzed and discussed the data; Z.W., L.L., S.L., S.J., P.C., and R.F. wrote the paper; X.T. and P.C. polished the English writing; all authors provided feedback. Acknowledgments This work was ﬁnancially supported by the National Key R&D Program of China (2016YFB0700600), the Guangdong Innovation Team Project (No. 2013N080), the Soft Science Research Project of Guangdong Province (No. 2017B030301013), the Shenzhen Science and Technology Research Grant (ZDSYS201707281026184 and JCYJ20170306165240649), and the Hong Kong Innovation and Technology Fund (ITF) ITS/452/17FP (CityU 9440179). We are very appreciative for the advices of Prof. Lei Jiang in the paper writing. Supplementary Materials Supplementary 1. Figure S1: transmission electron microscopy images and electron diﬀraction patterns of the powder stripped from the coating after the second stage of coating growth. Figure S2: contact angles dependence on pH and NaF concentration in the electrolyte. Figure S3: surface and cross-sectional morphologies of the superhydrophobic coating: A~C present irregular coral-like skeletons on the coating surface at diﬀerent magniﬁcations; D~F show cross-sectional images of the coating at diﬀerent magniﬁcations. Figure S4: (A1) XRD, (A2) SEM and EDS, and (B1-B4) XPS spectra of the superhydrophobic coating. Figure S5: hydrophobic characteristics of the disk made of powder scraped from the Research coating surface. Figure S6: the IR spectrums of samples after heating at diﬀerent temperatures. Figure S7: bouncing test: (A) superhydrophobic coating and (B) hydrophilic coating. Figure S8: high-temperature test at 300°C: (A) heating apparatus; (B) superhydrophobic coating; (C) hydrophilic coating. 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https://openalex.org/W2744436061	https://www.biorxiv.org/content/biorxiv/early/2017/08/11/173336.full.pdf	English	null	SMRT sequencing reveals differential patterns of methylation in two O111:H- Shiga toxigenic<i>Escherichia coli</i>isolates from a historic hemolytic uremic syndrome outbreak in Australia	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	18,768	. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint SMRT sequencing reveals differential patterns of 1 methylation in two O111:H- Shiga toxigenic Escherichia 2 coli isolates from a historic hemolytic uremic syndrome 3 outbreak in Australia 4 SMRT sequencing reveals differential patterns of 1 methylation in two O111:H- Shiga toxigenic Escherichia 2 coli isolates from a historic hemolytic uremic syndrome 3 outbreak in Australia 4 Brian M. Forde1,2,4, Lauren J. McAllister3, James C. Paton3, Adrienne W. Paton3, Scott A. 5 Beatson1,2,4* 6 1 Australian Infectious Diseases Centre, The University of Queensland, Brisbane, Australia 7 2The School of Chemistry and Molecular Biosciences, The University of Queensland, 8 Brisbane, Australia. 3Research Centre for Infectious Diseases, Department of Molecular and 9 Cellular Biology, University of Adelaide, Adelaide, SA 5005, Australia. 4Australian Centre 10 of Ecogenomics, The School of Chemistry and Molecular Biosciences, The University of 11 Queensland, Australia. 12 1 Australian Infectious Diseases Centre, The University of Queensland, Brisbane, Australia 7 2The School of Chemistry and Molecular Biosciences, The University of Queensland, 8 Brisbane, Australia. 3Research Centre for Infectious Diseases, Department of Molecular and 9 Cellular Biology, University of Adelaide, Adelaide, SA 5005, Australia. 4Australian Centre 10 of Ecogenomics, The School of Chemistry and Molecular Biosciences, The University of 11 Queensland, Australia. 12 * Scott A. Beatson, School of Chemistry and Molecular Biosciences, The University of 17 Queensland, Brisbane 4072, QLD, Australia; Telephone +61-7-33654863; email 18 s.beatson@uq.edu.au 19 20 1 1 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Abstract 21 Shiga toxigenic Escherichia coli (STEC) are important food-borne pathogens and a major 22 cause of haemorrhagic colitis and haemolytic-uremic syndrome (HUS) worldwide. In 1995 a 23 severe HUS outbreak in Adelaide occurred. A recent genomic analysis of STEC O111:H- 24 strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, 25 harboured two additional copies of the Shiga toxin 2 (Stx2) genes although the structure of 26 the Stx2-converting prophages could not be fully resolved due to the fragmented assembly. In 27 this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) long 28 read sequencing to characterise the complete epigenome (genome and methylome) of 95JB1 29 and 95NR1. Using long reads we completely resolved the structure of two, tandemly inserted, 30 stx2-converting phage in 95NR1. Our analysis of the methylome of 95NR1 and 95JB1 31 identified hemi-methylation of a novel motif (5’-CTGCm6AG-3’) in more than 4000 sites in 32 the 95NR1 genome. These sites were entirely unmethalyted in the 95JB1, including at least 33 180 potential promoter regions that could explain regulatory differences between the strains. 34 We identified a Type IIG methyltransferase encoded in both genomes in association with 35 three additional genes in an operon-like arrangement. IS1203 mediated disruption of this 36 operon in 95JB1 is the likely cause of the observed differential patterns of methylation 37 between 95NR1 and 95JB1. This study demonstrates the enormous potential of PacBio 38 SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic 39 heterogeneity within a clonal population of bacteria. 40 Shiga toxigenic Escherichia coli (STEC) are important food-borne pathogens and a major 22 cause of haemorrhagic colitis and haemolytic-uremic syndrome (HUS) worldwide. In 1995 a 23 severe HUS outbreak in Adelaide occurred. A recent genomic analysis of STEC O111:H- 24 strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, 25 harboured two additional copies of the Shiga toxin 2 (Stx2) genes although the structure of 26 the Stx2-converting prophages could not be fully resolved due to the fragmented assembly. In 27 this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) long 28 read sequencing to characterise the complete epigenome (genome and methylome) of 95JB1 29 and 95NR1. Using long reads we completely resolved the structure of two, tandemly inserted, 30 stx2-converting phage in 95NR1. Abstract 21 Our analysis of the methylome of 95NR1 and 95JB1 31 identified hemi-methylation of a novel motif (5’-CTGCm6AG-3’) in more than 4000 sites in 32 the 95NR1 genome. These sites were entirely unmethalyted in the 95JB1, including at least 33 180 potential promoter regions that could explain regulatory differences between the strains. 34 We identified a Type IIG methyltransferase encoded in both genomes in association with 35 three additional genes in an operon-like arrangement. IS1203 mediated disruption of this 36 operon in 95JB1 is the likely cause of the observed differential patterns of methylation 37 between 95NR1 and 95JB1. This study demonstrates the enormous potential of PacBio 38 SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic 39 heterogeneity within a clonal population of bacteria. 40 41 2 2 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Introduction 42 In 1995 a large outbreak of hemolytic uremic syndrome (HUS) occurred in Adelaide, South 43 Australia. This outbreak was attributed to uncooked, fermented, dry sausage contaminated 44 with Shiga toxigenic Escherichia coli (STEC) O111:H- (Paton et al. 1996). A total of 23 45 cases were confirmed, all in children aged 6 months - 14 years. As a result of infection 46 sixteen of the 23 children required dialysis and one 4 year old child died. STEC isolates from 47 the outbreak were found to be highly virulent with an infectious dose requiring as little as 1 48 organism per 10g of sausage. Interestingly, strains isolated after January 25th 1995 appeared 49 more virulent and, based on Southern blot analysis, were predicted to encode a second copy 50 of stx2AB (Centers for Disease and Prevention 1995; Paton et al. 1996). 51 Recently, the 1995 Adelaide outbreak was re-examined using genome sequencing 52 (McAllister et al. 2016). Two strains, 95JB1 and 95NR1 isolated pre and post January 25th 53 1995, respectively, were sequenced on the Illumina GAII platform and assembled de novo. 54 Comparison of the draft genomes of 95JB1 and 95NR1 identified a Stx1-converting prophage 55 and Stx2-converting phage shared by both strains as well as ~50 kb of phage-associated 56 sequence that was present in 95NR1 but absent in 95JB1. Based on read coverage and long- 57 range PCR it was inferred that there were two additional Stx2 prophage in 95NR1 when 58 compared to 95JB1 (McAllister et al. 2016). All single nucleotide polymorphisms (SNPs) 59 differentiating 95JB1 from 95NR1 had occurred in 95JB1 after divergence suggesting that the 60 two additional Stx2 prophages were likely deleted from 95JB1 rather than acquired by 61 95NR1 (McAllister et al. 2016). However, due to the inability of short read sequencing 62 technologies to accurately resolve repetitive loci, the structure and genomic context of these 63 prophages could not be unambiguously resolved. 64 3 3 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Introduction 42 It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint MTase are able to catalyse both restriction and methylation (Roberts et al. 2003). RM 90 systems are ubiquitous in bacteria and highly diverse, but until recently a small number have 91 been characterised. The primary role of RM systems is to protect the host genome from 92 invasion by foreign DNA, such as transposons, phage and plasmids (Takahashi et al. 2002). 93 MTase-genes can also occur independently of RM systems. These ‘orphan’ MTase are 94 common in bacterial genomes and can be involved in genome regulation (Blow et al. 2016). 95 The most well characterised orphan MTase is Dam (Casadesus and Low 2006). Dam is an 96 established regulator of gene expression in E. coli and also has a role in pathogenicity (Boye 97 and Lobner-Olesen 1990; Low et al. 2001; Casadesus and Low 2006). Less well studied 98 MTases that are components of RM systems may also have roles beyond genome defence. 99 For example, Type III (Res, Mod) RM systems are known to be involved in phase variation 100 in a number of bacterial species (de Vries et al. 2002; Fox et al. 2007; Seib et al. 2015) and 101 phase variation of Type IIG RM systems has been shown to alter gene expression in 102 Campylobacter jejuni (Anjum et al. 2016). 103 To investigate the differences between two STEC O111:H- outbreak strains 95JB1 and 104 95NR1 we determined their complete genome assemblies and methylomes using PacBio 105 SMRT sequencing. We completely resolve the genetic structure of all prophage-encoding 106 regions, including two distinct, but closely related, Stx2-converting prophage in 95NR1 that 107 are tandemly inserted in the same genomic location and appear to have been deleted from 108 95JB1. We identify all putative MTases in both strains, define their target sequences and 109 show the activity of a previously uncharacterised methyltransferase in 95NR1 that is not 110 active in 95JB1. Introduction 42 It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Due to the complexity of repeat sequences such as insertion sequences (IS), bacterial genome 65 assemblies generated from short sequencing reads are usually highly fragmented (Koren et al. 66 2013). Resolving these fragmented assemblies requires time consuming and costly finishing 67 procedures which are beyond the scope of most research projects (MacLean et al. 2009; 68 Nagarajan et al. 2010). Consequently, these genomes are often submitted as unfinished drafts 69 with many containing misassemblies and platform-specific sequencing errors (Phillippy et al. 70 2008; Chain et al. 2009). Fragmented assemblies are unsuitable for studying structural 71 variation and important information relating to the copy number and context of IS elements 72 and phage can be difficult to discern. The Pacific Biosciences (PacBio) Single Molecule Real 73 Time (SMRT) sequencing platform is able to completely resolve most bacterial genomes by 74 producing reads of sufficient length to span complex repeat loci and generate complete 75 assemblies without the need for costly manual finishing (Eid et al. 2009) (Chin et al. 2013; 76 Koren et al. 2013; Brown et al. 2014; Forde et al. 2014). 77 A remarkable feature of SMRT sequencing is the capacity to determine the methylation status 78 of every sequenced nucleotide (Clark et al. 2012). DNA methylation is the most common 79 post replicative modification in bacteria (Roberts et al. 2003) and is known to influence a 80 wide variety of host processes, including DNA replication, repair and transcriptional 81 regulation (Blow et al. 2016). Until recently the lack of a simple, efficient method to 82 determine the methylation status of DNA has resulted in these modifications being largely 83 ignored in the bacteria. However, by monitoring the actions of single DNA polymerase 84 enzymes in real-time SMRT sequencing can simultaneously determine nucleotide sequence 85 and the methylations status of each nucleotide. 86 4 Methylation is mediated in bacteria by methyltransferase (MTase) enzymes. MTase are found 87 as components of restriction-modifications (RM) systems, which in their simplest form are 88 generally composed of a MTase and a restriction enzyme (REase), although some classes of 89 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Introduction 42 Furthermore, we unambiguously determine the copy number and context of 111 all IS elements in the genomes of 95NR1 and 95JB1 and reveal that a single difference in the 112 IS complement could be directly responsible for differences in their methylome profiles. 113 114 5 5 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Complete genome assembly reveals full sequence of tandemly arrayed Stx2 116 prophages in 95NR1. 117 ; https://doi.org/10.1101/173336 doi: bioRxiv preprint including the Stx2 and Stx1 converting prophage (Phi10 and Phi11, respectively) carried by 138 both outbreak strains, (Table S1) were readily resolved in the complete PacBio assemblies. 139 Two plasmids, similar to the P1 and EHEC plasmids from E. coli 11128 (Ogura et al. 2009), 140 were completely assembled in the PacBio assemblies of 95JB1 and 95NR1 141 (p95NR1A/p95JB1A and p95NR1B/p95JB1B, respectively). In contrast to the draft Illumina 142 assemblies of 95JB1 and 95NR1 (McAllister et al. 2016), the small pO111_4 and pO111_5 143 colicin plasmids were not detected in the PacBio assemblies or in the raw PacBio read data 144 consistent with their exclusion during the library preparation process. 145 Complete genome assembly reveals full sequence of tandemly arrayed Stx2 116 prophages in 95NR1. 117 Complete genome assembly reveals full sequence of tandemly arrayed Stx2 116 prophages in 95NR1. 117 De novo assembly of E. coli O111:H- strains 95JB1 and 95NR1 generated single circular 118 chromosomal contigs of 5,347,879 bp and 5,467,946 bp, respectively. Previously reported 119 short read assemblies of 95NR1 (accession: AVDU00000000.1) and 95JB1 (accession: 120 AWFJ00000000.1) contained 182 and 179 contigs, respectively, with contig N50 sizes of less 121 than 100 kb (McAllister et al. 2016) (Table 1). A SNP comparison of the complete genomes 122 of 95JB1 and 95NR1 is in agreement with previous observations using Illumina data 123 (McAllister et al. 2016). Using E. coli O111:H- strain 11128 as a reference, McAllister et al 124 identified six SNPs, 5 on the chromosome and one plasmid encoded SNP, which discriminate 125 95JB1 from 95NR1 (McAllister et al. 2016). Our SNP comparison of the complete PacBio 126 assemblies of 95JB1 and 95NR1 identified four of the five chromosomally located 95JB1 127 SNPs and the single plasmid encoded SNP (Table 2). The remaining chromosomal SNP was 128 located in a phage tail protein and due to the repetitiveness of these proteins it was not 129 considered reliable for strain discrimination. 130 The ~120 kb difference in chromosome size between 95JB1 and 95NR1 was largely 131 attributed to two tandemly inserted Stx2-converting prophages in 95NR1 (Phi14 and Phi15) 132 that are absent from 95JB1 (Figure 1a). Phi14 and Phi15 from 95NR1 are highly conserved 133 and share 99% nucleotide sequence identity across 79% of their genomic sequence with 134 regions of difference largely confined to the 5’ end of the prophage sequences (Figure 1b). 135 This pattern of sequence identity explains why assembly of this region was not possible using 136 short read data alone. Indeed, the structure and context of all phage-encoding regions, 137 6 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. Insertion Sequence profiles differ between outbreak strains. 147 163 (EC95JB1_03895), suggesting a possible functional role due to a polar effect on 162 transcription. 163 Three additional MTases are encoded on the additional 95NR1 Stx2 prophages. 165 Bioinformatic characterisation of 95JB1 revealed that the strain encodes seven putative 166 MTases in addition to Dam (target site: 5’-Gm6ATC-3’) and Dcm (target site: 5’-Cm5CWGG- 167 3’). Five of these six putative MTases correspond to enzymes with known specificity that 168 have been previously characterised in the O104:H4 serotype E. coli outbreak strain C227-ll 169 (Fang et al. 2012). These include homologs of the C227-11 orphan MTases M.EcoGI, 170 M.EcoGII, M.EcoGVI and M.EcoGIX (M.Eco95JB1I, M.Eco95JB1II, M.Eco95JB1V and 171 M.Eco95JB1VII, respectively), the Stx phage-borne M.EcoGIII (M.Eco95JB1III), and the 172 Dam paralog M.EcoGV (M.Eco95JB1IV); the remaining MTase, M.Eco95JB1VI, is 173 homologous to Type IIG RM system SenTFIV from Salmonella enterica subsp. enterica 174 serovar Typhimurium (76% amino acid similarity)(Table 3). 175 176 In addition to encoding all MTase-encoding genes identified in 95JB1, 95NR1 also encodes 177 three additional Dam homologs encoded on its additional Stx-converting prophages (Table 178 3). These comprise: (i) an additional copy of the Dam homolog M.EcoGV located on Stx2- 179 converting prophage Phi14 (M.Eco95NR1VI), (ii) an identical copy of the orphan MTase 180 M.EcoVT2Dam of E. coli prophage VT2-Sa on Stx2 prophage Phi14 (M.Eco95NR1VII) and 181 (iii) a second identical copy of M.EcoVT2Dam on Stx2 prophage Phi15 (M.Eco95NR1VIII). 182 183 The methylome of 95JB1 and 95NR1 show remarkably different methylation 184 patterns 185 In addition to encoding all MTase-encoding genes identified in 95JB1, 95NR1 also encodes 177 three additional Dam homologs encoded on its additional Stx-converting prophages (Table 178 3). These comprise: (i) an additional copy of the Dam homolog M.EcoGV located on Stx2- 179 converting prophage Phi14 (M.Eco95NR1VI), (ii) an identical copy of the orphan MTase 180 M.EcoVT2Dam of E. coli prophage VT2-Sa on Stx2 prophage Phi14 (M.Eco95NR1VII) and 181 (iii) a second identical copy of M.EcoVT2Dam on Stx2 prophage Phi15 (M.Eco95NR1VIII). 182 Insertion Sequence profiles differ between outbreak strains. 147 To explore if there were any additional mobile genetic element differences between the 148 outbreak strains we first compared the complete plasmids of each strain. Whereas the P1 149 plasmids were identical in both strains, the EHEC plasmid, p95JB1B, contained an additional 150 IS3 ssgr IS51-family insertion sequence (99% nucleotide sequence identity to IS1203 from E. 151 coli O111:H- PH) that was not present in p95NR1B (data not shown). This IS1203-like 152 element has inserted into the 3’ end of the transposase EC95JB1_B00047 within an IS91-like 153 insertion element. 154 To explore if there were any additional mobile genetic element differences between the 148 outbreak strains we first compared the complete plasmids of each strain. Whereas the P1 149 plasmids were identical in both strains, the EHEC plasmid, p95JB1B, contained an additional 150 IS3 ssgr IS51-family insertion sequence (99% nucleotide sequence identity to IS1203 from E. 151 coli O111:H- PH) that was not present in p95NR1B (data not shown). This IS1203-like 152 element has inserted into the 3’ end of the transposase EC95JB1_B00047 within an IS91-like 153 insertion element. 154 We next surveyed the chromosomal Insertion Sequence (IS) profiles of each outbreak strain 155 (Figure 2). Both 95JB1 and 95NR1 contain 17 different families of IS elements with two or 156 more copies on their respective chromosomes (Table S2). Notably, 95JB1 encodes an 157 additional chromosomal copy of an IS3 ssgr IS51-family element (100% identical to the 158 additional IS1203-like element on p95JB1B) inserted at the 3’ end of EC95JB1_03899 (Table 159 S3). EC95JB1_03899 is predicted to encode an ATPase. However, the IS has inserted ~150 160 bp upstream of the translational start site of a putative methyltransferase (MTase) gene 161 7 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint (EC95JB1_03895), suggesting a possible functional role due to a polar effect on 162 transcription. The methylome of 95JB1 and 95NR1 show remarkably different methylation 184 patterns 185 8 8 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Using the in-built capacity of PacBio to detect methylated nucleotides we detected two 186 distinct recognition motifs (5’-Gm6ATC-3’ and 5’-CTGCm6AG-3’) in 95JB1 and 95NR1, that 187 match MTases with known specificities. 5’-GATC-3’ is a well characterised methylation 188 motif, routinely identified in E. coli methylome analyses (Fang et al. 2012; Powers et al. 189 2013; Cooper et al. 2014; Forde et al. 2015) and known to be targeted by Dam (Geier and 190 Modrich 1979). We predict that the 5’-CTGCAG-3’ motif is targeted by 191 EC95JB1_EC95NR1_MTases in 95JB1 and 95NR1 (M.Eco95JB1III/M.Eco95NR1III) that 192 share 99% amino acid identity with M.EcoGIII, a previously characterised PstI homolog 193 shown to methylate 5’-CTGCAG-3’ (Fang et al. 2012). No additional methylated motifs in 194 95JB1 were detected suggesting that the remaining putative MTases are inactive under the 195 tested conditions (Table 4). Dcm-mediated methylation of cytosine (5’-Cm5CWGG-3’) could 196 not be detected using the method used in this study. 197 Remarkably, 95NR1 also contained more than 4000 methylated bases corresponding to a 199 third motif, 5’-CRARCm6AG-3’ (Table 4). As no methylation was detected at the 200 corresponding motifs in 95JB1 we considered that this new activity must relate to an MTase 201 that is not present or non-functional in 95JB1. Screening the 5’-CRARCAG-3’ motif against 202 REBASE (Roberts et al. 2010) confirmed that a cognate MTase enzyme has not been 203 previously characterised and to the best of our knowledge it represents a novel MTase target 204 recognition site (Table 4). 205 A novel MTase responsible for methylation of the CRARCAG motif 207 In order to identify the MTase responsible for methylation of the 5’-CRARCAG-3’ motif in 208 95NR1 (but not 95JB1) we first examined the specificities of the experimentally determined 209 E. coli C227-11 MTases for which there are five close homologs in 95NR1/95JB1 (>92% 210 9 9 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint amino acid sequence identity; Table 3) (Fang et al. 2012). Based on the high amino acid 211 identity between these homologs it is highly unlikely that any C227-11 MTase homologs 212 could be responsible for methylation of the 5’-CRARCAG-3’ motif. Similarly, 213 M.EcoVT2Dam is known to target 5’-GATC-3’; thus both homologs of M.EcoVT2Dam in 214 95NR1 (99% amino acid identity) would also be expected to target 5’-GATC-3’ (Radlinska 215 and Bujnicki 2001). The remaining candidate MTase encoded by M.Eco95NR1IX shares 216 76% amino acid identity to SenTFIV, a Type IIG R-M system which targets the GATCAG 217 recognition site. Minor difference in the target recognition domain (TRD) of homologous 218 MTase enzymes can result in major differences in target specificity as exemplified in E. coli 219 EC958 where the Type IIG R-M system M.EcoMVII and SenTFIV share 68% amino acid 220 identity, but their target sites are very different (5’-CANCATC-3’ and 5’-GATCAG-3’, 221 respectively). Furthermore, 100% hemi-methylation of the 5’-CRARCAG-3’ motif as 222 observed in 95NR1 is characteristic of the Type IIG family of MTases. Based on these 223 observations, we propose that the MTase M.EcoEC95NR1_IX catalyses CRARCAG 224 modification in 95NR1 and that the lack of corresponding methylation in 95JB1 is due to IS- 225 mediated transcriptional inactivation of the orthologous M.Eco95JB1VI gene. 226 IS insertion results in inactivation of M.Eco95JB1IX 228 BLAST comparison (blastp, E-value cutoff=1e-3) of M.Eco95NR1IX against a database of 229 complete E. coli genomes identified homologs of this MTase in four additional E. coli strains: 230 E. coli str. HS (EcHS_A0339; 76% amino acid sequence identity), E. coli O111:H- str. 11128 231 (ECO111_5156; 100% amino acid sequence identity), E. coli str. ‘clone i2’ (i02_4877; 76% 232 amino acid sequence identity) and E. coli str. ‘clone i14’ (i14_4877; 76% amino acid 233 sequence identity). With the exception of ECO111_5156, homologs of M.Eco95NR1IX 234 exhibited extensive variation in their TRDs and are likely to have different target specificities 235 10 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint (Figure S1a). Interestingly genes upstream of M.Eco95NR1IX are highly conserved in all 236 strains (Figure S1b) and suggest that M.Eco95NR1IX could be transcribed as part of a large 237 8.5 kb operon encoding a Type IIG MTase (M.Eco95NR1IX), a putative ATPase 238 (EC95NR1_04072) and two hypothetical proteins whose function is currently unknown 239 (EC95NR1_04073 and EC95NR1_04074) (Figure S2; Table S4). A putative promoter region 240 (5’-TGGCAT-14 bp-CATTACAAT-3’) was identified 50 bp upstream of EC95NR1_04074 241 and could indicate the primary transcriptional start site for this operon. Predicted promoter 242 regions were also identified 68 bp upstream of EC95NR1_04072 and overlapping the 243 predicted start codon of M.Eco95NR1IX. An additional predicted promoter region was 244 observed in 95JB1 which was located on the boundary of the IS insertion into 245 EC95JB1_03895 (Figure S2). Based on these observations we propose that the IS insertion 246 into EC95JB1_03899 results in premature transcriptional termination of the operon leaving 247 M.Eco95JB1IX untranscribed. 248 The functional characterisation of CRARCAG methylation 250 To determine if methylation of 5’-CRARCAG-3’ motifs could have a functional role in the 251 genome of 95NR1 we analysed the distribution of sites located within 300 bp upstream of an 252 annotated start codon. We identified 871 candidate genes where 5’-CRARCAG-3’ 253 methylation might have a regulatory role (Table S5). These include the flagellar genes 254 fliCDSHIJKQPR, flgCJG and the flagellar regulator flk; Type II secretion associated genes 255 epsMLJHGE; the pilus associated genes papD and pilN, Shiga toxin gene stx1B (Phi11); the 256 two component regulatory system BaeSR, and EutR, a transcriptional regulator associated 257 with EHEC pathogenesis (Luzader et al. 2013). Upstream sequences were further analysed 258 for the presence of putative promoter regions using Neural Network Promoter Prediction. A 259 total of 601 of the 871 candidate genes were found to contain putative promoter regions 260 11 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint within 300 bp of their start codon, 177 of which contained a methylated 5’-CRARCAG-3’ 261 motif (Table S5). Clustering of these genes based on their functional class revealed no 262 significant enrichment in any functional category when compared to the functional clustering 263 of all genes (Figure S3). 264 265 Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 There was an observed difference in the distribution of the 5’-GATC-3’ motif between the 268 core genome and the prophages in both 95NR1 and 95JB1 (Figure 3). This difference is 269 attributable to the presence of GATC-free regions within the prophages and is suggestive of 270 selection against Dam methylation in certain phage genes. Differences in the distribution of 271 GATC sites between the core and accessory genome have previously be described in E. coli 272 K12 (Henaut et al. 1996) and more recently in E. coli EC958 (Forde et al. 2015). The functional characterisation of CRARCAG methylation 250 273 Interestingly, these prophage-associated GATC-free regions are enriched for the 5’- 274 CTGCAG-3’ motif in most prophages in 95JB1 and 95NR1 with the exception of Phi9 275 (unclassified Myoviridae) and Phi12 (Mu-like Myoviridae) where the 5’-CTGCAG-3’ motif 276 is significantly under-represented compared to the core genome (P <= 0.0001). In 95NR1 the 277 5’-CRARCAG-3’ motif appears to be randomly distributed throughout the genome and 278 exhibited no significant enrichment bias to either the core or accessory genome. 279 280 within 300 bp of their start codon, 177 of which contained a methylated 5’-CRARCAG-3’ 261 motif (Table S5). Clustering of these genes based on their functional class revealed no 262 significant enrichment in any functional category when compared to the functional clustering 263 of all genes (Figure S3). 264 Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 In this 291 study we hypothesised that there were other genetic differences between the strains that may 292 account for the different virulence profiles. Using SMRT sequencing to produce complete 293 genomes of these important historical isolates enabled the complete definition of the structure 294 and context of all prophages (including the tandemly arranged Stx prophages in 95NR1). By 295 analysing the kinetic variation data produced during sequencing on the SMRT platform we 296 found a difference between the methylomes of 95JB1 and 95NR1. By identifying the position 297 and context of all IS elements in both genomes we identified an additional chromosomal copy 298 of an IS1203-like element in 95JB1 that likely prevents transcription of a Type IIG MTase 299 encoded nearby. As we are able to exclude all other MTases encoded by 95NR1 we conclude 300 that this IS difference accounts for the methylation patterns between the strains. It has been 301 well established that DNA methylation can play an active role in virulence and gene 302 regulation (Heithoff et al. 1999; Low et al. 2001; Wallecha et al. 2002; Casadesus and Low 303 2006). By examining methylated bases that may affect promoter regions in 95NR1, but not 304 95JB1, we have identified a number of potential promoters that could contribute, directly or 305 indirectly, to the increased virulence observed in 95NR1. 306 E. coli 95JB1 and 95NR1 are two isolates from a historical HUS outbreak in Adelaide, 284 Australia in 1995 (Centers for Disease and Prevention 1995; Paton et al. 1996). 95NR1, the 285 more virulent of the two strains, is characterised by the presence of two additional Stx2 286 prophages and two additional copies of stx2AB when compared to 95JB1 (McAllister et al. 287 2016). Previous analysis of the Illumina draft genomes of these outbreak strains showed that, 288 relative to 95NR1, 95JB1 had acquired a handful of SNPs and had lost two Stx2 prophages. 289 Despite these findings it was not possible to resolve the sequences of most mobile genetic 290 elements (including prophages) due to the fragmented nature of the draft assembly. In this 291 study we hypothesised that there were other genetic differences between the strains that may 292 account for the different virulence profiles. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 There was an observed difference in the distribution of the 5’-GATC-3’ motif between the 268 core genome and the prophages in both 95NR1 and 95JB1 (Figure 3). This difference is 269 attributable to the presence of GATC-free regions within the prophages and is suggestive of 270 selection against Dam methylation in certain phage genes. Differences in the distribution of 271 GATC sites between the core and accessory genome have previously be described in E. coli 272 K12 (Henaut et al. 1996) and more recently in E. coli EC958 (Forde et al. 2015). 273 Interestingly, these prophage-associated GATC-free regions are enriched for the 5’- 274 CTGCAG-3’ motif in most prophages in 95JB1 and 95NR1 with the exception of Phi9 275 (unclassified Myoviridae) and Phi12 (Mu-like Myoviridae) where the 5’-CTGCAG-3’ motif 276 is significantly under-represented compared to the core genome (P <= 0.0001). In 95NR1 the 277 5’-CRARCAG-3’ motif appears to be randomly distributed throughout the genome and 278 exhibited no significant enrichment bias to either the core or accessory genome. 279 283 12 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint E. coli 95JB1 and 95NR1 are two isolates from a historical HUS outbreak in Adelaide, 284 Australia in 1995 (Centers for Disease and Prevention 1995; Paton et al. 1996). 95NR1, the 285 more virulent of the two strains, is characterised by the presence of two additional Stx2 286 prophages and two additional copies of stx2AB when compared to 95JB1 (McAllister et al. 287 2016). Previous analysis of the Illumina draft genomes of these outbreak strains showed that, 288 relative to 95NR1, 95JB1 had acquired a handful of SNPs and had lost two Stx2 prophages. 289 Despite these findings it was not possible to resolve the sequences of most mobile genetic 290 elements (including prophages) due to the fragmented nature of the draft assembly. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 Using SMRT sequencing to produce complete 293 genomes of these important historical isolates enabled the complete definition of the structure 294 and context of all prophages (including the tandemly arranged Stx prophages in 95NR1). By 295 analysing the kinetic variation data produced during sequencing on the SMRT platform we 296 found a difference between the methylomes of 95JB1 and 95NR1. By identifying the position 297 and context of all IS elements in both genomes we identified an additional chromosomal copy 298 of an IS1203-like element in 95JB1 that likely prevents transcription of a Type IIG MTase 299 encoded nearby. As we are able to exclude all other MTases encoded by 95NR1 we conclude 300 that this IS difference accounts for the methylation patterns between the strains. It has been 301 well established that DNA methylation can play an active role in virulence and gene 302 regulation (Heithoff et al. 1999; Low et al. 2001; Wallecha et al. 2002; Casadesus and Low 303 2006). By examining methylated bases that may affect promoter regions in 95NR1, but not 304 95JB1, we have identified a number of potential promoters that could contribute, directly or 305 indirectly, to the increased virulence observed in 95NR1. 306 E. coli 95JB1 and 95NR1 are two isolates from a historical HUS outbreak in Adelaide, 284 Australia in 1995 (Centers for Disease and Prevention 1995; Paton et al. 1996). 95NR1, the 285 more virulent of the two strains, is characterised by the presence of two additional Stx2 286 prophages and two additional copies of stx2AB when compared to 95JB1 (McAllister et al. 287 2016). Previous analysis of the Illumina draft genomes of these outbreak strains showed that, 288 relative to 95NR1, 95JB1 had acquired a handful of SNPs and had lost two Stx2 prophages. 289 Despite these findings it was not possible to resolve the sequences of most mobile genetic 290 elements (including prophages) due to the fragmented nature of the draft assembly. In this 291 study we hypothesised that there were other genetic differences between the strains that may 292 account for the different virulence profiles. Using SMRT sequencing to produce complete 293 genomes of these important historical isolates enabled the complete definition of the structure 294 and context of all prophages (including the tandemly arranged Stx prophages in 95NR1). Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 By 295 analysing the kinetic variation data produced during sequencing on the SMRT platform we 296 found a difference between the methylomes of 95JB1 and 95NR1. By identifying the position 297 and context of all IS elements in both genomes we identified an additional chromosomal copy 298 of an IS1203-like element in 95JB1 that likely prevents transcription of a Type IIG MTase 299 encoded nearby. As we are able to exclude all other MTases encoded by 95NR1 we conclude 300 that this IS difference accounts for the methylation patterns between the strains. It has been 301 well established that DNA methylation can play an active role in virulence and gene 302 regulation (Heithoff et al. 1999; Low et al. 2001; Wallecha et al. 2002; Casadesus and Low 303 2006). By examining methylated bases that may affect promoter regions in 95NR1, but not 304 95JB1, we have identified a number of potential promoters that could contribute, directly or 305 indirectly, to the increased virulence observed in 95NR1. 306 307 13 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Previously, a detailed analysis of the Illumina draft genome assemblies of 95JB1 and 95NR1 308 enabled accurate determination of prophage insertion sites, plasmid content and the 309 multiplicity of stx genes (McAllister et al. 2016). As expected, repeat elements were the 310 major cause of fragmentation in the draft genomes of 95JB1 and 95NR1, with the majority of 311 contigs terminating at IS elements or within prophage regions. Long SMRT sequencing reads 312 easily bridge these repeat regions generating complete, high quality assemblies with little or 313 no manual intervention. Complete genomes are a vital resource when characterising the 314 genetic variation which exists between strains, as the positions of IS and other mobile 315 elements can influence host biological processes, including pathogenicity and antibiotic 316 resistance (Conlan et al. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint is characteristic of the Type IIG family of MTases. Formal demonstration that the 5’- 333 CRARCAG-3’ recognition motif is methylated by M.Eco95NR1IX would require methylome 334 profiling of a 95NR1 M.Eco95NR1IX deletion mutant. 335 336 In this study we identified two active MTases common to both 95JB1 and 95NR1: Dam and 337 the recently characterised Stx phage-encoded RM system methyltransferase M.EcoGIII, 338 which recognises the CTGCAG motif. M.EcoGIII is known to affect the expression of 1,951 339 genes in E.coli C227-ll, provide resistance to infection by other lambda-like phage and 340 influence growth. It is likely that M.EcoGIII occupies a similar regulatory role in 95JB1 and 341 95NR1 as it does in C227-11. However, determining the functional consequences of 342 CTGCAG methylation in 95JB1 and 95NR1 requires further analysis. Interestingly, three 343 prophage loci in C227-11 were found to be enriched for the CTGCAG motif (Fang et al. 344 2012) and a similar enrichment of the CTGCAG motif was also observed in most prophages 345 of 95JB1 and 95NR1. In contrast, two prophage regions in 95JB1 and 95NR1 (Phi12 and 346 Phi16) contained no CTGCAG motifs, suggesting selection against the presence of these 347 sites. In C227-11 the M.EcoGIII RM system was shown to protect against infection by other 348 lambda-like phages, but T4 phages were resistant to restriction, which could be attributed to 349 heavy modification of their genomes (Fang et al. 2012). 95NR1 and 95JB1 do not contain 350 any T4 phage, although Phi12 and Phi16 are members of the Myoviridae, the class of virus to 351 which the T4 phage belong. The lack of any CTGCAG motifs in these Phi12 and Phi16 352 is characteristic of the Type IIG family of MTases. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 2014; Cameron et al. 2015; He et al. 2015; Yan et al. 2015; Zowawi 317 et al. 2015). 318 319 Determining the context of all IS in 95JB1 and 95NR1 was crucial for characterising 320 methylome differences between both strains. Surprisingly, we found that the most likely 321 explanation for the observed differences in methylation between the outbreak strains was the 322 insertion of an additional IS1203-like element upstream of the gene encoding 323 M.Eco95JB1IXin 95JB1. This particular IS element was first identified in E. coli O111:H- 324 strain PH (Paton and Paton 1994) and has previously been associated with insertional 325 inactivation of genes in STEC (Kusumoto et al. 1999; Okitsu et al. 2001; Suzuki et al. 2004). 326 Several lines of evidence support our contention that M.Eco95NR1IX/M.Eco95JB1IX 327 catalyses the methylation of the 5’-CRARCm6AG-3’ motif. Other than M.Eco95NR1IX there 328 were no candidate MTases in the 95NR1 or 95JB1 genomes that could account for the 329 methylation of the 5’-CRARCAG-3’ motif in 95NR1 alone. M.Eco95NR1IX and SenTFIV 330 share 76% amino acid identity consistent with a different recognition site. Furthermore, the 331 distinctive pattern of hemi-methylation of the 5’-CRARCAG-3’ motif as observed in 95NR1 332 Determining the context of all IS in 95JB1 and 95NR1 was crucial for characterising 320 methylome differences between both strains. Surprisingly, we found that the most likely 321 explanation for the observed differences in methylation between the outbreak strains was the 322 insertion of an additional IS1203-like element upstream of the gene encoding 323 M.Eco95JB1IXin 95JB1. This particular IS element was first identified in E. coli O111:H- 324 strain PH (Paton and Paton 1994) and has previously been associated with insertional 325 inactivation of genes in STEC (Kusumoto et al. 1999; Okitsu et al. 2001; Suzuki et al. 2004). 326 Several lines of evidence support our contention that M.Eco95NR1IX/M.Eco95JB1IX 327 catalyses the methylation of the 5’-CRARCm6AG-3’ motif. Other than M.Eco95NR1IX there 328 were no candidate MTases in the 95NR1 or 95JB1 genomes that could account for the 329 methylation of the 5’-CRARCAG-3’ motif in 95NR1 alone. M.Eco95NR1IX and SenTFIV 330 share 76% amino acid identity consistent with a different recognition site. Furthermore, the 331 distinctive pattern of hemi-methylation of the 5’-CRARCAG-3’ motif as observed in 95NR1 332 14 14 . Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 Formal demonstration that the 5’- 333 CRARCAG-3’ recognition motif is methylated by M.Eco95NR1IX would require methylome 334 profiling of a 95NR1 M.Eco95NR1IX deletion mutant. 335 is characteristic of the Type IIG family of MTases. Formal demonstration that the 5’- 333 CRARCAG-3’ recognition motif is methylated by M.Eco95NR1IX would require methylome 334 profiling of a 95NR1 M.Eco95NR1IX deletion mutant. 335 In this study we identified two active MTases common to both 95JB1 and 95NR1: Dam and 337 the recently characterised Stx phage-encoded RM system methyltransferase M.EcoGIII, 338 which recognises the CTGCAG motif. M.EcoGIII is known to affect the expression of 1,951 339 genes in E.coli C227-ll, provide resistance to infection by other lambda-like phage and 340 influence growth. It is likely that M.EcoGIII occupies a similar regulatory role in 95JB1 and 341 95NR1 as it does in C227-11. However, determining the functional consequences of 342 CTGCAG methylation in 95JB1 and 95NR1 requires further analysis. Interestingly, three 343 prophage loci in C227-11 were found to be enriched for the CTGCAG motif (Fang et al. 344 2012) and a similar enrichment of the CTGCAG motif was also observed in most prophages 345 of 95JB1 and 95NR1. In contrast, two prophage regions in 95JB1 and 95NR1 (Phi12 and 346 Phi16) contained no CTGCAG motifs, suggesting selection against the presence of these 347 sites. In C227-11 the M.EcoGIII RM system was shown to protect against infection by other 348 lambda-like phages, but T4 phages were resistant to restriction, which could be attributed to 349 heavy modification of their genomes (Fang et al. 2012). 95NR1 and 95JB1 do not contain 350 any T4 phage, although Phi12 and Phi16 are members of the Myoviridae, the class of virus to 351 which the T4 phage belong. The lack of any CTGCAG motifs in these Phi12 and Phi16 352 (CTGCAG motif is expected to occur by chance every 4064 bp) suggests active selection 353 against this motif in the Myoviridae, which would presumably render them immune to 354 digestion by M.EcoGIII. 355 356 15 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 We have identified several operons and regulators that may be likely 365 candidates for differential expression, although it should be noted that putative promoter sites 366 were predicted in silico and may not reflect true promotor regions. It is also worth noting that 367 95NR1 and 95JB1 have been classifed as non-motile (H-) according to their original 368 serotyping results suggesting that differential expression of flagellar loci is unlikely to result 369 in phenotypic difference (Centers for Disease and Prevention 1995). Analysis of the 370 transcriptome using RNA-Seq will be necessary to fully define the influence of methylation 371 on differential regulation between these otherwise very similar strains. 372 373 DNA modifications are an established cause of phenotypic heterogeneity in both isogenic and 374 clonal populations (Casadesus and Low 2013). Phase variation or ON/OFF switching of the 375 pyelonephritis-associated pili (Pap) operon and antigen 43 (Ag43) are two of the most well 376 characterised examples of methylation-mediated intercellular heterogeneity in E. coli (van 377 der Woude et al. 1992; van der Woude and Henderson 2008). In these examples, differences 378 in the methylation status of GATC motifs (mediated by Dam) in the promoter regions of pap 379 and ag43 control ON/OFF expression of these loci and intercellular heterogeneity within the 380 clonal population. Differences in the expression of the same MTase in 95NR1 and 95JB1 381 DNA modifications are an established cause of phenotypic heterogeneity in both isogenic and 374 clonal populations (Casadesus and Low 2013). Phase variation or ON/OFF switching of the 375 pyelonephritis-associated pili (Pap) operon and antigen 43 (Ag43) are two of the most well 376 characterised examples of methylation-mediated intercellular heterogeneity in E. coli (van 377 der Woude et al. 1992; van der Woude and Henderson 2008). In these examples, differences 378 in the methylation status of GATC motifs (mediated by Dam) in the promoter regions of pap 379 and ag43 control ON/OFF expression of these loci and intercellular heterogeneity within the 380 clonal population. Differences in the expression of the same MTase in 95NR1 and 95JB1 381 DNA modifications are an established cause of phenotypic heterogeneity in both isogenic and 374 clonal populations (Casadesus and Low 2013). Phase variation or ON/OFF switching of the 375 pyelonephritis-associated pili (Pap) operon and antigen 43 (Ag43) are two of the most well 376 characterised examples of methylation-mediated intercellular heterogeneity in E. coli (van 377 der Woude et al. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Bacterial MTases are known to have diverse roles which include regulation of gene 357 expression. For example, the orphan MTase Dam has been established as regulator of gene 358 expression in other E. coli species and recently the Type II RM system EcoGIII was shown to 359 directly or indirectly affect the expression of ~1900 E. coli genes (Low et al. 2001; Casadesus 360 and Low 2006). Similarly, it has been suggested that Type IIG RM systems might have a role 361 in bacterial genomes other than protection (Loenen et al. 2014). The proximity of methylated 362 5’-CRARCAG-3’ sites to the predicted promotor regions of 180 genes in 95NR1 suggests 363 that methylation of this motif could directly cause differential gene expression between 364 95NR1 and 95JB1. We have identified several operons and regulators that may be likely 365 candidates for differential expression, although it should be noted that putative promoter sites 366 were predicted in silico and may not reflect true promotor regions. It is also worth noting that 367 95NR1 and 95JB1 have been classifed as non-motile (H-) according to their original 368 serotyping results suggesting that differential expression of flagellar loci is unlikely to result 369 in phenotypic difference (Centers for Disease and Prevention 1995). Analysis of the 370 transcriptome using RNA-Seq will be necessary to fully define the influence of methylation 371 on differential regulation between these otherwise very similar strains. 372 expression. For example, the orphan MTase Dam has been established as regulator of gene 358 expression in other E. coli species and recently the Type II RM system EcoGIII was shown to 359 directly or indirectly affect the expression of ~1900 E. coli genes (Low et al. 2001; Casadesus 360 and Low 2006). Similarly, it has been suggested that Type IIG RM systems might have a role 361 in bacterial genomes other than protection (Loenen et al. 2014). The proximity of methylated 362 5’-CRARCAG-3’ sites to the predicted promotor regions of 180 genes in 95NR1 suggests 363 that methylation of this motif could directly cause differential gene expression between 364 95NR1 and 95JB1. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 1992; van der Woude and Henderson 2008). In these examples, differences 378 in the methylation status of GATC motifs (mediated by Dam) in the promoter regions of pap 379 and ag43 control ON/OFF expression of these loci and intercellular heterogeneity within the 380 clonal population. Differences in the expression of the same MTase in 95NR1 and 95JB1 381 16 16 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint (M.Eco95NR1IX and M.Eco95JB1VI, respectively) is a clear example of epigenetic 382 heterogeneity which has arisen within this clonal population. Whether this observed 383 epigenetic heterogeneity is driving phenotypic difference within the population is currently 384 unknown and represents an interesting avenue for further research. 385 386 During our characterisation of the 95NR1 and 95JB1 methylomes we made the surprising 387 discovery that a Type IIG MTase carried by 95NR1 and 95JB1 is likely encoded as part of an 388 operon. To the best of our knowledge a Type IIG MTase has not previously been reported as 389 a component of a multi-gene system. The function of the other proteins in this putative 390 operon and whether they are linked to the activity of the MTase is currently not known and 391 requires further research which is beyond the scope of this study. Despite identifying a 392 putative primary promoter at the 5’ operon boundary, the presence of several additional 393 promoters distributed through the operon raises the possibility that some genes are 394 transcribed separately. Future analysis of the transcriptome of 95NR1 or other E. coli that 395 carry homologs of M.Eco95NR1IX will be necessary to correctly determine transcriptional 396 start-sites. Notably, SMRT sequencing also offers the potential for characterising complete 397 polycistronic mRNAs using the Iso-Seq protocol (Sharon et al. 2013; Tilgner et al. 2014). Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 398 399 Characterising the genetic differences between strains is highly important for determining the 400 (M.Eco95NR1IX and M.Eco95JB1VI, respectively) is a clear example of epigenetic 382 heterogeneity which has arisen within this clonal population. Whether this observed 383 epigenetic heterogeneity is driving phenotypic difference within the population is currently 384 unknown and represents an interesting avenue for further research. 385 (M.Eco95NR1IX and M.Eco95JB1VI, respectively) is a clear example of epigenetic 382 heterogeneity which has arisen within this clonal population. Whether this observed 383 epigenetic heterogeneity is driving phenotypic difference within the population is currently 384 unknown and represents an interesting avenue for further research. 385 During our characterisation of the 95NR1 and 95JB1 methylomes we made the surprising 387 discovery that a Type IIG MTase carried by 95NR1 and 95JB1 is likely encoded as part of an 388 operon. To the best of our knowledge a Type IIG MTase has not previously been reported as 389 a component of a multi-gene system. The function of the other proteins in this putative 390 operon and whether they are linked to the activity of the MTase is currently not known and 391 requires further research which is beyond the scope of this study. Despite identifying a 392 putative primary promoter at the 5’ operon boundary, the presence of several additional 393 promoters distributed through the operon raises the possibility that some genes are 394 transcribed separately. Future analysis of the transcriptome of 95NR1 or other E. coli that 395 carry homologs of M.Eco95NR1IX will be necessary to correctly determine transcriptional 396 start-sites. Notably, SMRT sequencing also offers the potential for characterising complete 397 polycistronic mRNAs using the Iso-Seq protocol (Sharon et al. 2013; Tilgner et al. 2014). 398 Characterising the genetic differences between strains is highly important for determining the 400 evolutionary history of bacterial populations, tracking clinical outbreaks and identifying 401 functional mutations which contribute to virulence and antibiotic resistance (Holt et al. 2008; 402 Loman and Pallen 2008; Petty et al. 2014; Bartley et al. 2016). Currently, Illumina is the 403 platform of choice for studying single nucleotide variation, due to its capacity for accurate 404 high throughput sequencing of hundreds or thousands of strains. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 Of particular 407 importance to our understanding of STEC biology was the complete resolution of two 408 tandemly arrayed Stx2 prophages encoded by 95NR1 and not 95JB1. The complete sequence 409 of three full-length Stx2 prophages was determined in 95NR1 highlighting the difficulty of 410 resolving multiple similar prophage genomes within a draft assembly. Although a highly 411 accurate WGS method for subtyping stx genes has been developed (Ashton et al. 2015), the 412 reliance of this method on short read sequencing data means it lacks the discriminatory power 413 to determine whether multiple copies of the same subtype are due to multiple insertions by 414 different Stx-converting phage or as a result of gene duplication. Our study highlights how 415 population genomic studies of STEC outbreaks or global collections could benefit from 416 SMRT sequencing and/or bioinformatics approaches that take into account mobile genetic 417 element heterogeneity. 418 419 Although the ability of SMRT sequencing to resolve large mobile genetic elements is 420 currently unparalleled, it is important to recognise that small plasmids are easily lost during 421 the library preparation for PacBio. On the PacBio instruments the size distribution of the 422 SMRTBell sequencing libraries influences read length performance. For example, short DNA 423 library fragments preferentially load into the sequencing wells (Zero-mode wave guides) on 424 the SMRT Cell limiting the long read potential of the sequencing library. In order to 425 maximise the lengths of reads prior to sequencing it is necessary to filter small fragments 426 from the library and narrow its size distribution. In this study targeted DNA size selection 427 was achieved using the BluePippin instrument (http://www.sagescience.com). Using 428 BluePippin small DNA fragments are separated from larger fragments enabling the collection 429 of sequencing libraries with narrower size distributions. Both 95NR1 and 95JB1 were 430 sequenced using BluePippin size-selected 20 kb SMRTBell libraries. As small plasmid DNA 431 Although the ability of SMRT sequencing to resolve large mobile genetic elements is 420 currently unparalleled, it is important to recognise that small plasmids are easily lost during 421 the library preparation for PacBio. On the PacBio instruments the size distribution of the 422 SMRTBell sequencing libraries influences read length performance. For example, short DNA 423 library fragments preferentially load into the sequencing wells (Zero-mode wave guides) on 424 the SMRT Cell limiting the long read potential of the sequencing library. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 In this study the SNP profile 405 between 95JB1 and 95NR1 was identical using PacBio or Illumina data, whereas only PacBio 406 Characterising the genetic differences between strains is highly important for determining the 400 evolutionary history of bacterial populations, tracking clinical outbreaks and identifying 401 functional mutations which contribute to virulence and antibiotic resistance (Holt et al. 2008; 402 Loman and Pallen 2008; Petty et al. 2014; Bartley et al. 2016). Currently, Illumina is the 403 platform of choice for studying single nucleotide variation, due to its capacity for accurate 404 high throughput sequencing of hundreds or thousands of strains. In this study the SNP profile 405 between 95JB1 and 95NR1 was identical using PacBio or Illumina data, whereas only PacBio 406 17 17 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint could accurately resolve the mobile genetic element content of both strains. Of particular 407 importance to our understanding of STEC biology was the complete resolution of two 408 tandemly arrayed Stx2 prophages encoded by 95NR1 and not 95JB1. The complete sequence 409 of three full-length Stx2 prophages was determined in 95NR1 highlighting the difficulty of 410 resolving multiple similar prophage genomes within a draft assembly. Although a highly 411 accurate WGS method for subtyping stx genes has been developed (Ashton et al. 2015), the 412 reliance of this method on short read sequencing data means it lacks the discriminatory power 413 to determine whether multiple copies of the same subtype are due to multiple insertions by 414 different Stx-converting phage or as a result of gene duplication. Our study highlights how 415 population genomic studies of STEC outbreaks or global collections could benefit from 416 SMRT sequencing and/or bioinformatics approaches that take into account mobile genetic 417 element heterogeneity. 418 could accurately resolve the mobile genetic element content of both strains. Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 These 442 results reveal that in addition to acquiring a small number of SNPs and losing two tandemly 443 arranged Stx2 prophages, 95JB1 has also lost the activity of a novel MTase that may 444 influence the transcription of several hundred genes. PacBio SMRT sequencing has enormous 445 potential to reveal the genetic and epigenetic heterogeneity within a clonal population. 446 Further genomic analysis of IS and prophages within closely related STEC strains will further 447 build our understanding of short-term evolution within the context of an outbreak. 448 449 Methods 450 451 Genome Sequencing and assembly. 452 Genomic DNA (gDNA) from the Escherichia coli strains 95NR1 and 95JB1 was sequenced 453 on a PacBio RSII instrument (University of Queensland Centre for Clinical Genomics; 454 UQCCG) using two SMRT cells, a 20 kb insert library and the P6 polymerase and C4 455 sequencing chemistry. De novo assembly of the raw PacBio sequencing data was done using 456 Here we have analysed the complete genome sequences of two clonal STEC O111:H- strains 436 from a historical outbreak using PacBio sequencing. In addition to resolving complex 437 prophage regions to highlight the precise genetic differences between these strains, we also 438 identified more than 4000 differentially methylated chromosomal sites implicating a MTase 439 that was active in 95NR1 but not 95JB1. We linked the differences in methylome to the 440 mobilization of an IS1203-like insertion sequence in 95JB1, highlighting the importance of 441 characterising IS differences when comparing two closely related bacterial strains. These 442 results reveal that in addition to acquiring a small number of SNPs and losing two tandemly 443 arranged Stx2 prophages, 95JB1 has also lost the activity of a novel MTase that may 444 influence the transcription of several hundred genes. PacBio SMRT sequencing has enormous 445 potential to reveal the genetic and epigenetic heterogeneity within a clonal population. 446 Further genomic analysis of IS and prophages within closely related STEC strains will further 447 build our understanding of short-term evolution within the context of an outbreak. 448 Distribution of Methylated motifs reveals difference in methylation patterns 266 within prophage sequences 267 In order to 425 maximise the lengths of reads prior to sequencing it is necessary to filter small fragments 426 from the library and narrow its size distribution. In this study targeted DNA size selection 427 was achieved using the BluePippin instrument (http://www.sagescience.com). Using 428 BluePippin small DNA fragments are separated from larger fragments enabling the collection 429 of sequencing libraries with narrower size distributions. Both 95NR1 and 95JB1 were 430 sequenced using BluePippin size-selected 20 kb SMRTBell libraries. As small plasmid DNA 431 18 18 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint was visible in the original DNA extraction (data not shown) it appears that DNA fragments 432 representing the missing colicin plasmids of 95NR1 and 95JB1 were filtered from the 433 sequencing library during targeted size selection. 434 was visible in the original DNA extraction (data not shown) it appears that DNA fragments 432 representing the missing colicin plasmids of 95NR1 and 95JB1 were filtered from the 433 sequencing library during targeted size selection. 434 q g y g g 435 Here we have analysed the complete genome sequences of two clonal STEC O111:H- strains 436 from a historical outbreak using PacBio sequencing. In addition to resolving complex 437 prophage regions to highlight the precise genetic differences between these strains, we also 438 identified more than 4000 differentially methylated chromosomal sites implicating a MTase 439 that was active in 95NR1 but not 95JB1. We linked the differences in methylome to the 440 mobilization of an IS1203-like insertion sequence in 95JB1, highlighting the importance of 441 characterising IS differences when comparing two closely related bacterial strains. Genome Sequencing and assembly. 452 Genomic DNA (gDNA) from the Escherichia coli strains 95NR1 and 95JB1 was sequenced 453 on a PacBio RSII instrument (University of Queensland Centre for Clinical Genomics; 454 UQCCG) using two SMRT cells, a 20 kb insert library and the P6 polymerase and C4 455 sequencing chemistry. De novo assembly of the raw PacBio sequencing data was done using 456 19 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint the hierarchical genome assembly process (HGAP version 2) and Quiver (Chin et al. 2013) 457 from the SMRT Analysis software suite (version 2.3.0 – http://www.pacb.com/devnet/) with 458 default parameters. Following de novo assembly all contigs were visually screened for 459 overlapping sequences on their 5' and 3' ends using contiguity 460 (https://github.com/mjsull/Contiguity)(Sullivan et al. 2015). Overlapping ends, a 461 characteristic feature of the HGAP assembly process, were manually trimmed based on 462 sequence similarity and the contigs were circularised. Circularised contigs (Chromosome and 463 plasmids) were then subjected to a polishing phase, were the raw PacBio sequencing reads 464 were mapped back onto the assembled circular contigs (BLASR (Chaisson and Tesler 2012) 465 and quiver) to validate the assembly and resolve any remaining errors. Following multiple 466 rounds of polishing an additional improvement step was required to resolve single nucleotide 467 insertion and deletions errors associated homopolyer tracts. Reads from the publically 468 available Illumina sequence data for 95NR1 (SRA accession: SRR953500) and 95JB1 (SRA 469 accession: SRR954273) were aligned to their respective genomes using bwa version: 0.7.12- 470 r1039(Li and Durbin 2009) and a corrected consensus was called using Pilon version 1.18 471 (Walker et al. 2014). A total of 42 indels were corrected in 95NR1 and 30 indels in 95JB1. 472 95NR1 was further processed as the incorrect distribution of reads between two tandemly, 473 integrated Stx prophages initially resulted in a misassembly characterised by a contig break. Genome Sequencing and assembly. 452 474 This misassembly was manually corrected, and verified by re-aligning the raw reads to the 475 complete chromosome. 476 the hierarchical genome assembly process (HGAP version 2) and Quiver (Chin et al. 2013) 457 from the SMRT Analysis software suite (version 2.3.0 – http://www.pacb.com/devnet/) with 458 default parameters. Following de novo assembly all contigs were visually screened for 459 overlapping sequences on their 5' and 3' ends using contiguity 460 (https://github.com/mjsull/Contiguity)(Sullivan et al. 2015). Overlapping ends, a 461 characteristic feature of the HGAP assembly process, were manually trimmed based on 462 sequence similarity and the contigs were circularised. Circularised contigs (Chromosome and 463 plasmids) were then subjected to a polishing phase, were the raw PacBio sequencing reads 464 were mapped back onto the assembled circular contigs (BLASR (Chaisson and Tesler 2012) 465 and quiver) to validate the assembly and resolve any remaining errors. Following multiple 466 rounds of polishing an additional improvement step was required to resolve single nucleotide 467 insertion and deletions errors associated homopolyer tracts. Reads from the publically 468 available Illumina sequence data for 95NR1 (SRA accession: SRR953500) and 95JB1 (SRA 469 accession: SRR954273) were aligned to their respective genomes using bwa version: 0.7.12- 470 r1039(Li and Durbin 2009) and a corrected consensus was called using Pilon version 1.18 471 (Walker et al. 2014). A total of 42 indels were corrected in 95NR1 and 30 indels in 95JB1. 472 95NR1 was further processed as the incorrect distribution of reads between two tandemly, 473 integrated Stx prophages initially resulted in a misassembly characterised by a contig break. 474 This misassembly was manually corrected, and verified by re-aligning the raw reads to the 475 complete chromosome. 476 The detection of methylated bases and clustering of modified sites to identify methylation- 479 associated motifs was performed using the RS_Modification_and_Motif_analysis.1 tool from 480 20 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. Genome Sequencing and assembly. 452 ; https://doi.org/10.1101/173336 doi: bioRxiv preprint the SMRT analysis package version 2.3.0. Raw reads were aligned to the complete genomes 481 of 95JB1 and 95NR1 and interpulse duration (IPD) ratios were calculated using PacBio’s in 482 silico kinetic reference computational model (http://www.pacb.com/wp- 483 content/uploads/2015/09/WP_Detecting_DNA_Base_Modifications_Using_SMRT_Sequenci 484 ng.pdf). 485 486 To compare the CTGCAG motif distribution of Phi9 and Phi12 with the rest of the 487 chromosome, the sequence for each strand was split into 1000 bp segments with a 250 bp 488 overlap using Bedtools v2.17.0 (Quinlan and Hall 2010). Analysis of the mean distribution of 489 CTGCAG motifs per segment within these genomic regions was performed as previously 490 described using a custom analysis of variance (ANOVA) R script, which adjusts for 491 heteroscedasticity (Forde et al. 2015). 492 493 SNP analysis of 95NR1 and 95JB1 494 To determine the number and position of unique SNPs that differentiate 95JB1 and 95NR1, 495 Illumina reads were simulated from the complete genomes of 95NR1 and 95JB1 and aligned 496 to the genome of E. coli O111:H- strain 11128. SNP calling and Indel prediction was 497 performed using Nesoni and the Nesoni n-way pairwise comparison method was used to 498 identify SNPs conserved in all three strains 499 (http://www.vicbioinformatics.com/software.nesoni.shtml). Additionally, a reference free 500 SNP analysis was performed by direct comparison of the complete genome of 95JB1 and 501 95NR1 using MUMmer version 3.2.3 (Kurtz et al. 2004). 502 503 Genome annotation and comparative genomics 504 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made the SMRT analysis package version 2.3.0. Raw reads were aligned to the complete genomes 481 of 95JB1 and 95NR1 and interpulse duration (IPD) ratios were calculated using PacBio’s in 482 silico kinetic reference computational model (http://www.pacb.com/wp- 483 content/uploads/2015/09/WP_Detecting_DNA_Base_Modifications_Using_SMRT_Sequenci 484 ng.pdf). 485 486 To compare the CTGCAG motif distribution of Phi9 and Phi12 with the rest of the 487 chromosome, the sequence for each strand was split into 1000 bp segments with a 250 bp 488 overlap using Bedtools v2.17.0 (Quinlan and Hall 2010). Analysis of the mean distribution of 489 CTGCAG motifs per segment within these genomic regions was performed as previously 490 described using a custom analysis of variance (ANOVA) R script, which adjusts for 491 heteroscedasticity (Forde et al. 2015). 492 SNP analysis of 95NR1 and 95JB1 494 2009) were used to visually compare the genomes 511 and methylomes of 95NR1 and 95JB1 and identify regions of similarity and difference. 512 Methytransferase genes and restriction modification systems were identified and annotated by 513 comparison of all coding sequences from 95JB1 and 95NR1 against the REBASE database 514 (Roberts et al. 2010). Promoter sequences were predicted using BPROM(Li 2011). 515 516 The functional characterisation of CRARCAG methylation 517 In silico prediction of promoter sequences was done using Neural Network Promoter 518 Prediction version 2.2(Reese 2001) and KEGG gene ontologies (KOs) were assigned using 519 BlastKOALA (Kanehisa et al. 2016). The proximity of the CRARCAG motifs to all protein 520 coding regions was determined using custom python scripts. Protein coding regions that did 521 not contain a methylated CRARCAG motif within 300 bp of its start codon were excluded 522 from the analysis. For the remaining protein coding regions, 300 bp of sequence upstream of 523 their respective start codons was screened for putative promoter regions. CRARCAG motifs 524 located within predicated promotor regions where identified, and KOs were assigned using 525 BlastKOALA. 526 527 Initial gene calling and automated functional annotation of 95NR1 and 95JB1 were 505 performed using Prokka (Prokka: Prokaryotic Genome Annotation System – 506 http://vicbioinformatics.com/) using a custom Escherichia genus database consisting of 507 protein sequences from the EcoCyc website (http://ecocyc.org/). Putative phage encoding loci 508 and IS elements were identified using PHAST (Zhou et al. 2011) and ISfinder (Siguier et al. 509 2006) respectively. Artemis Comparison Tool (ACT) (Carver et al. 2005), EasyFig (Sullivan 510 et al. 2011) and Circos (Krzywinski et al. 2009) were used to visually compare the genomes 511 and methylomes of 95NR1 and 95JB1 and identify regions of similarity and difference. 512 Methytransferase genes and restriction modification systems were identified and annotated by 513 comparison of all coding sequences from 95JB1 and 95NR1 against the REBASE database 514 (Roberts et al. 2010). Promoter sequences were predicted using BPROM(Li 2011). 515 SNP analysis of 95NR1 and 95JB1 494 To determine the number and position of unique SNPs that differentiate 95JB1 and 95NR1, 495 Illumina reads were simulated from the complete genomes of 95NR1 and 95JB1 and aligned 496 to the genome of E. coli O111:H- strain 11128. SNP calling and Indel prediction was 497 performed using Nesoni and the Nesoni n-way pairwise comparison method was used to 498 identify SNPs conserved in all three strains 499 (http://www.vicbioinformatics.com/software.nesoni.shtml). Additionally, a reference free 500 SNP analysis was performed by direct comparison of the complete genome of 95JB1 and 501 95NR1 using MUMmer version 3.2.3 (Kurtz et al. 2004). 502 503 To determine the number and position of unique SNPs that differentiate 95JB1 and 95NR1, 495 Illumina reads were simulated from the complete genomes of 95NR1 and 95JB1 and aligned 496 to the genome of E. coli O111:H- strain 11128. SNP calling and Indel prediction was 497 performed using Nesoni and the Nesoni n-way pairwise comparison method was used to 498 identify SNPs conserved in all three strains 499 (http://www.vicbioinformatics.com/software.nesoni.shtml). Additionally, a reference free 500 SNP analysis was performed by direct comparison of the complete genome of 95JB1 and 501 95NR1 using MUMmer version 3.2.3 (Kurtz et al. 2004). 502 Genome annotation and comparative genomics 504 21 21 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Initial gene calling and automated functional annotation of 95NR1 and 95JB1 were 505 performed using Prokka (Prokka: Prokaryotic Genome Annotation System – 506 http://vicbioinformatics.com/) using a custom Escherichia genus database consisting of 507 protein sequences from the EcoCyc website (http://ecocyc.org/). Putative phage encoding loci 508 and IS elements were identified using PHAST (Zhou et al. 2011) and ISfinder (Siguier et al. 509 2006) respectively. Artemis Comparison Tool (ACT) (Carver et al. 2005), EasyFig (Sullivan 510 et al. 2011) and Circos (Krzywinski et al. References 528 Vol Neural Network Promoter Prediction http://www.fruitfly.org/seq_tools/promoter.html. Vol 529 2015. 530 Anjum A, Brathwaite KJ, Aidley J, Connerton PL, Cummings NJ, Parkhill J, Connerton I, 531 Bayliss CD. 2016. 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CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Sullivan MJ, Petty NK, Beatson SA. 2011. Easyfig: a genome comparison visualizer. 724 Bioinformatics 27: 1009-1010. 725 Suzuki M, Kondo F, Ito Y, Matsumoto M, Hata M, Oka H, Takahashi M, Sakae K. 2004. 726 Identification of a Shiga-toxin type I variant containing an IS1203-like element, from 727 Shiga-toxin producing Escherichia coli O157:H7. FEMS microbiology letters 234: 63- 728 67. 729 Takahashi N, Naito Y, Handa N, Kobayashi I. 2002. A DNA methyltransferase can protect 730 the genome from postdisturbance attack by a restriction-modification gene complex. 731 Journal of bacteriology 184: 6100-6108. 732 f gy Tilgner H, Grubert F, Sharon D, Snyder MP. 2014. Defining a personal, allele-specific, and 733 single-molecule long-read transcriptome. Proceedings of the National Academy of 734 Sciences of the United States of America 111: 9869-9874. Acknowledgements: 758 769 (b) Pairwise nucleotide comparison of three Stx2-converting phage from 95NR1. Phi10 , 770 Phi14 and Phi15 are represented to scale. Prophage encoded proteins are represented by 771 arrows coloured as per the legend. Yellow and grey shading between phage represent regions 772 of homology from 71% (yellow) to 100% (grey) nucleotide sequence identity. Prophage 773 genes are coloured as per the legend. 774 (b) Pairwise nucleotide comparison of three Stx2-converting phage from 95NR1. Phi10 , 770 Phi14 and Phi15 are represented to scale. Prophage encoded proteins are represented by 771 arrows coloured as per the legend. Yellow and grey shading between phage represent regions 772 of homology from 71% (yellow) to 100% (grey) nucleotide sequence identity. Prophage 773 genes are coloured as per the legend. 774 (b) Pairwise nucleotide comparison of three Stx2-converting phage from 95NR1. Phi10 , 770 Phi14 and Phi15 are represented to scale. Prophage encoded proteins are represented by 771 arrows coloured as per the legend. Yellow and grey shading between phage represent regions 772 of homology from 71% (yellow) to 100% (grey) nucleotide sequence identity. Prophage 773 genes are coloured as per the legend. 774 References 528 coli 95JB1 and 95NR1 highlighting the position and context 764 of Stx-carrying prophage. (a) Pairwise nucleotide comparison of 95NR1 (top) and 95JB1 765 (bottom) chromosomes. The chromosomes of 95NR1 and 95JB1 are represented to scale by 766 the black bar with the Stx-carrying prophage insertion points indicated with red rectangles. 767 Popouts display schematic representations of the four Stx-carrying phage carried on one or 768 both strains. 769 (b) Pairwise nucleotide comparison of three Stx2-converting phage from 95NR1. Phi10 , 770 Phi14 and Phi15 are represented to scale. Prophage encoded proteins are represented by 771 arrows coloured as per the legend. Yellow and grey shading between phage represent regions 772 of homology from 71% (yellow) to 100% (grey) nucleotide sequence identity. Prophage 773 genes are coloured as per the legend. 774 775 Figure 2: Comparison of 95JB1 and 95NR1 genome assemblies. 776 Circos plot comparing prophage and IS content of 95JB1 (left) and 95NR1 (right). Putative 777 prophage regions are highlighted on the outer most track by coloured rectangles: Stx- 778 converting (Red), myoviriadae (yellow), other (orange). Small, degenerate phage–associated 779 regions are highlighted on the outer most track in grey. The position and context of all IS 780 Acknowledgements: 758 Acknowledgements: 758 This work was supported by Discovery Grant DP170103962 from the Australian Research 759 Council. J.C.P. is a National Health and Medical Research (NHMRC) Senior Principal 760 Research Fellow; S.A.B. is a NHMRC Career Development Fellow. 761 762 Figure legends: 763 Figure 1: Comparison of E. coli 95JB1 and 95NR1 highlighting the position and context 764 of Stx-carrying prophage. (a) Pairwise nucleotide comparison of 95NR1 (top) and 95JB1 765 (bottom) chromosomes. The chromosomes of 95NR1 and 95JB1 are represented to scale by 766 the black bar with the Stx-carrying prophage insertion points indicated with red rectangles. 767 Popouts display schematic representations of the four Stx-carrying phage carried on one or 768 both strains. 769 (b) Pairwise nucleotide comparison of three Stx2-converting phage from 95NR1. Phi10 , 770 Phi14 and Phi15 are represented to scale. Prophage encoded proteins are represented by 771 arrows coloured as per the legend. Yellow and grey shading between phage represent regions 772 of homology from 71% (yellow) to 100% (grey) nucleotide sequence identity. Prophage 773 genes are coloured as per the legend. 774 This work was supported by Discovery Grant DP170103962 from the Australian Research 759 Council. J.C.P. is a National Health and Medical Research (NHMRC) Senior Principal 760 Research Fellow; S.A.B. is a NHMRC Career Development Fellow. 761 This work was supported by Discovery Grant DP170103962 from the Australian Research 759 Council. J.C.P. is a National Health and Medical Research (NHMRC) Senior Principal 760 Research Fellow; S.A.B. is a NHMRC Career Development Fellow. 761 Figure 1: Comparison of E. coli 95JB1 and 95NR1 highlighting the position and context 764 of Stx-carrying prophage. (a) Pairwise nucleotide comparison of 95NR1 (top) and 95JB1 765 (bottom) chromosomes. The chromosomes of 95NR1 and 95JB1 are represented to scale by 766 the black bar with the Stx-carrying prophage insertion points indicated with red rectangles. 767 Popouts display schematic representations of the four Stx-carrying phage carried on one or 768 both strains. 769 Figure 1: Comparison of E. coli 95JB1 and 95NR1 highlighting the position and context 764 of Stx-carrying prophage. (a) Pairwise nucleotide comparison of 95NR1 (top) and 95JB1 765 (bottom) chromosomes. The chromosomes of 95NR1 and 95JB1 are represented to scale by 766 the black bar with the Stx-carrying prophage insertion points indicated with red rectangles. 767 Popouts display schematic representations of the four Stx-carrying phage carried on one or 768 both strains. References 528 735 van der Woude MW, Braaten BA, Low DA. 1992. Evidence for global regulatory control of 736 pilus expression in Escherichia coli by Lrp and DNA methylation: model building 737 based on analysis of pap. Molecular microbiology 6: 2429-2435. 738 van der Woude MW, Henderson IR. 2008. Regulation and function of Ag43 (flu). Annual 739 review of microbiology 62: 153-169. 740 Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, 741 Wortman J, Young SK et al. 2014. Pilon: an integrated tool for comprehensive 742 microbial variant detection and genome assembly improvement. PloS one 9: e112963. 743 Wallecha A, Munster V, Correnti J, Chan T, van der Woude M. 2002. Dam- and OxyR- 744 dependent phase variation of agn43: essential elements and evidence for a new role of 745 DNA methylation. Journal of bacteriology 184: 3338-3347. 746 y f gy Yan X, Fratamico PM, Bono JL, Baranzoni GM, Chen CY. 2015. Genome sequencing and 747 comparative genomics provides insights on the evolutionary dynamics and pathogenic 748 potential of different H-serotypes of Shiga toxin-producing Escherichia coli O104. 749 BMC microbiology 15: 83. 750 Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. 751 Nucleic acids research 39: W347-352. 752 Zowawi HM, Forde BM, Alfaresi M, Alzarouni A, Farahat Y, Chong TM, Yin WF, Chan 753 KG, Li J, Schembri MA et al. 2015. Stepwise evolution of pandrug-resistance in 754 Klebsiella pneumoniae. Scientific reports 5: 15082. 755 757 27 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Acknowledgements: 758 This work was supported by Discovery Grant DP170103962 from the Australian Research 759 Council. J.C.P. is a National Health and Medical Research (NHMRC) Senior Principal 760 Research Fellow; S.A.B. is a NHMRC Career Development Fellow. 761 762 Figure legends: 763 Figure 1: Comparison of E. Figure 2: Comparison of 95JB1 and 95NR1 genome assemblies. 776 The 789 remaining coloured tracks highlight the chromosomal positions of all methylated sites for 790 each motif. Tracks are coloured as per the legend; GATC, red; CTGCAG, green; 791 CRARCAG, red. 792 and 95NR1, mapped to their complete genomes, are represented in black on the inner most 782 ring where assembly gaps are shown as white space. Green ribbons connect prophage 783 sequences common to both strains. Red lines connect IS that are common to both strains. 784 ring where assembly gaps are shown as white space. Green ribbons connect prophag 783 sequences common to both strains. Red lines connect IS that are common to both strains. 784 785 Figure 3: Comparison of methylated DNA on the chromosomes of 95JB1 and 95NR1. 786 Circos plot displaying the distribution of methylated nucleotides on the chromosomes of E 787 coli 95JB1 (left) and 95NR1 (right). Prophage insertion points are highlighted on the outer 788 Figure 2: Comparison of 95JB1 and 95NR1 genome assemblies. 776 Circos plot comparing prophage and IS content of 95JB1 (left) and 95NR1 (right). Putative 777 prophage regions are highlighted on the outer most track by coloured rectangles: Stx- 778 converting (Red), myoviriadae (yellow), other (orange). Small, degenerate phage–associated 779 regions are highlighted on the outer most track in grey. The position and context of all IS 780 elements are represented by red bars on the middle track. Draft Illumina assemblies of 95JB1 781 Circos plot comparing prophage and IS content of 95JB1 (left) and 95NR1 (right). Putative 777 prophage regions are highlighted on the outer most track by coloured rectangles: Stx- 778 converting (Red), myoviriadae (yellow), other (orange). Small, degenerate phage–associated 779 regions are highlighted on the outer most track in grey. The position and context of all IS 780 elements are represented by red bars on the middle track. Draft Illumina assemblies of 95JB1 781 28 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint and 95NR1, mapped to their complete genomes, are represented in black on the inner most 782 ring where assembly gaps are shown as white space. Green ribbons connect prophage 783 sequences common to both strains. Red lines connect IS that are common to both strains. 784 and 95NR1, mapped to their complete genomes, are represented in black on the inner most 782 ring where assembly gaps are shown as white space. Green ribbons connect prophage 783 sequences common to both strains. Red lines connect IS that are common to both strains. 784 785 Figure 3: Comparison of methylated DNA on the chromosomes of 95JB1 and 95NR1. 786 Circos plot displaying the distribution of methylated nucleotides on the chromosomes of E. 787 coli 95JB1 (left) and 95NR1 (right). Prophage insertion points are highlighted on the outer- 788 most track in orange with Stx-carrying prophage in red and myoviriadae in yellow. Figure 3: Comparison of methylated DNA on the chromosomes of 95JB1 and 95NR1. 786 Circos plot displaying the distribution of methylated nucleotides on the chromosomes of E. 787 coli 95JB1 (left) and 95NR1 (right). Prophage insertion points are highlighted on the outer- 788 most track in orange with Stx-carrying prophage in red and myoviriadae in yellow. The 789 remaining coloured tracks highlight the chromosomal positions of all methylated sites for 790 each motif. Tracks are coloured as per the legend; GATC, red; CTGCAG, green; 791 CRARCAG, red. 792 Circos plot displaying the distribution of methylated nucleotides on the chromosomes of E. 787 coli 95JB1 (left) and 95NR1 (right). Prophage insertion points are highlighted on the outer- 788 most track in orange with Stx-carrying prophage in red and myoviriadae in yellow. The 789 remaining coloured tracks highlight the chromosomal positions of all methylated sites for 790 each motif. Tracks are coloured as per the legend; GATC, red; CTGCAG, green; 791 CRARCAG, red. 792 793 29 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Table 1: Assembly Statistics 794 Strain Number of SMRTCells Number of PacBio contigs PacBio contig N50 (bp) Number of Illumina Contigs Illumina contig N50 (bp) 95JB1 2 4 5373164 179 91606 95NR1 2 3 5462770 182 94405 795 Table 2. SNPs differentiating 95NR1 and 95JB1 796 Strain Base Change 95JB1 site 95NR1 site Amino Acid Change Illumina* PacBio Annotation 95JB1 C-T 587038 587038 P-L + + glxK (Glycerate kinase II 95JB1 G-T 3227026 3227373 V-F + + End of Stx1 prophage 95JB1 G-A 3602596 3602945 E-K + + metK (Methionine adenosyltransferase 1) 95JB1 G-C 3813994 3814343 Stop codon-Y + + fadH (2,4-dienoyl- CoA reductase) p95JB1B G-C 20482 20477 P-A + + * SNPs identified by McAllister et al (McAllister et al. Predicted recognition motif based on the in silico bioinformatic characterisation of all MTase in 95JB1 and 95NR1 804 Figure 3: Comparison of methylated DNA on the chromosomes of 95JB1 and 95NR1. 786 2016) 797 Table 1: Assembly Statistics 794 Strain Number of SMRTCells Number of PacBio contigs PacBio contig N50 (bp) Number of Illumina Contigs Illumina contig N50 (bp) 95JB1 2 4 5373164 179 91606 95NR1 2 3 5462770 182 94405 795 Table 2. SNPs differentiating 95NR1 and 95JB1 796 797 800 30 Table 3. Summary of Methyltransferase genes from the chromosomes of 95JB1 and 95NR1 01 MTase# Typea 95JB1 Locus_tag 95NR1 Locus_tag Predicted Specificityb Homologa (% aa similarity) I IIb M.Eco95JB1I M.Eco95NR1I nonspecific M.EcoGI/GII(92.8/92.4) II IIb M.Eco95JB1II M.Eco95NR1II nonspecific M.EcoGI/GII(93.6/93.1) Dcm II M.Eco95JB1Dcm M.Eco95NR1Dcm CCWGG M.EcoGDcm (98.5) III IIP M.Eco95JB1III M.Eco95NR1III CTGCAG M.EcoGIII (99.7) IV IIP M.Eco95JB1IV M.Eco95NR1IV GATC M.EcoGV (99.6) V IIb M.Eco95JB1V M.Eco95NR1V ATGCAT M.EcoGVI (98.6) Dam IIa M.Eco95JB1Dam M.Eco95NR1Dam GATC M.EcoGDam (99.7 ) VI II - M.Eco95NR1VI GATC M.EcoGV (99.4) VII II - M.Eco95NR1VII GATC M.EcoVT2 (99.2) VIII II - M.Eco95NR1VIII GATC M.EcoVT2 (99.2) IX IIG M.Eco95JB1IX M.Eco95NR1IX CRARCAG SenTFIV (76) X IIb M.Eco95JB1X M.Eco95NR1X SAY M.EcoGIX (96) a Methyltransferases were classified based on similarity searches with the Rebase database 02 bSpecificities only included if determined by PacBio 03 *Predicted recognition motif based on the in silico bioinformatic characterisation of all MTase in 95JB1 and 95NR1 04 801 805 31 31 . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted August 11, 2017. ; https://doi.org/10.1101/173336 doi: bioRxiv preprint Table 4. MTase recognition motifs identified in 95JB1 and 95NR1 Motif Modification Type Number Detected Number in Chromosome Methylated (%) Mean IPD Ratio 95JB1 CTGCAG m6A 2390 2390 100.0 6.9934 053 GATC m6A 41658 41686 99.9 5.6757 846 95NR1 CTGCAG m6A 2434 2434 100.0 7.1674 423 CRARCAG m6A 4074 4074 100.0 7.1986 117 GATC m6A 42242 42270 99.9 5.7941 27 Table 4. MTase recognition motifs identified in 95JB1 and 95NR1
https://openalex.org/W4244152745	https://www.qeios.com/read/FXVIL1/pdf	English	null	Gonococcal Ophthalmia Neonatorum	Definitions	2,020	cc-by	62	Open Peer Review on Qeios Open Peer Review on Qeios Gonococcal Ophthalmia Neonatorum National Cancer Institute Qeios ID: FXVIL1 · https://doi.org/10.32388/FXVIL1 Qeios · Definition, February 2, 2020 Source National Cancer Institute. Gonococcal Ophthalmia Neonatorum. NCI Thesaurus. Code C116816. Inflammation of the conjunctiva in a newborn due to Neisseria gonorrhoeae which was acquired during labor and delivery. Qeios ID: FXVIL1 · https://doi.org/10.32388/FXVIL1 1/1
https://openalex.org/W2924899502	https://europepmc.org/articles/pmc6427035?pdf=render	English	null	Glucose tolerance in Canadian and French cystic fibrosis adult patients	Scientific reports	2,019	cc-by	7,944	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Glucose tolerance in Canadian and French cystic fibrosis adult patients Quitterie Reynaud 1,2, Valérie Boudreau3,4, Sandrine Touzet5,6, Katherine Desjardins3, Stéphanie Poupon Bourdy5,6, Emilie Blond7,8, Yves Berthiaume3,9, Rémi Rabasa-Lhoret3,4,9 & Isabelle Durieu1,2,5,6 Glucose tolerance in Canadian and French cystic fibrosis adult patients Received: 4 June 2018 Accepted: 4 February 2019 Published: xx xx xxxx Received: 4 June 2018 Accepted: 4 February 2019 Published: xx xx xxxx Cystic fibrosis (CF)-related diabetes is associated with increased mortality. We analysed the clinical and glycemic profiles of two cohorts of patients treated according to the same guidelines in France and Canada. To investigate incidence differences in phenotypic and glucose abnormalities and to explore the evolution over a 4-year follow-up period, two cohorts of 224 Canadian and 147 French adult CF patients (≥18 years) without treated CF-related diabetes (CFRD) were followed over a 4 year period. In each of these groups, we investigated the longitudinal relationship between glucose tolerance and pulmonary function. An annual 2-hour oral glucose tolerance test was performed: fasting blood glucose (G0) and 2-h blood glucose (G2) were measured. Patients were classified at inclusion according to their glucose tolerance status: Normal glucose tolerant, abnormal glucose tolerant or de novo CFRD. Age, sex ratio and proportion of F508del homozygous patients were not statistically different between both cohorts. Canadian patients had better pulmonary function (median %FEV1 (IQR): 71.0 (55.0–82.0) vs. 64.0 (40.0–78.0), p < 0.001) and greater body mass index (BMI; median BMI in kg/m2) (IQR) 21.1 (19.5–22.8) vs. 19.9 (18.4–21.4), p < 0.001). Glucose values: G0 (5.4 (5.0–5.9) vs. 4.8 (4.5–5.1) mmol/L, p < 0.001) and G2 (7.6 (5.8–9.7) vs. 6.5 (5.2–8.5) mmol/L, p = 0.001) were higher in the Canadian cohort translating into a higher incidence of de novo CFRD diagnosis (19.2 vs. 9.8%, p = 0.003). Decline in FEV1 over time was not different between patients according to glucose tolerance groups. Despite higher glucose levels and incidence of de novo CFRD, Canadian CF patients have a better lung function and a higher BMI than French patients. In spite of these differences between the cohorts, the decline in FEV1 in patients with abnormal glucose tolerance is similar between these groups. In the last decade, life expectancy of patients with cystic fibrosis (CF) has significantly improved. While the median life expectancy was 10 years of age in the 1960’s, it is now estimated to greater than 50 years both in Canada and France1,2. However, along with this better life expectancy, new complications have emerged. Received: 4 June 2018 Accepted: 4 February 2019 Published: xx xx xxxx Methods S d Study population. Data were obtained from the Montreal Cystic fibrosis Cohort (MCFC) for Canada and from the Lyon Cystic Fibrosis Cohort (DIAMUCO) for France. Both research cohorts are investigating mecha- nisms of glucose intolerance as well as associations between glucose intolerance and clinical outcomes in adult patients with CF. The Montreal Cystic Fibrosis Cohort was established in 2004 and all available data at inclusion from patients included between 2004 to 2016 were considered for this analysis. The DIAMUCO Cohort was established in 2009 and all available data at inlusion from patients included between 2009 to 2012 were considered for this analysis. All available follow-up pulmonary function data were included in both cohorts over a 4-year period.h Informed consents have been obtained for all subjects included. The institutional review board of each partic- ipating hospital and research ethics board authorized the cohorts in accordance with the current ethical stand- ards (Comité de Protection des Personnes in France, Comité d’éthique de la recherche in Canada), as well as the French data Protection Agency for DIAMUCO Cohort (Comission Nationale de l’Informatique et des Libertés CNIL).fi For both cohorts, all adult patients (>18 years) with CF, pancreatic insufficiency and no previous history of treated CFRD were included. Patients who received a de novo CFRD diagnosis during recruitment were however included. Main exclusion criteria were pancreatic sufficiency and previous pulmonary transplantation. In patients with ongoing pregnancy, pulmonary exacerbation or current treatment with medication known to interfere with glucose metabolism (e.g. high dose oral steroids and enteral tube feeding), OGTT was delayed upon resolu- tion of this temporary exclusion factor. OGTT results were accepted in patients taking stable, long-term (≥1 year) low-dose oral corticosteroids (maximal dose of 10 mg prednisone per day). At the time of the OGTT, all patients were clinically stable with no recent (<1 month) pulmonary exacerbation or symptoms of acute infec- tion. Included patients represent around 80% of all patients with CF followed at our respective clinics. Clinical and biological data. In both cohorts, a harmonized data collection process was used to extract required information from medical charts at inclusion. This included age, sex, CF related genotype, chronic bac- teriological colonization and number of intravenous antibiotics courses in the previous year. Chronic bacterio- logical colonization was defined as follow: 50% or more of samples being positive for a specific bacteria in the preceding 12 months18. Methods S d Other data such as BMI, FEV1, glycosylated hemoglobin (HbA1c) were also collected at inclusion and then between 12 and 18 months during a 4-year follow-up for FEV1. BMI was calculated using weight in kilograms divided by height in square meter (kg/m2). Pulmonary function was measured by spirom- etry using the forced expiratory volume in 1 second in L (FEV1) using Hankinson 1999 formula for FEV1(%)19. HbA1c was measured using HPLC automate variant II (Biorad) in France, and immunotubidimeter (Bayer Health Care diagnosis) in Canada. Both HbA1c are aligned with international standards. Oral glucose tolerance test (OGTT). OGTT was realized at the inclusion in both cohorts and was carried out after an overnight fast. Patients were given glucose (1.75 g per kg bodyweight, maximum 75 g) then plasma glucose was measured at start (G0), 1 hour (G1) and 2 hour (G2)20. Patients diagnosed with de novo CFRD under- went within 2 months a second OGTT to confirm the diagnosis. Plasma insulin was also measured at start (I0), 1 hour (I1) and 2 hour (I2) by immunoradiometric assay (BI-INS-IRMA, Cisbio Bioassays, France) and measures were centralized in Quebec, to obtain comparable values.if Depending on the results of the OGTT at inclusion, patients were classified into different subgroups of glu- cose tolerance according to international guidelines10: Normal Glucose Tolerance (NGT) (G0 ≤ 7.0 mmol/L and G2 ≤ 7.7 mmol/L), Abnormal Glucose Tolerance (AGT) defined as having either indeterminate glucose toler- ance (INDET) (G0 ≤ 7.0 mmol/L and G2 ≤ 7.7 mmol/L, but G1 ≥ 11.1 mmol/L) or impaired glucose tolerance (IGT) (G0 ≤ 7.0 mmol/L and G2 > 7.7 mmol/L but <11.1 mmol/L) or de novo CFRD (G0 > 7.0 mmol/L or G2 ≥ 11.1 mmol/L). Statistical methods. Values are expressed as median (interquartile range [IQR]) or percentage, as appro- priate. Demographic and clinical data, insulin and glucose values from both cohorts were compared at inclusion of patients of each cohort, using non parametric tests (Chi2 or Mann-Whitney tests, as appropriate). Subgroups analyses determined by glucose tolerance subgroup classification were then performed to compare clinical status, glucose and insulin values of both cohorts using non parametric tests (Chi2 or Mann-Whitney tests, as appro- priate). Correlation analysis between BMI and pulmonary function (FEV1), AUC glycemia and AUC insulin were made with Spearman’s correlation. Glucose tolerance in Canadian and French cystic fibrosis adult patients Abnormalities in glycemic status and CF-related diabetes (CFRD) have become the main complications after respiratory disease and exocrine pancreatic insufficiency3.hf In the last decade, life expectancy of patients with cystic fibrosis (CF) has significantly improved. While the median life expectancy was 10 years of age in the 1960’s, it is now estimated to greater than 50 years both in Canada and France1,2. However, along with this better life expectancy, new complications have emerged. Abnormalities in glycemic status and CF-related diabetes (CFRD) have become the main complications after respiratory disease and exocrine pancreatic insufficiency3.hf yfi y The prevalence of CFRD increases with age, affecting around 50% of adult CF patients. In adult patients with- out CFRD, the prevalence of abnormal glucose tolerance is 35%4. A progressive loss of beta cell mass with a direct effect from CFTR-mutation and a possible contributory role of insulin resistance lead to abnormal glucose levels and development of CFRD5–8. Because of the insidious onset of CFRD and the fact that standard simple screening tests are less reliable for CF patients (e.g. fasting glucose, glycosylated hemoglobin, etc.)9, annual screening for CFRD is recommended starting at ten years of age, using the 2-h Oral Glucose Tolerance Test (OGTT)10. CFRD is still associated with increased mortality despite the progressive improvement in screening and management11. 1Centre de référence Adulte de la Mucoviscidose, Service de médecine interne, Hospices civils de Lyon, F-69495, Pierre Bénite, France. 2Université de Lyon, Équipe d’Accueil Health Services and Performance Research (HESPER) 7425, F-69003, Lyon, France. 3Montreal Clinical Research Institute, Québec, Canada. 4Université de Montréal, Département de nutrition et de Médecine, Montréal, Québec, Canada. 5Hospices Civils de Lyon, Pôle de Santé Publique, Lyon, F-69003, France. 6Université de Lyon, Équipe d’Accueil Health Services and Performance Research (HESPER) 7425, Lyon, France. 7Service de Biochimie et Biologie Moléculaire, Hospices Civils de Lyon, F-69495, Pierre Bénite, France. 8Université de Lyon, INSERM U1060, Laboratoire CarMen, F-69003, Lyon, France. 9Cystic fibrosis clinic, Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada. Quitterie Reynaud, Valérie Boudreau contribution equally. Correspondence and requests for materials should be addressed to Q.R. (email: quitterie.reynaud@chu-lyon.fr) Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 1 www.nature.com/scientificreports/ Studies have compared demographic data of European and North American CF children patients using CF registries12,13, suggesting differences in demographic data (height and weight) between populations. Glucose tolerance in Canadian and French cystic fibrosis adult patients No data are available regarding difference in CFRD prevalence between Europe and North America except data from annual registry reports14. Furthermore, registry data concerning specific comorbidities like CFRD are not homogene- ously collected, and most of the time registries do not collect data about other glucose metabolic abnormalitites. It is thus possible that both prevalence of CFRD and its impact on clinical CF condition differ between these two populations. The consequences of different glucose tolerance profiles on respiratory function have not been evalu- ated in large cohorts and over long periods. Similarly, the potential role of insulin deficiency and insulin resistance have not been described and compared in large cohorts15–17.h p g The objective of this study was to compare the following parameters between the Canadian and French cohorts: (1) clinical characteristics, (2) glucose and insulin values as well as CFRD incidence and (3) evolution of pulmonary function over a 4-year follow-up period. Results Ch t Characteristics at baseline. Data of 224 Canadian and 147 French patients were included (See Table 1). Demographic and clinical data are detailed in Table 1. No difference of sex ratio, proportion of F508del homozy- gous patients and age was observed between the 2 cohorts. The clinical status of Canadian group was better with higher BMI (median in kg/m2 [IQR]) 21.1 [19.5–22.8] vs. 19.9 [18.4–21.4], p < 0.001 and higher FEV1 (median in % [IQR]) 71.0 [55.0–82.0] vs. 64.0 [40.0–78.0], p < 0.001. Accordingly, a higher proportion of patients with mild to normal predicted FEV1 (>70%) was observed in Canadians cohort (51.3% vs. 38.1%, p = 0.012). The propor- tion of patients colonized with Pseudomonas aeruginosa (PA), Burkholderia cepacia, and Aspergillus was not sta- tistically different between both cohorts. There was a higher proportion of patients colonized with Staphylococcus aureus (SA) in the French cohort (70.1% vs. 55.8%, p < 0.006). The number of intravenous antibiotics courses in the year preceding the OGTT was not statistically different between the 2 groups.h y p g yf g p The incidence of de novo CFRD diagnosis was higher in the Canadian population (19.2% vs. 9.8%, p = 0.003). Canadian cohort displayed a lower proportion of patients with normal glucose tolerance (36.6% vs. 53.4%, p = 0.003). Both fasting (G0) and 2-hours (G2) OGTT values (median in mmol/L [IQR]) were higher in the Canadian cohort: 5.4 [5.0–5.9] vs 4.8 [4.5–5.1], p < 0.001 for G0; 7.6 [5.8–9.7] vs. 6.5 [5.2–8.5], p = 0.001 for G2. The AUC OGTT curve for glucose values is higher in Canadian patients (p < 0.001). However, there was no statistical difference in HbA1c values between the two groups. Regarding insulin values (median μU/dl [IQR]) in Canadian compared to French patients, fasting (I0) and I2 values were higher in the Canadian cohort: 3.8 [2.3–5.7] vs. 3.2 [2.2–5.0], p = 0.031 for I0; and 27.4 [16.6–42.3] vs. 18.1 [11.0–33.0], p < 0.001 for I2. For the entire OGTT test area under the curve for insulin values is higher in Canadian patients (p < 0.001). Clinical status comparison among the different glucose tolerance groups. Subgroup analyses determined by glucose tolerance subgroup classification were performed (Table 2). Fourteen French patients were excluded from the analyses because data for either G1 or G2 were not available and their data did not allow classi- fication into glucose tolerance subgroups. Results Ch t %FEV1 (median in % [IQR]) was higher for Canadian patients for the NGT group compared to NGT French patients: 72.0 [56.0–86.0] vs. 64.0[38.0–80.0], p = 0.006, while no difference was observed for %FEV1 between Canada and France for the AGT and CFRD groups. BMI (median in kg/m2 [IQR]) was also significantly higher for NGT and AGT Canadian patients compared to the French patients: 20.8 [19.5–22.7] vs. 20.2 [18.4–21.4], p = 0.010 for NGT and 21.1 [19.5–23.1] vs. 19.8 [18.5–21.6], p = 0.002 for AGT. Glucose values according to glucose tolerance group. For each glucose tolerance category (see Table 2), Canadian patients displayed higher G0 median values and higher glucose median AUC as compared to French patients: p < 0.001 and p = 0.005 in NGT group, p < 0.001 and p < 0.001 in AGT group, and p = 0.005 and p = 0.006 in CFRD group. Insulin values according to glucose tolerance group. NGT and AGT Canadian patients displayed higher insulin median AUC than French patients (p < 0.001 and p = 0.040), but this difference no longer exists for patients with de novo CFRD, p = 0.278 (see Table 2). Insulin values according to glucose tolerance group. NGT and AGT Canadian patients displayed higher insulin median AUC than French patients (p < 0.001 and p = 0.040), but this difference no longer exists for patients with de novo CFRD, p = 0.278 (see Table 2). p Insulin sensitivity (Stumvoll index) was higher for NGT French patients (p = 0.039) compared to Canadian NGT patients, but no difference was observed in the 2 other subgroups. Insulin resistance (HOMA-IR) was higher for Canadian NGT and AGT patients (p = 0.004 and p = 0.048) compared to French patients.i g p (p p ) p p Despite higher level of insulin values for Canadian patients, the trends of the curve of insulin profile is similar for both Canadian and French NGT and AGT patients. Patients of both cohorts with NGT present a plasma insu- lin rise during the first hour of the OGTT (Fig. 1a), followed by moderated reduction at 2-h when glucose levels are trending downward. For all AGT patients, a similar insulin profile as for NGT-patients is observed for the 1st hour of the OGTT (Fig. 1b), but then insulin levels remain high at the second hour. For CFRD patients (Fig. www.nature.com/scientificreports/ FEV1 change over 4 years. Effect of glucose tolerance subgroup was controlled for the cohort and age. Interaction between covariates (cohort, NGT and AGT) and time were tested to characterize differences in longitudinal rates of change. The relationship between FEV1 decline and CFRD subgroup (56 patients) was not analyzed due to the small sample size and no available clinical data in the canadian group since confirmed de novo CFRD patients are excluded from the cohort after diagnosis. t g Analyses were performed using SPSS software (version 24 by IBM, Chicago, USA) and SAS® software (version 9.4, SAS® Institute Inc., Cary, NC, USA). Area under the curve (AUC) for glucose and insulin was calculated using the software GraphPad Prism (GraphPad Software Inc; CA, USA). A probability value ≤ 0.05 was consid- ered as statistically significant. Methods S d A linear mixed regression model with random intercept and random slope was fitted to assess the effect of glucose tolerance subgroup at entry in the cohort on the mean slope of Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 2 www.nature.com/scientificreports/ Results Ch t 1c), if insulin values are similar at the end of the test for both cohorts, it is slightly higher in Canadian patients at 1-h. However, the 1 h insulin peak observed in NGT and AGT patients is reduced by approximatively 30% for CFRD patients. Insulin values keep rising during the second hour of the test for Canadian and French CFRD patients. Insulin sensitivity and resistance and correlation analysis between BMI and pulmonary function, and between glycemic and clinical parameters in the global cohort. We observe higher insulin sensitivity during the OGTT using the Stumvoll index (median [IQR]) in NGT patients 0.111 [0.102–0.119] vs. both AGT 0.093 [0.085–0.104] and CFRD 0.059 [0.047–0.071] groups and in AGT patients 0.093 [0.085–0.104] vs. CFRD 0.059 [0.047–0.071] (p < 0.001). For fasting insulin resistance (HOMA-IR, median [IQR]), it is higher in CFRD 1.15 [0.71–1.45] than in NGT 0.76 [0.47–1.16] and AGT patients 0.82 [0.51–1.22] (p = 0.013).ti p p After controlling for cohort, we observed a significant positive correlation (Fig. 2) between BMI and pul- monary function (FEV1) for all glucose tolerance groups (respectively p < 0.001, p = 0.001, p < 0.001 for NGT, AGT and CFRD). No significant correlation was observed for all subgroups between BMI and AUC glycemia. Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 3 www.nature.com/scientificreports/ Canada France p value N = 224 N = 147 Gender: woman, % 42.0 43.5 0.764* Age in year, median (IQR) 22.0 (19.0–28.0) 22.5 (19.0–28.7) 0.831 ∆F508 homozygous, % 57.9 55.1 0.818* %FEV1, median (IQR) 71.0 (55.0–82.0) 64.0 (40.0–78.0) 0.001 %FEV1 > 70%, % 51.3 38.1 0.012* BMI in kg/m2, median (IQR) 21.1 (19.5–22.8) 19.9 (18.4–21.4) <0.001 Colonized with P. Aeruginosa, % 74.5 67.3 0.155* Colonized with B. Cepacia, % 2.8 2.7 0.968* Colonized with S. Results Ch t Aureus, % 55.8 70.1 0.006* Colonized with Aspergillus, % 42.3 33.3 0.084 Patients requiring IV antibiotics in the year prior the OGTT, % 41.1 49.0 0.238* Glycemia G0 in mmol/L, median (IQR) 5.4 (5.0–5.9) 4.8 (4.5–5.1) <0.001 Glycemia G2 in mmol/L, median (IQR) 7.6 (5.8–9.7) 6.5 (5.2–8.5) 0.001 AUC Glycemia (G0, G1, G2), median (IQR) 1059.5 (914.9–1239.0) 913.5 (761.2–1043.2) <0.001 NGT, % 36.6 53.4 0.003* INDET, % 16.5 9.0 IGT, % 27.7 27.8 De novo CFRD, % 19.2 9.8 HbA1c in %, median (IQR) 5.8 (5.5–6.1) 5.7 (5.5–6.0) 0.825 Insulin I0 in μU/dl, median (IQR) 3.8 (2.3–5.7) 3.2 (2.2–5.0) 0.031 Insulin I2 in μU/dl, median (IQR) 27.4 (16.6–42.3) 18.1 (11.0–33.0) <0.001 AUC Insulin (I0, I1, I2), median (IQR) 2530.0 (1837.0–3649.0) 1974.0 (1302.0–2910.0) <0.001 Stumvoll Index, median (IQR) 0.096 (0.078–0.111) 0.103 (0.088–0.116) <0.001 HOMA-IR, median (IQR) 0.93 (0.55–1.39) 0.68 (0.47–1.07) <0.001 Table 1. Comparison of demographic characteristics and clinical data at inclusion of the Canadian and French patients. Bold values represent significant differences. Abbreviations: AGT: Abnormal glucose tolerance (INDET: indeterminate glucose tolerance + IGT: impaired glucose tolerance), AUC: area under the curve, BMI: body mass index, CFRD: cystic fibrosis-related diabetes, CRP: C reactive protein, FEV1: predicted forced expiratory volume in 1 second, G0: plasma glucose measured at start of OGTT, G2: plasma glucose measured at 2 hours of OGTT, HbA1c: glycated hemoglobin, HOMA-IR: Homeostasis model assessment of insulin resistance, IV antibiotics: number of days of intravenous antibiotics in the year of OGTT, NGT: normal glucose tolerance, P. Aeruginosa: Pseudomonas aeruginosa, S. Aureus: Staphylococcus aureus, : p value was determined by chi2. Concerning BMI and AUC insulin, a significant correlation was observed in NGT subgroup (p = 0.004) but not in AGT and CFRD patients. No significant correlation between pulmonary function (FEV1) and AUC glycemia were observed for all glucose tolerance groups (data not shown). FEV1 evolution during follow-up according to glucose tolerance subgroup. FEV1 measurements during the 4-year follow-up period were obtained in 301 patients (81% of overall cohort: 181 French and 120 Canadian). No inter- action for glucose tolerance subgroup with time were observed, indicating that the longitudinal changes in FEV1 were not different between NGT and AGT groups (difference in mean annual FEV1 change in NGT compared to AGT group (0.4% 95% CI [−0.5–1.3], p = 0.375). Results Ch t However in all glucose tolerance subgroup a significant dif- ference in mean change in FEV1 per year was observed when French and Canadian patients were compared with Canadian patients having a slower mean annual decline of their FEV1 (difference in mean annual FEV1 change 0.89%, 95% CI [0.0;1.77], p = 0.049), Fig. 3. Discussion Recently, insulin resistance variations has Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 4 www.nature.com/scientificreports/ NGT AGT (INDET + IGT) De Novo CFRD Canada France P value Canada France P value Canada France P value n 82 71 99 49 43 13 Age in year, median (IQR) 22.0 (20.0–25.0) 24.0 (19.0–31.0) 0.063 22.0 (19.0–27.0) 22.0 (19.0–26.0) 0.591 25.0 (20.0–30.0) 27.0 (20.0.−37.0) 0.736 %FEV1, median (IQR) 72.0 (56.0–86.0) 64.0 (38.0–80.0) 0.006 73.0 (54.7–82.0) 65.0 (42.5–81.5) 0.143 61.0 (51.0–79.0) 51.0 (42.5–67.0) 0.098 %FEV1 > 70%, % 53.6 39.4 0.079 56.1 42.8 0.129* 35.7 23.1 0.396* BMI in kg/m2, median (IQR) 20.8 (19.5–22.7) 20.2 (18.4–21.4) 0.010 21.1 (19.5–23.1) 19.8 (18.5–21.6) 0.002 21.1 (19.3–22.9) 20.2 (18.8–21.1) 0.178 HbA1c in %, median (IQR) 5.7 (5.3–5.9) 5.7 (5.5–6.0) 0.325 5.7 (5.5–6.0) 5.8 (5.5–6.0) 0.759 6.1 (5.8–6.9) 6.3 (5.6–6.7) 0.796 G0 in mmol/L, median (IQR) 5.2 (4.9–5.5) 4.7 (4.4–5.0) <0.001 5.4 (5.0–5.8) 4.8 (4.5–5.2) <0.001 6.3 (5.4–7.4) 5.0 (4.7–6.3) 0.005 G2 in mmol/L, median (IQR) 5.6 (4.8–6.7) 5.4 (4.6–6.5) 0.234 8.3 (6.8–9.5) 8.3 (7.6–9.2) 0.851 13.6 (11.5–16.7) 13.0 (12.3–14.2) 0.437 AUC Glycemia (G0, G1, G2), median (IQR) 881 (783–943) 834 (735–918) 0.005 1105 (1041–1212) 1026 (951–1122) <0.001 1513 (1342–1776) 1293 (1212–1447) 0.006 AUC Insulin (I0, I1, I2), median (IQR) 2483 (1906–3768) 1866 (1257–2649) <0.001 2652 (2022–3678) 2283 (1318–3247) 0.040 2008 (1520–3255) 1833 (1083–2856) 0.278 Stumvoll index, median (IQR) 0.110 (0.099–0.117) 0.114 (0.103–0.121) 0.039 0.093 (0.085–0.105) 0.092 (0.087–0.104) 0.735 0.060 (0.048–0.071) 0.065 (0.050–0.074) 0.432 HOMA-IR, median (IQR) 0.88 (0.53–1.40) 0.64 (0.39–1.04) 0.004 0.91 (0.54–1.32) 0.65 (0.49–1.04) 0.048 1.17 (0.69–1.49) 1.00 (0.53–1.32) 0.331 Table 2. Comparison between glucose tolerance groups of the Canadian and French patients. Bold values represent significant differences. Abbreviations: AGT: Abnormal glucose tolerance (INDET: indeterminate glucose tolerance + IGT: impaired glucose tolerance), AUC: area under the curve, BMI: body mass index, CFRD: cystic fibrosis-related diabetes, CRP: C reactive protein,FEV1: predicted forced expiratory volume in 1 second, HbA1c: glycated hemoglobin, HOMA-IR: Homeostasis model assessment of insulin resistance, NGT: normal glucose tolerance. Mann-Whitney analysis were performed except for p value with * that were determined by chi2. Figure 1. Insulin secretion (μU/dl) at start, 1-h and 2-h of the OGTT for. Discussion Abbreviations: AGT: Abnormal glucose tolerance (INDET: indeterminate glucose tolerance + IGT: impaired glucose tolerance), AUC: area under the curve, BMI: body mass index, CFRD: cystic fibrosis-related diabetes, CRP: C reactive protein,FEV1: predicted forced expiratory volume in 1 second, HbA1c: glycated hemoglobin, HOMA-IR: Homeostasis model assessment of insulin resistance, NGT: normal glucose tolerance. Mann-Whitney analysis were performed except for p value with * that were determined by chi2. Table 2. Comparison between glucose tolerance groups of the Canadian and French patients. Bold values represent significant differences. Abbreviations: AGT: Abnormal glucose tolerance (INDET: indeterminate glucose tolerance + IGT: impaired glucose tolerance), AUC: area under the curve, BMI: body mass index, CFRD: cystic fibrosis-related diabetes, CRP: C reactive protein,FEV1: predicted forced expiratory volume in 1 second, HbA1c: glycated hemoglobin, HOMA-IR: Homeostasis model assessment of insulin resistance, NGT: normal glucose tolerance. Mann-Whitney analysis were performed except for p value with * that were determined by chi2. glucose tolerance + IGT: impaired glucose tolerance), AUC: area under the curve, BMI: body mass index, CFRD: cystic fibrosis-related diabetes, CRP: C reactive protein,FEV1: predicted forced expiratory volume in 1 second, HbA1c: glycated hemoglobin, HOMA-IR: Homeostasis model assessment of insulin resistance, NGT: normal glucose tolerance. Mann-Whitney analysis were performed except for p value with * that were determined by chi2. Figure 1. Insulin secretion (μU/dl) at start, 1-h and 2-h of the OGTT for. (a) NGT, (b) AGT and (c) CFRD patients according to their respective cohorts: black dot (•) for Canadian patients and black square (◼) for French patients. Values are presented as mean ± SEM. Abbreviations: AGT: abnormal glucose tolerance, CA: Canadian patients, CFRD: cystic fibrosis-related diabetes, FR: French patients, NGT: normal glucose tolerance. Figure 1. Insulin secretion (μU/dl) at start, 1-h and 2-h of the OGTT for. (a) NGT, (b) AGT and (c) CFRD patients according to their respective cohorts: black dot (•) for Canadian patients and black square (◼) for French patients. Values are presented as mean ± SEM. Abbreviations: AGT: abnormal glucose tolerance, CA: Canadian patients, CFRD: cystic fibrosis-related diabetes, FR: French patients, NGT: normal glucose tolerance. emerged as a possible contributor to hyperglycemia for patients with CF8,24. Higher BMI could contribute to higher insulin resistance in Canadian patients25,26. As insulin is an anabolic hormone, as long as a certain degree of insulin secretion is preserved, this could allow a higher BMI27. Discussion To our knowledge, this is the first study that compares the incidence of glucose abnormalities in adult patients with CF, in two large cohorts and their association with clinical status. Despite higher glucose levels and incidence of de novo CFRD, Canadian CF patients secrete more insulin and have a better pulmonary and nutritional status according to their FEV1 and BMI than French patients. To explore the mechanisms of these differences, we con- ducted correlation analyses but we observed no correlation between BMI and glycemia AUC. Canadian patients also have slower annual decrease of their FEV1 than French patients during follow-up. No significant difference was observed for pulmonary function change over time between AGT and NGT patients. These observations challenge the concept of a possible causal role of hyperglycemia and/or hypoinsulinemia favouring clinical status (BMI and/or FEV1) degradation. In the context of a well-established limited insulin secretion in adult CF patients21,22, three key factors could contribute to the development of hyperglycemia: progression of insulin secretory deficiency, higher insulin resistance or higher insulin requirements22,23. In Canadian patients, we observe higher insulin secretory capacity, higher estimated insulin resistance but also higher CFRD incidence. Discussion (a) NGT, (b) AGT and (c) CFRD i di h i i h bl k d ( ) f C di i d bl k (◼) f NGT AGT (INDET + IGT) De Novo CFRD Canada France P value Canada France P value Canada France P value n 82 71 99 49 43 13 Age in year, median (IQR) 22.0 (20.0–25.0) 24.0 (19.0–31.0) 0.063 22.0 (19.0–27.0) 22.0 (19.0–26.0) 0.591 25.0 (20.0–30.0) 27.0 (20.0.−37.0) 0.736 %FEV1, median (IQR) 72.0 (56.0–86.0) 64.0 (38.0–80.0) 0.006 73.0 (54.7–82.0) 65.0 (42.5–81.5) 0.143 61.0 (51.0–79.0) 51.0 (42.5–67.0) 0.098 %FEV1 > 70%, % 53.6 39.4 0.079* 56.1 42.8 0.129* 35.7 23.1 0.396* BMI in kg/m2, median (IQR) 20.8 (19.5–22.7) 20.2 (18.4–21.4) 0.010 21.1 (19.5–23.1) 19.8 (18.5–21.6) 0.002 21.1 (19.3–22.9) 20.2 (18.8–21.1) 0.178 HbA1c in %, median (IQR) 5.7 (5.3–5.9) 5.7 (5.5–6.0) 0.325 5.7 (5.5–6.0) 5.8 (5.5–6.0) 0.759 6.1 (5.8–6.9) 6.3 (5.6–6.7) 0.796 G0 in mmol/L, median (IQR) 5.2 (4.9–5.5) 4.7 (4.4–5.0) <0.001 5.4 (5.0–5.8) 4.8 (4.5–5.2) <0.001 6.3 (5.4–7.4) 5.0 (4.7–6.3) 0.005 G2 in mmol/L, median (IQR) 5.6 (4.8–6.7) 5.4 (4.6–6.5) 0.234 8.3 (6.8–9.5) 8.3 (7.6–9.2) 0.851 13.6 (11.5–16.7) 13.0 (12.3–14.2) 0.437 AUC Glycemia (G0, G1, G2), median (IQR) 881 (783–943) 834 (735–918) 0.005 1105 (1041–1212) 1026 (951–1122) <0.001 1513 (1342–1776) 1293 (1212–1447) 0.006 AUC Insulin (I0, I1, I2), median (IQR) 2483 (1906–3768) 1866 (1257–2649) <0.001 2652 (2022–3678) 2283 (1318–3247) 0.040 2008 (1520–3255) 1833 (1083–2856) 0.278 Stumvoll index, median (IQR) 0.110 (0.099–0.117) 0.114 (0.103–0.121) 0.039 0.093 (0.085–0.105) 0.092 (0.087–0.104) 0.735 0.060 (0.048–0.071) 0.065 (0.050–0.074) 0.432 HOMA-IR, median (IQR) 0.88 (0.53–1.40) 0.64 (0.39–1.04) 0.004 0.91 (0.54–1.32) 0.65 (0.49–1.04) 0.048 1.17 (0.69–1.49) 1.00 (0.53–1.32) 0.331 Table 2. Comparison between glucose tolerance groups of the Canadian and French patients. Bold values represent significant differences. Abbreviations: AGT: Abnormal glucose tolerance (INDET: indeterminate glucose tolerance + IGT: impaired glucose tolerance), AUC: area under the curve, BMI: body mass index, CFRD: cystic fibrosis-related diabetes, CRP: C reactive protein,FEV1: predicted forced expiratory volume in 1 second, HbA1c: glycated hemoglobin, HOMA-IR: Homeostasis model assessment of insulin resistance, NGT: normal glucose tolerance. Mann-Whitney analysis were performed except for p value with * that were determined by chi2. Table 2. Comparison between glucose tolerance groups of the Canadian and French patients. Bold values represent significant differences. Discussion Indeed, when insulin secretion further deteri- orates leading to de novo CFRD, both cohorts do not present anymore differences for BMI and insulin secretion. The frequency of exacerbations which is a good marker of respiratory function stability does not seem to be impli- cated in observed insulin resistance differences as the number of antibiotic courses is similar in both cohorts. It is also possible that insulin secretion itself might explain clinical differences between cohorts rather than blood glucose. A higher insulin secretion level might allow to reach and/or maintain a higher weight28. In a context of a limited insulin secretion capacity, hyperglycemia could also play a role by favoring pulmonary exacerbations29 and promoting oxidative stress30. Both higher insulin as well as lower glucose values can thus contribute to better lung function. g In contrast to previous reports, our results highlight that despite higher glucose values, Canadian CF patients have better pulmonary function and BMI. Despite these higher glucose values, Canadian patients also presented a slower annual FEV1 decline. When both cohorts are combined and the pulmonary evolution of patients with AGT is compared to patients with NGT, there is no difference in annual FEV1 decline. Various factors not related Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 5 www.nature.com/scientificreports/ Figure 2. Spearman’s correlation for (a) BMI and FEV1in NGT patients (b) BMI and FEV1 in AGT patients and (c) BMI and FEV1 in CFRD patients (d) BMI and AUC glucose in NGT patients (e) BMI and AUC glucose in AGT patients (f) BMI and AUC glucose in CFRD patients (g) BMI and AUC insulin in NGT patients (h) BMI and AUC insulin in AGT patients (i) BMI and AUC insulin in CFRD patients. Blue diamond: Canadian cohort and Red square: French cohort. Figure 2. Spearman’s correlation for (a) BMI and FEV1in NGT patients (b) BMI and FEV1 in AGT patients and (c) BMI and FEV1 in CFRD patients (d) BMI and AUC glucose in NGT patients (e) BMI and AUC glucose in AGT patients (f) BMI and AUC glucose in CFRD patients (g) BMI and AUC insulin in NGT patients (h) BMI and AUC insulin in AGT patients (i) BMI and AUC insulin in CFRD patients. Blue diamond: Canadian cohort and Red square: French cohort. Discussion 0 1 2 0 1 0 2 0 3 0 4 0 N G T O G T T tim e p o in t (h o u rs ) In s u lin s e c re tio n ( U /d l) 0 1 2 0 1 0 2 0 3 0 4 0 A G T O G T T tim e p o in t (h o u rs ) 0 1 2 0 1 0 2 0 3 0 4 0 C F R D O G T T tim e p o in t (h o u rs ) C a n a d ia n p a tie n ts F re n c h P a tie n ts Figure 3. Mean FEV1 change in FEV1 according to glucose tolerance subgroup and cohort. Values are presented as mean ± SEM. Abbreviations: AGT: abnormal glucose tolerance, CA: Canadian patients, FR: French patients, NGT: normal glucose tolerance. 1 2 N G T tim e p o in t (h o u rs ) 0 1 2 0 1 0 2 0 3 0 4 0 A G T O G T T tim e p o in t (h o u rs ) 0 1 2 0 1 0 2 0 3 0 4 0 C F R D O G T T tim e p o in t (h o u rs ) C a n a d ia n p a tie n ts F re n c h P a tie n ts Figure 3. Mean FEV1 change in FEV1 according to glucose tolerance subgroup and cohort. Values are presented as mean ± SEM. Abbreviations: AGT: abnormal glucose tolerance, CA: Canadian patients, FR: French patients, NGT: normal glucose tolerance. to cystic fibrosis can also explain this observation. Among them, the role of genetic factors other than CFTR mutations may explain the observed differences between these two cohorts (modifier genes). In the present study, the distribution for F508del CFTR mutation proportions is similar between the two cohorts but it is now well established that other mutations are associated, for some of them, with lung disease severity and for others, with diabetes susceptibility31,32. This emerging important factor may also have a role in observed glucose and BMI differences. www.nature.com/scientificreports/ limiting the ability to compare different populations33. In addition, despite merging two large and well character- ized cohorts, the sample size of some subgroups, such as de novo CFRD, remains small thus limiting our ability to explore some differences. pf In order to interpret our results, the potential mechanisms of higher BMI in Canadian patients should be explored. Key factors involved in energy balance and nutritional intake, absorption and energy expenditure (physical activity, energy demand related to CF exacerbations, etc.) should be evaluated. Nutritional recommen- dations are similar in North America and Europe with a recommended energy intake range from 120 to 150% of energy needs for the healthy population of similar age, sex and size. Patients included in both countries are also exposed to similar pancreatic enzyme replacement therapy (PERT) protocol, starting at 500 U lipase/kg/meal to a maximal dose of 1000–2500 U lipase/kg/meal which should lead to similar nutrient absorption capacity. Thus the two cohorts should be exposed to similar quantitative nutritional intake and absorption. The frequency of exacerbations necessitating intravenous antibiotics does not seem to play a role on BMI values as the number of antibiotics course is similar in both cohorts. International guidelines for antibiotic use in CF are worldwide applied and this may contribute to the very close use of antibiotics in Canadian and French cohorts. However, chronic higher caloric intake and/or differences in physical activity may still be important factors in explaining the differences and may play a role in the higher BMI of Canadian patients. In addition, backgroud population differences in diabetes and obesity, which are both higher in Canada compared to France34–37, could also explain the disparities between French and Canadians independently of CF status. As previously reported herein, there is a positive correlation between BMI and FEV1 which could explain the higher FEV1 observed in Canadian patients as well as their lower mean annual FEV1 decline. Observed association and differences do not imply causality and despite our careful assessement of two large nd well characterized cohorts important underlying mechanistic factors were not measured in that study. In conclusion, Canadian patients present a better clinical status (higher BMI, insulin secretion and FEV1) than French patients, but unexpectedly they also present a higher incidence of glucose abnormalities. In addition, patients in the abnormal glucose tolerance group do not have worse mean FEV1 decline over observed time than patients with normal glucose tolerance. www.nature.com/scientificreports/ To better understand the complex interplay between glucose tolerance and clincal status (BMI and/or FEV1) of adult patients with CF, further investigations should focus on potential underlying factors that may play a role in the observed differences. References 1. Cystic Fibrosis Canada. Focus on a cure 2014–2015 Annual Report. 1. Cystic Fibrosis Canada. Focus on a cure 2014–2015 Annual Report. y p 2. Registre Français de la Mucoviscidose. Bilan des données 2014. Vaincre la Mucoviscidose et Institut national d’étude démographiques (Ined). Paris, mars 2016.i g p q ( ) 3. Costa, M. et al. Diabetes: a major co-morbidity of cystic fibrosis. Diabetes Metab. 31, 221–232 (2005).i g p q 3. Costa, M. et al. Diabetes: a major co-morbidity of cystic fibrosis. Diabetes Metab. 31, 221–232 (2005).i j y yi 4. O’Shea, D. & O’Connell, J. Cystic fibrosis related diabetes. Curr. Diab. Rep. 14, 511 (2014).i i 5. Ode, K. L. & Moran, A. New insights into cystic fibrosis-related diabetes in children. Lancet Diabetes Endocrinol. 1, 52–58 (20if 6. Street, M. E. et al. Insulin production and resistance in cystic fibrosis: effect of age, disease activity, and genotype. J. Endocrinol Invest. 35, 246–253 (2012).i 7. Ntimbane, T. et al. Cystic fibrosis-related diabetes: from CFTR dysfunction to oxidative stress. Clin. Biochem. Rev. 30, 153–177 (2009). ( ) 8. Boudreau, V. et al. Variation of glucose tolerance in adult patients with cystic fib sensitivity? J. Cyst. Fibros., https://doi.org/10.1016/j.jcf.2016.04.004 (2016). 8. Boudreau, V. et al. Variation of glucose tolerance in adult patients with cystic fibrosis: What is the potential contribution of insulin eau, V. et al. Variation of glucose tolerance in adult patients with cystic fibrosis: What is the potential contribution of insulin vity? J Cyst Fibros https //doi org/10 1016/j jcf 2016 04 004 (2016) 8. Boudreau, V. et al. Variation of glucose tolerance in adult patients with cystic fibrosis: What is the potential cont sensitivity? J. Cyst. Fibros., https://doi.org/10.1016/j.jcf.2016.04.004 (2016). oudreau, V. et al. Variation of glucose tolerance in adult patients with cystic fibrosis: What is the potential contribution of insulin nsitivity? J. Cyst. Fibros., https://doi.org/10.1016/j.jcf.2016.04.004 (2016). i sensitivity? J. Cyst. Fibros., https://doi.org/10.1016/j.jcf.2016.04.004 (2016). i sensitivity? J. Cyst. Fibros., https://doi.org/10.1016/j.jcf.2016.04.004 (2016). 9. Boudreau, V. et al. Screening for Cystic Fibrosis-Related Diabetes: Matching Pathophysiology and Addressing Current Challenges. Can. J. Diabetes 40, 466–470 (2016). 10. Moran, A. et al. Clinical care guidelines for cystic fibrosis-related diabetes: a position statement of the American Diabetes Association and a clinical practice guideline of the Cystic Fibrosis Foundation, endorsed by the Pediatric Endocrine Society. Diabetes Care. 33, 2697–2708 (2010).i 11. Lewis, C. et al. Diabetes-related mortality in adults with cystic fibrosis. Discussion For example, some mutations in genes involved in a higher risk for type 2 diabetes (e.g. TCF7L2) are also associated with a higher and earlier CFRD prevalence32. Further investigations need to be done to compare modifier genes between the 2 cohorts. Secondly, despite comparable nutritional and clinical recommendations for CF care as well as health care systems (universal access) between the 2 cohorts, other factors can still influence glucose values as well as the nutritional status of CF-patients. For example, differences in qualitative and quanti- tative nutritional intake could impact both glucose tolerance and BMI. Unfortunately, nutritional intake was not assessed in the present study. If done in the future, such assessment could be limited by the precision of available tools (e.g. food journals, 24-hour dietary recall, etc.) as well as the fact that most tools are country specific thus to cystic fibrosis can also explain this observation. Among them, the role of genetic factors other than CFTR mutations may explain the observed differences between these two cohorts (modifier genes). In the present study, the distribution for F508del CFTR mutation proportions is similar between the two cohorts but it is now well established that other mutations are associated, for some of them, with lung disease severity and for others, with diabetes susceptibility31,32. This emerging important factor may also have a role in observed glucose and BMI differences. For example, some mutations in genes involved in a higher risk for type 2 diabetes (e.g. TCF7L2) are also associated with a higher and earlier CFRD prevalence32. Further investigations need to be done to compare modifier genes between the 2 cohorts. Secondly, despite comparable nutritional and clinical recommendations for CF care as well as health care systems (universal access) between the 2 cohorts, other factors can still influence glucose values as well as the nutritional status of CF-patients. For example, differences in qualitative and quanti- tative nutritional intake could impact both glucose tolerance and BMI. Unfortunately, nutritional intake was not assessed in the present study. If done in the future, such assessment could be limited by the precision of available tools (e.g. food journals, 24-hour dietary recall, etc.) as well as the fact that most tools are country specific thus Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 6 www.nature.com/scientificreports/ References Diagn. Res. 7, 1764–1766 (2013).i l 6. Williams, C. A., Saynor, Z. L., Tomlinson, O. W. & Barker, A. R. Cystic fibrosis and physiological responses to exercise. Expert Rev Respir. Med. 8, 751–762 (2014). p 7. Peterson, M. L., Jacobs, D. R. & Milla, C. E. Longitudinal changes in growth parameters are correlated with changes in pulmonary function in children with cystic fibrosis. Pediatrics. 112, 588–592 (2003).h yi ( ) 28. Coriati, A., Ziai, S., Lavoie, A., Berthiaume, Y. & Rabasa-Lhoret, R. The 1-h oral glucose tolerance test glucose and insulin value associated with markers of clinical deterioration in cystic fibrosis. Acta Diabetol. 53, 359–66 (2016).fi k yi , ( ) 29. Brennan, A. L. et al. Airway glucose concentrations and effect on growth of respiratory pathogens in cystic fibrosis. J. Cyst. Fibros. 6, 101–9 (2007). i 29. Brennan, A. L. et al. Airway glucose concentrations and effect on growth of respiratory pathogens in cystic fibrosis. J. Cyst. Fibr 101–9 (2007).i 30. Ntimbane, T. et al. Oxidative stress and cystic fibrosis-related diabetes: a pilot study in children. J. Cyst. Fibros. 7, 373–84 (200ii yi p y y 31. Guillot, L. et al. Lung disease modifier genes in cystic fibrosis. Int. J. Biochem. Cell Biol. 52, 83–93 (2014).ii . Blackman, S. M. et al. Genetic modifiers of cystic fibrosis-related i yi 3. Thompson, F. E. et al. The National Cancer Institute’s Dietary Assessment Primer: A Resource for Diet Research. J. Acad. Nutr. Diet 115, 1986–1995 (2015). 4. OECD, “Overweight and obesity among adults”, in Health at a Glance 2017: OECD Indicators, OECD Publishing, Paris (2017).i g y g g 35. Public Health agency of Canada. Diabetes in Canada: Facts and figures from a public health perspective. (July 2011). g yi g p p p (J y ) 36. Matta, J. et al. Overweight, obesity and cardiometabolic risk factors prevalence in france: the constances cohort. Bu Epidémiologique. Institut National de Veille Sanitaire (Invs). Santé publique France. (Octobre 2016). p g q p q 37. Mandereau-Bruno, L. et al. Prévalence du diabète traité pharmacologiquement et disparités territoriales en 2012. Bulletin Epidémiologique. 30-31, 493–99 (2012). 7. Mandereau-Bruno, L. et al. Prévalence du diabète traité pharmacologiquement et disparités territoriales en 2012. Bulletin Epidémiologique. 30-31, 493–99 (2012). Acknowledgements g We would like to thank all CF patients for their participation as well as both CF and diabetes clinics’ teams in France and Canada for their help to collect data and to perform OGTTs. This study was supported by a CF Canada operating grant (to RRL), a Vaincre la Mucoviscidose operating grant (to ID), an Université de Montreal gant for international collaboration (to RRL) and the J-A DeSève chair (to RRL). VB has a doctoral Banting and Best scholarship from the Canadian Institutes of Health Research and a scholarship from the Fonds de Recherche en Santé du Québec. We also would like to thank Angélique Denis who largely contributed to this work. This work was supported by a grant to ID from the association Vaincre la Mucoviscidose (#RC20140501069), an operating grant to RRL from the Canadian Cystic Fibrosis Foundation (#2883), and funds to RRL: the Direction des relations internationales (DRI) of the Université de Montréal and the J-A DeSève Diabetes research chair. VB was supported by the Canadian Institute of Health Research and by the Fonds de recherche du Québec – Santé scholarships. Author Contributions Substantial contributions to the conception or design of the work: Q.R., V.B., S.T., K.D., S.P.B., Y.B., R.R.L. and I.D. Drafting the work: Q.R.,V.B., R.R.L. and I.D. Final approval of the version to be published: Q.R., V.B., S.T. and K.D., S.P.B., E.B., Y.B., R.R.L. and I.D. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: R.R.L. and I.D. References Role of genotype and sex. Am. J. Respir. Crit. Care Med. 191, 194–200 (2015). 2. McCormick, J. et al. Comparative analysis of Cystic Fibrosis Registry data from the UK with USA, France and Australasia. J. Cyst Fibros. 4, 115–122 (2005).i 3. McCormick, J. et al. 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F., Coriati, A., Rabasa-Lhoret, R. & Berthiaume, Y. Combined Exercise Training Improves Glycemic Control in Adult With Cystic Fibrosis. Med. Sci. Sports Exerc., https://doi.org/10.1249/MSS.0000000000001104 (2016). Scientific Reports \| (2019) 9:4763 \| https://doi.org/10.1038/s41598-019-40592-9 7 www.nature.com/scientificreports/ 25. Venkatasamy, V. V., Pericherla, S., Manthuruthil, S., Mishra, S. & Hanno, R. Effect of Physical activity on Insulin Resista Inflammation and Oxidative Stress in Diabetes Mellitus. J. Clin. Additional Informationh Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W2980603910	https://europepmc.org/articles/pmc6843026?pdf=render	English	null	Ablation or Resection for Colorectal Liver Metastases? A Systematic Review of the Literature	Frontiers in oncology	2,019	cc-by	9,520	Philipp Kron 1†, Michael Linecker 2†, Robert P. Jones 1, Giles J. Toogood 1, Pierre-Alain Clavien 2 and J. P. A. Lodge 1* Philipp Kron 1†, Michael Linecker 2†, Robert P. Jones 1, Giles J. Toogood 1, Pierre-Alain Clavien 2 and J. P. A. Lodge 1* 1 Department of HPB and Transplant Surgery, St. James’s University Hospital, NHS Trust, Leeds, United Kingdom, 2 Department of Surgery and Transplantation, Swiss HPB and Transplant Center, University Hospital Zurich, Zurich, Switzerland Background: Successful use of ablation for small hepatocellular carcinomas (HCC) has led to interest in the role of ablation for colorectal liver metastases (CRLM). However, there remains a lack of clarity about the use of ablation for colorectal liver metastases (CRLM), speciﬁcally its efﬁcacy compared with hepatic resection. Edited by: Edited by: Mark Girgis, University of California, Los Angeles, United States University of California, Los Angeles, United States Reviewed by: Luca Antonio Aldrighetti, San Raffaele Hospital (IRCCS), Italy Florian Primavesi, Innsbruck Medical University, Austria Correspondence: J. P. A. Lodge peter.lodge@nhs.net Results: This review identiﬁed 1,773 studies of which 18 were eligible for inclusion. In the majority of the studies, overall survival (OS) and disease-free survival (DFS) were signiﬁcantly higher and local recurrence (LR) rates were signiﬁcantly lower in the resection groups. On subgroup analysis of solitary CRLM, resection was associated with improved OS, DFS, and reduced LR. Three series assessed the outcome of resection vs. ablation for technically resectable CRLM, and showed improved outcome in the resection group. In fact, there were no studies showing a survival advantage of ablation compared to resection in the treatment of CRLM. Reviewed by: Luca Antonio Aldrighetti, San Raffaele Hospital (IRCCS), Italy Florian Primavesi, Innsbruck Medical University, Austria Correspondence: J. P. A. Lodge peter.lodge@nhs.net †These authors have contributed equally to this work Conclusions: Resection remains the “gold standard” in the treatment of CRLM and should not be replaced by ablation at present. This review supports the use of ablation only as an adjunct to resection and as a single treatment option when resection is not safely possible. Specialty section: This article was submitted to Surgical Oncology, a section of the journal Frontiers in Oncology Keywords: liver surgery, liver resection, colorectal liver metastases, radiofrequency ablation, systematic review Received: 22 July 2019 Accepted: 27 September 2019 Published: 16 October 2019 SYSTEMATIC REVIEW published: 16 October 2019 doi: 10.3389/fonc.2019.01052 Methods: A systematic review of the literature on ablation or resection of colorectal liver metastases was performed using MEDLINE, Cochrane Library, and Embase until December 2018. The aim of this study was to summarize the evidence for ablation vs. resection in the treatment of CRLM. Study Design Since the majority of the available evidence involved RFA (as opposed to other forms of thermal ablation) only, this reviewed considered reports comparing RFA vs. resection in the treatment of CRLM. The study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement standards (21). Studies were identiﬁed by searching the electronic databases EMBASE, MEDLINE, and Cochrane Library. A professional experienced information specialist from the Main Library of the University of Zurich performed the literature search for published data, between September and December 2018. A combination of subject headings and keywords for liver resection, liver metastases, ablation therapy, cryoablation, thermoablation electrocoagulation, radiofrequency ablation, rectum tumor, colon tumor, and colorectal liver metastases was used for the literature search. One of the main goals of liver resection for CRLM is to achieve a complete tumor removal with cancer free resection margins (6, 7). With the introduction of better imaging, potent chemotherapy and new surgical approaches, the boundaries of treatment have been expanded in CRLM (6–8). Patients that formerly seemed to be unresectable, nowadays have a chance to undergo potentially curative resection. Even in patients with extensive, bilobar CRLM and an expected marginal future liver remnant (FLR), newly introduced multi-stage resection strategies oﬀer a potential opportunity for cure by allowing time for the liver to regenerate between the stages (6, 9, 10). Existing approaches for multi- stage liver resections are the classical two-stage hepatectomy (TSH) approach and the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) approach. In the “classical” two stage approach, portal vein ligation (PVL) or portal vein embolization (PVE) is included in the ﬁrst stage to stimulate liver hypertrophy of the planned FLR, followed by resection in the second step, most usually 4– 8 weeks later (after a conﬁrmed appropriate volume increase of the FLR) (11). The other two- stage approach, namely ALPPS, was introduced more recently (10). Besides PVL/PVE, the ﬁrst step in ALPPS includes transection of the liver parenchyma (12). ALPPS is able to accelerate liver growth facilitating the second step within a shorter period of time, keeping the inter-stage interval short and providing the potential beneﬁt of a higher resection rate compared to the classical two stage hepatectomy approach in extensive colorectal liver disease (8, 13, 14). Objectives Two reviewers independently reviewed the abstracts. Suitable abstracts were identiﬁed and full text analysis was performed. Discrepancies between reviewers were resolved after discussion between them and the senior authors. These encouraging data led to the demand of directly comparing resection and ablation in CRLM to deﬁne the roles of the two treatment modalities in the treatment algorithm of CRLM (20). MATERIALS AND METHODS in the treatment of CRLM was recognized very early (5). Richard Cattel performed the ﬁrst resection of colorectal liver metastases in 1940. However, it took several decades for the impact of liver surgery on overall survival (OS) and disease free survival (DFS) to be recognized (5). Rationale Kron P, Linecker M, Jones RP, Toogood GJ, Clavien P-A and Lodge JPA (2019) Ablation or Resection for Colorectal Liver Metastases? A Systematic Review of the Literature. Front. Oncol. 9:1052. doi: 10.3389/fonc.2019.01052 Colorectal cancer (CRC) is the third most common cancer worldwide (1). At the time of diagnosis, 30–50% of the patients already have (synchronous) or will develop (metachronous) colorectal liver metastases (CRLM) in the further course of their disease (2). In metastatic CRC limited to the liver without extrahepatic disease, resection of liver lesions remains the gold standard with 5 year survivals reported to be over 60% for selected patients (3, 4). The importance of surgery October 2019 \| Volume 9 \| Article 1052 Frontiers in Oncology \| www.frontiersin.org Kron et al. Ablation or Resection for CRLM gy Keywords for the Search exp Liver Neoplasms/sc or (exp neoplasm metastases/ or exp neoplasm recurrence, local/) and ((hepatic or liver or hepatocellular or hepato-cellular).mp.) or (hepatic or liver or hepatocellular or hepato-cellular) adj3 (metasta∗or secundar∗or spread or advanced)).ti,ab. or (hepatic or liver or hepatocellular or hepato-cellular) adj3 ((neoplasm∗or cancer∗or carcinom∗ or tumo∗or malign∗) and metasta∗).ti,ab. or (recurren∗adj9 (liver or hepat∗) adj1 (neoplasm∗or cancer∗or carcinom∗or tumo∗or metasta∗or malign∗).ti,ab. exp colorectal neoplasms/ or (colorectal or colon∗or rect∗) adj3 (neoplasm∗or cancer∗ or carcinom∗or adenocarcinom∗or tumo∗or malign∗).ti,ab. exp Ablation Techniques/ or (ablation or cryotherapy or thermoablati∗or “thermo destruc∗” or “thermal destruc∗” or “thermocoag∗” or “thermo coag∗” or “thermal coag∗” or electrocoagulation or radiofrequ∗or radio-frequ∗or rfa or pei or PAI).ti,ab. or (ablati∗adj1 (therap∗or method∗or treatment∗ or procedure∗or surgery or technique∗).ti,ab. or (injection adj5 (ethanol or “acetic acid”)).ti,ab. Hepatectomy/or exp Liver Neoplasms/su or (liver or hepat∗ or surgical) adj3 (resect∗or surgery).ti,ab. or (hepatectomy or lobectomy).ti,ab. 1 and 2 and 3 and 4. The PRISMA ﬂow chart is shown in Figure 1. The last electronic literature search was performed on 20 December 2018. PRISMA checklist is shown in Supplementary Figure 1. Study Design Despite these developments, extending the limits of resectability, a high percentage of patients with CRLM remain unresectable either due to extensive liver disease or due to comorbidities precluding resection (15). Therefore, a variety of local ablative approaches have evolved to either complement resection or as a single treatment modality for otherwise unresectable CRLM, most commonly radiofrequency ablation (RFA) and microwave ablation (MWA) (16, 17). These local ablative strategies have shown to be safe and feasible in selected patient subpopulations and the approach is well-accepted for patients who are not candidates for resection (18). Over the last years the local ablative strategies have shown promising results, with response rates up to 95% and median survival rates up to 36 months (19). Study Selection and Characteristics Study Selection and Characteristics The systematic literature search identiﬁed 2,548 records. Excluding duplicates, 1,773 publications were screened according to title and abstract. Eight hundred and nine abstracts were excluded due to language, topic, tumor entity, type of manuscript, or because the full text was not available. In total, 964 publications were eligible for full-text review. A further 946 were excluded due to language, topic or tumor type. A total of 18 studies were therefore included in the ﬁnal analysis (Figure 1) (22–38). Data Selection Since the evidence on this topic is scarce, the aim of this study was, based on a discussion at the EAHPBA 2019 in Amsterdam, the Netherlands, to assess the evidence comparing resection and RFA for the treatment of CRLM. For ﬁnal inclusion, studies had to compare the eﬀects of ablation (RFA) and hepatic resection (HR) in the treatment of CRLM. Studies dealing with primary cancer or other ablation strategies than RFA were excluded. Editorials, letters, abstracts, case reports October 2019 \| Volume 9 \| Article 1052 Frontiers in Oncology \| www.frontiersin.org 2 Kron et al. Ablation or Resection for CRLM FIGURE 1 \| PRISMA ﬂow chart of literature search. FIGURE 1 \| PRISMA ﬂow chart of literature search. CRLM. In total, 2,667 patients were treated either with RFA (n = 998, 37%) or hepatic resection (n = 1669, 63%). In 11.1% of the studies included RFA was performed by surgeons. In 33.3% of the cases RFA was performed by radiologists. In 55.6% of the manuscripts included it was not clearly stated who performed the intervention. Gender was reported for 2,420 patients, with 1,505 males (62.2%) and 915 (37.8%) females. The patient study and detailed characteristics of the interventions are summarized in Table 1. The study cohorts except one manuscript were not matched. RFA was in the majority of the studies performed when surgical resection was not an option due to insuﬃcient future liver remnant, unresectable disease or underlying patient comorbidities precluding surgery. In one study (6%) RFA was the ﬁrst line treatment oﬀered to all patients (35). Ko et al. performed RFA in all patients declining HR as a ﬁrst line treatment (38). Wang et al. performed RFA if a complete necrosis based on tumor size and position could be achieved, patients comorbidities that precluded general anesthesia or surgery, and relying on patient choice (40). In this analysis the cohorts were matched according to the propensity score (40). were not included. All included studies had to be available in English. Non-randomized Studies and Patients Demographics Eighteen non-randomized studies were identiﬁed. Seventeen (94%) of the eighteen studies included were retrospective studies, and only one was prospective. Among the 18 studies, seven (39%) compared the outcomes following RFA vs. resection in solitary CRLM (23, 24, 26, 28, 34, 37, 38). Three of the 18 studies (17%) compared the two treatment modalities in potentially resectable Overall Survival (OS) RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year; PFS, Progression free survival; US, Ultrasound third (28%) of the studies did not show any signiﬁcance in favor of one group (Table 2). resection compared to RFA (22, 23, 25, 27, 28, 30–32; Table 2). Ten studies (56%) did not show any diﬀerence in terms of overall survival in favor of either treatment modality. Overall Survival (OS) Overall Survival (OS) Of the 18 studies included, eight (44%) studies showed a signiﬁcantly better overall survival in patients treated with October 2019 \| Volume 9 \| Article 1052 Frontiers in Oncology \| www.frontiersin.org 3 Ablation or Resection for CRLM Kron et al. TABLE 1 \| Identiﬁed studies for the systematic review according to PRISMA guidelines. References Study Modality Number of patients Gender m/f RFA via US/CT Open/percutaneous Abdalla et al. (22) Retrospective RFA 57 Unclear US All RFA open during Resection 190 Unclear Laparotomy Agcaoglu et al. (25) Retrospective RFA 295 196/99 US All RFA done during Resection 94 50/44 Laparoscopy Aliyev et al. (26) Retrospective RFA 44 24/20 US All RFA done during Resetion 60 34/26 Laparoscopy Aloia et al. (23) Retrospective RFA 30 23/7 US Percutaneous/intraop Resection 150 85/65 Gleisner et al. (27) Retrospective RFA 11 7/4 US All RFA open during Resection 192 121/71 Laparotomy Hur et al. (28) Retrospective RFA 25 15/10 US Intraop/percutaneous Resection 42 27/15 Kim et al. (36) Retrospective RFA 177 121/56 US Intraop/percutaneous Resetion 278 168/110 Kim et al. (29) Retrospective RFA 17 12/5 US Intraop/percutaneous Resection 43 28/15 Ko et al. (38) Retrospective RFA 17 9/8 Modality unclear Intraop/percutaneous Resection 12 9/3 Lee et al. (30) Retrospective RFA 37 26/11 US Percutaneous Resection 116 76/40 Lee et al. (37) Retrospective RFA 28 23/5 US Percutaneous/intraop Resection 25 14/11 McKay et al. (31) Retrospective RFA 43 25/18 US All RFA open during Resection 58 29/29 Laparotomy Oshowo et al. (24) Retrospective RFA 25 11/14 US/CT/MRI Percutaneous Resection 20 10/10 Otto et al. (35) Prospective RFA 28 20/8 CT Percutaneous Resection 82 49/33 Park et al. (32) Retrospective RFA 30 22/8 US/CT Percutaneous Resection 59 41/18 Reuter et al. (33) Retrospective RFA 66 46/20 US Intraop Resection 126 69/57 Wang et al. (39) Retrospective RFA 46 29/17 US Unclear Resection 92 58/34 White et al. (34) Retrospective RFA 22 8/14 CT Unclear Resection 30 20/10 RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year; PFS, Progression free survival; US, Ultrasound. TABLE 1 \| Identiﬁed studies for the systematic review according to PRISMA guidelines. ies for the systematic review according to PRISMA guidelines. Local Recurrence Rate In 10 (56%) of the 18 included studies the local recurrence rate was signiﬁcantly lower in patients treated with hepatic resection compared to RFA. Three (16%) studies included did not report their local recurrence rate following treatment with RFA or hepatic resection. Five (28%) studies did not show any signiﬁcant diﬀerences in terms of local recurrence rate following the two treatment modalities in favor of either group. Details are shown in Table 2. Eight (44%) studies showed a signiﬁcantly longer DFS for patients undergoing hepatic resection compared to RFA. One study (6%) showed a signiﬁcant better progression free survival (PFS) in the resection group (Table 2). Half of the studies showed a signiﬁcantly better DFS/PFS beneﬁt for resection (Table 2). In four (22%) of the 18 studies included, the DFS or the signiﬁcance between the two treatment groups are not reported. Almost a October 2019 \| Volume 9 \| Article 1052 Frontiers in Oncology \| www.frontiersin.org 4 ncluded. onths Median OS in months 5y OS in % Local recurrence rate in % Signiﬁcant 25 Signiﬁcant 21 Signiﬁcant 44 Signiﬁcant 72 58 11 Signiﬁcant 31 Signiﬁcant 17 Signiﬁcant 69 ns 60 58 40 22 ns 47 ns 18 Signiﬁcant 25 57 4 Signiﬁcant 47 Signiﬁcant 27 Signiﬁcant 37 Signiﬁcant 126 71 5 Signiﬁcant 38.1 Signiﬁcant 28.3 Signiﬁcant 41.3 Signiﬁcant 73.4 57.4 2 not reported 25.5 Signiﬁcant 28 ns not reported 50.1 9.5 Signiﬁcant not reported 14.3 ns Not reported not reported 34.6 Not reported ns 30 ns 47.1 ns 76.5 ns 3 y DFS in % 57 53.3 3y OS 60.2 ns na 37.8 ns Not reported na 66.7 Not reported ns 40 ns 48.5 ns 29.7 Signiﬁcant 44.7 65.7 6.9 Signiﬁcant 24 Signiﬁcant Not reported 42.9 Signiﬁcant 41 Not reported 8 Recurrence at margin ns 30 Signiﬁcant 23 Signiﬁcant 60 Signiﬁcant 44 43 7 34 ns 52.6 ns Not reported 41 55.5 3 y OS Not reported 45 ns 60 ns 32 Signiﬁcant 56 67 3 y OS 4 Signiﬁcant 36 Signiﬁcant Not reported Signiﬁcant 23.3 Signiﬁcant 56 Not reported 1.7 Recurrence at margin 27 ns 21 ns 17 Signiﬁcant 36.4 23 2 Signiﬁcant 74 ns 71.7 ns 15.2 ns 59 66.8 3 y OS 6.5 Signiﬁcant 31 Not clearly stated Not reported 55 Not reported PFS 80 Not reported 12 Local disease progression survival; OS, overall survival; y, year; PFS, Progression free survival. The bold represent signiﬁcant values. Local Recurrence Rate igniﬁcant 25 Signiﬁcant 21 Signiﬁcant 44 Signiﬁcant 72 58 11 igniﬁcant 31 Signiﬁcant 17 Signiﬁcant 69 ns 60 58 40 22 ns 47 ns 18 Signiﬁcant 25 57 4 igniﬁcant 47 Signiﬁcant 27 Signiﬁcant 37 Signiﬁcant 126 71 5 igniﬁcant 38.1 Signiﬁcant 28.3 Signiﬁcant 41.3 Signiﬁcant 73.4 57.4 2 not reported 25.5 Signiﬁcant 28 ns not reported 50.1 9.5 igniﬁcant not reported 14.3 ns Not reported not reported 34.6 Not reported s 30 ns 47.1 ns 76.5 ns y DFS in % 57 53.3 3y OS 60.2 s na 37.8 ns Not reported na 66.7 Not reported s 40 ns 48.5 ns 29.7 Signiﬁcant 44.7 65.7 6.9 igniﬁcant 24 Signiﬁcant Not reported 42.9 Signiﬁcant 41 Not reported 8 Recurrence at margin s 30 Signiﬁcant 23 Signiﬁcant 60 Signiﬁcant 44 43 7 34 ns 52.6 ns Not reported 41 55.5 3 y OS Not reported 45 ns 60 ns 32 Signiﬁcant 56 67 3 y OS 4 igniﬁcant 36 Signiﬁcant Not reported Signiﬁcant 23.3 Signiﬁcant 56 Not reported 1.7 Recurrence at margin 27 ns 21 ns 17 Signiﬁcant 36.4 23 2 igniﬁcant 74 ns 71.7 ns 15.2 ns 59 66.8 3 y OS 6.5 igniﬁcant 31 Not clearly stated Not reported 55 Not reported FS 80 Not reported 12 Local disease progression urvival; OS, overall survival; y, year; PFS, Progression free survival. The bold represent signiﬁcant values. Ablation or Resection for CRLM Kron et al. TABLE 3 \| DFS, OS, and Local recurrence rate of the studies included with solitary CRLM. References Modality DFS in months/5 y DFS in % Median OS in months 5 y OS in % Local recurrence rate in % Aliyev et al. (26) RFA Not reported ns 22 ns 47 ns 18 Signiﬁcant Resection Not reported 25 57 4 Aloia et al. (23) RFA 0 Signiﬁcant 47 Signiﬁcant 27 Signiﬁcant 37 Signiﬁcant Resection 50 126 71 5 Hur et al. (28) RFA Not reported Not reported 25.5 Signiﬁcant 28 ns Resection Not reported Not reported 50.1 9.5 Ko et al. (38) RFA 17.6 ns Not reported 37.8 ns na Resection 22.2 Not reported 66.7 na Lee et al. (37) RFA 21.1 ns 40 ns 48.5 ns 29.7 Signiﬁcant Resection 23.7 44.7 65.7 6.9 Oshowo et al. (24) RFA Not reported 34 ns 52.6 ns Not reported Resection Not reported 41 55.5 3 y OS Not reported White et al. Local Recurrence Rate (34) RFA 17 Signiﬁcant 31 Signiﬁcant Not reported 55 Not reported Resection 68 PFS 80 Not reported 12 Local disease progression RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year; PFS, Progression free survival. The bold represent signiﬁcant values. October 2019 \| Volume 9 \| Article 10 g Solitary Colorectal Liver Metastases There were seven studies which assessed outcomes in patients with only a solitary liver metastases (23, 24, 26, 28, 34, 37, 38). In total 630 patients were treated for solitary CRLM. Sixty eight percent (n = 430) of patients were treated with resection and 32% (n = 200) of patients were treated with ablation. Of the patients treated with ablation, 116 (58%) were male and 84 (42%) female. In the resection group 61% (n = 261) were male and 39% (n = 169) female. Of the seven studies, only one (14.3%) showed a signiﬁcant advantage for resection in terms of 5 year DFS (50 vs. 0%; p = 0.001). White et al. demonstrated a signiﬁcantly longer PFS in patients undergoing resection (17 vs. 68 months, p < 0.01). Two studies (28.6%) showed a signiﬁcantly higher 5 year overall survival in the hepatic resection (HR) group (Aloia et al. 27 vs. 71%; p < 0.001; Hur et al. 26 vs. 50%, p = 0.026) (23, 28); Ko et al. showed a higher DFS and 5 year OS in the HR group, but these diﬀerences were not signiﬁcant (5 year DFS 18 vs. 22%; 5 year OS 38 vs. 67%) (38). Three studies (43%) showed a signiﬁcantly higher local recurrence rate in the RFA group [Table 3; (23, 26, 37)]. Two additional studies also showed a signiﬁcant advantage for HR in terms of 5 year recurrence free survival/PFS over RFA in the treatment of solitary colorectal liver metastasis (28, 34, 37). In total, three studies (43%) showed a signiﬁcant advantage for HR compared to RFA on recurrence. Subgroup Analysis Solitary Colorectal Liver Metastases g p y Solitary Colorectal Liver Metastases Frontiers in Oncology \| www.frontiersin.org Local recurrence rate in % g g p In a subgroup analysis, four out of these seven studies (57%) provided enough data to assess the eﬀects of RFA vs. HR for solitary CRLM lesions ≤3 cm (23, 26, 28, 37, 38). A total of 255 patients were included, 125 (49%) underwent RFA and 130 (51%) patients underwent resection for solitary CRLM ≤3 cm in size. Aloia et al. demonstrated a signiﬁcantly higher 5 year OS in the HR group (71 vs. 18%; p = 0.006; Table 4) and a signiﬁcantly lower local recurrence rate for the resection group in this subgroup analysis (3 vs. 31%; p < 0.001) (23). Aliyev et al. showed a signiﬁcant advantage for resection compared to RFA in terms of local recurrence (4 vs.18%; p = 0.012) (26, 34). Hur et al. failed to show any signiﬁcance for one of the treatment groups when analyzing this particular subpopulation but did show a signiﬁcant higher survival rate (42 vs. 30%; p < 0.001) as well as a signiﬁcant longer LRFS (81 vs. 50 months; p = 0.013) in the group of solitary CRLM > 3 cm in the resection group (28, 38). These observations are further supported by the results from Ko et al. (38). This group showed a signiﬁcant lower 5 year OS as well as a signiﬁcantly lower 5 year DFS in the RFA group for patients with solitary CRLM > 3 cm (5 year OS: 0 vs. 57%; p = 0.005 5 year DFS: 0 vs. 38.1%; p = 0.013). October 2019 \| Volume 9 \| Article 1052 RFA vs. Resection for resectable CRLM indications for RFA were as follows: complete necrosis of the CRLM feasible, tumor size and position, comorbidities precluding HR and patient choice (39). of adequate data resection should remain the gold standard treatment for resectable disease (43). Despite these concerns, ablation may still have a role as an adjunct to resection. Patients with small volume resectable metastases who are not suﬃciently ﬁt to undergo liver resection can be considered for ablation as should those with limited liver metastases who have insuﬃcient liver volume to undergo resection due to tumor position (24, 44). g p Based on a discussion at the EAHPBA 2019 in Amsterdam, the Netherlands, this systematic review was conducted to investigate the contemporary evidence on ablation vs. resection for the treatment of CRLM. So far there are no completed randomized studies on this topic. The COLLISION trial is a randomized controlled trial (RCT) comparing the eﬃcacy of ablation and resection in patients with at least one resectable and ablatable CRLM with a maximal diameter of not more than 3 cm (45). The protocol of this trial was published in 2018 but results are still pending. Another ongoing RCT is the HELARC trial (Trial ID NCT02886104). This study aims to compare surgical and ablative strategies for the treatment of resectable synchronous CRLM, with patients randomized to resection of the primary tumor and resection or ablation of the liver metastases. The study has an estimated completion date of 2026. A major UK trial (LAVA) comparing the impact of resection and ablation on disease free survival was aimed at patients mainly selected on grounds of age and comorbidities and this recently closed due to failure to recruit adequate patient numbers (46). One interesting, unpublished study on this topic was presented at the EAHPBA 2019, by Engstrand et al. comparing the results of MWA and resection in CRLM in a propensity score matched cohort with promising results for MWA. Due to the limited evidence of Wang et al. and Otto et al. demonstrated a signiﬁcant beneﬁt of resection in terms of local recurrence rate and time to local recurrence compared to RFA in resectable CRLM (35, 39). The local recurrence rate in in the resection group was 4 vs. 32%; p < 0.001 in the RFA group (35). Wang et al. showed a signiﬁcant higher intrahepatic recurrence (37 vs. RFA vs. Resection for resectable CRLM In three of the 18 studies (16.7%) hepatic resection was compared to RFA in potentially resectable CRLM (35, 38, 39). Two of the studies were retrospective studies, with one prospective series. In total 277 patients were included in this subgroup analysis. One hundred and eighty-six patients (67%) underwent resection and 91 patients (33%) ablation. In the resection group 116 (63%) were male and 70 (38%) female. Sixty four percent of patients undergoing RFA were male and thirty six percent female. In the study by Otto et al. RFA was considered as ﬁrst line treatment. Frontiers in Oncology \| www.frontiersin.org October 2019 \| Volume 9 \| Article 1052 6 Ablation or Resection for CRLM Kron et al. TABLE 4 \| DFS, OS, and Local recurrence rate of the studies included with solitary CRLM ≤3cm. References Modality DFS in months Median OS in months 5 y OS in % Local recurrence rate in % Aliyev et al. (26) RFA Not reported 22 ns 47 ns 18 Signiﬁcant Resection Not reported 25 57 4 Aloia et al. (23) RFA Not reported Not reported 18 Signiﬁcant 31 Signiﬁcant Resection Not reported Not reported 71 3 Hur et al. (28) RFA Not reported Not reported 55.4 ns 13.3 ns Resection Not reported Not reported 56.1 4.3 Ko et al. (38) RFA Not reported Not reported 80 ns Not reported Resection Not reported Not reported 49.5 Not reported RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year. The bold represent signiﬁcant values. TABLE 5 \| DFS, OS, and Local recurrence rate of the studies included with technically resectable CRLM. References Modality DFS in months/5 y DFS in % Median OS in months 5 y OS in % Local recurrence rate in % Ko et al. (38) RFA 17.6 ns Not reported 37.8 ns Not reported Resection 22.2 Not reported 66.7 Not reported Otto et al. (35) RFA not reported Not reported 67 ns 32 Signiﬁcant Resection not reported Not reported 60 3 y OS 4 Wang et al. (39) RFA 14 Signiﬁcant 74 ns 71.7 ns 15.2 ns Resection 22 59 66.8 3 y OS 6.5 RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year. The bold represent signiﬁcant values. TABLE 4 \| DFS, OS, and Local recurrence rate of the studies included with solitary CRLM ≤3cm. RFA vs. Resection for resectable CRLM 12%; p = 0.001) in the RFA group as well as a signiﬁcant shorter time to recurrence in the RFA group. Furthermore, they demonstrated a signiﬁcant longer survival in the resection group (22 vs. 14 months; p = 0.032; Table 5) (39). RFA vs. Resection for resectable CRLM References Modality DFS in months Median OS in months 5 y OS in % Local recurrence rate in % Aliyev et al. (26) RFA Not reported 22 ns 47 ns 18 Signiﬁcant Resection Not reported 25 57 4 Aloia et al. (23) RFA Not reported Not reported 18 Signiﬁcant 31 Signiﬁcant Resection Not reported Not reported 71 3 Hur et al. (28) RFA Not reported Not reported 55.4 ns 13.3 ns Resection Not reported Not reported 56.1 4.3 Ko et al. (38) RFA Not reported Not reported 80 ns Not reported Resection Not reported Not reported 49.5 Not reported RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year. The bold represent signiﬁcant values. TABLE 4 \| DFS, OS, and Local recurrence rate of the studies included with solitary CRLM ≤3cm. TABLE 5 \| DFS, OS, and Local recurrence rate of the studies included with technically resectable CRLM. References Modality DFS in months/5 y DFS in % Median OS in months 5 y OS in % Local recurrence rate in % Ko et al. (38) RFA 17.6 ns Not reported 37.8 ns Not reported Resection 22.2 Not reported 66.7 Not reported Otto et al. (35) RFA not reported Not reported 67 ns 32 Signiﬁcant Resection not reported Not reported 60 3 y OS 4 Wang et al. (39) RFA 14 Signiﬁcant 74 ns 71.7 ns 15.2 ns Resection 22 59 66.8 3 y OS 6.5 RFA, Radiofrequency ablation; ns, not signiﬁcant; DFS, disease free survival; OS, overall survival; y, year. The bold represent signiﬁcant values. Surgery was only performed in patients not suitable for RFA due to number, size, or location of the metastases (35). Ko et al. only performed RFA for CRLM when patients refused HR after being informed about the two diﬀerent treatment options, potential complications and survival rates (38). In the manuscript by Wang et al. indications for RFA were as follows: complete necrosis of the CRLM feasible, tumor size and position, comorbidities precluding HR and patient choice (39). Surgery was only performed in patients not suitable for RFA due to number, size, or location of the metastases (35). Ko et al. only performed RFA for CRLM when patients refused HR after being informed about the two diﬀerent treatment options, potential complications and survival rates (38). In the manuscript by Wang et al. Frontiers in Oncology \| www.frontiersin.org Summary of Main Findings y RFA gained popularity as an interventional treatment option for small hepatocellular carcinomas and on this background of relative success RFA was postulated to be an eﬀective alternative also in CRLM, especially in the treatment of CRLM ≤3 cm (42, 47–50). However, in the absence of evidence, this was a dangerous assumption. RFA is attractive in that it oﬀers a minimally invasive treatment alternative compared to HR, in conjunction with lower post-interventional morbidity, lower complication rates and shorter hospital stays compared to resection (22, 51–53). In a review from Weng et al. overall complication rates following RFA were signiﬁcantly lower compared to hepatic resection (3.9 vs. 18.3%; p < 0.01). Van Amerong et al. support these results in a meta-analysis published in 2017 (48). But these short- term advantages do not translate into an oncological beneﬁt. This systematic review identiﬁed a higher OS in the resection group compared to RFA. Eight (44%) of the 18 non-randomized studies included in this analysis showed a signiﬁcantly higher OS following resection compared to RFA (22, 23, 25, 27, 28, 30–32). These results are similar to the results of other meta-analyses (48, 49). The diﬀerence in success rate for RFA for CRLM ≤3 cm compared to the initial very promising treatment outcomes in HCC might be explained by a diﬀerent biological behavior among the two tumor entities. There is existing evidence that CRLM are assessed as an independent risk factor, negatively inﬂuencing the outcome following treatment. These ﬁndings of superiority of HR in the treatment of CRLM, even in metastases ≤3 cm, are supported by other studies as well, although some groups claim that tumor size ≤3 cm and solitary metastases are prognostic characteristics favorable to RFA (23, 48, 49). Outcome is not only predicted by tumor size alone. The approach of doing RFA itself has a signiﬁcant impact on the outcome. There is no consensus among experts which treatment approach is most favorable: percutaneous, laparoscopic, or open (laparotomy). Existing evidence suggests that open RFA is associated with a signiﬁcantly lower risk of LR compared to percutaneous treatment (54–56). In a meta- analysis done by Mulier et al., the percutaneous approach was identiﬁed as an independent risk factor for poor outcome, independent of tumor size. Summary of Main Findings The present systematic review has shown a superior oncological outcome following hepatic resection in comparison to RFA in the treatment of CRLM. This systematic review has identiﬁed a higher overall- and DFS in the HR group. RFA as a single treatment was associated with a signiﬁcantly higher local recurrence. Even in a subgroup analysis of solitary CRLM and solitary CRLM ≤3 cm HR showed oncological beneﬁts in DFS, OS and local recurrence rate. Within a subgroup analysis comparing the eﬀects of HR and RFA in technically resectable CRLM, RFA produced inferior results. Currently there are no results from RCTs available, directly comparing the outcome of the two treatment modalities. There are RCTs registered and protocols of these trials published and their results are eagerly awaited. The failure of the UK LAVA trial to recruit patients also highlights a potential equipoise amongst surgeons regarding ablation vs. resection for resectable disease. Although screening data has yet to be released for this study, it may also be that patients were unwilling to be randomized to ablation when surgery was an option. These data will be vital to assess the feasibility of future trials. Since Interventional treatment modalities have gained popularity in the treatment of small tumors, RFA has been postulated to be an eﬀective treatment alternative, especially in the treatment of CRLM ≤3 cm (42, 47–50). However, this systematic review has shown in a subgroup analysis that HR is still superior compared to RFA even for solitary CRLM ≤3 cm. Aloia et al. also showed a signiﬁcant advantage of HR compared to RFA in this subgroup analysis in terms of LR rate (3 vs. 31%; p < 0.001), 5 year LRFS (97 vs. 66%; p < 0.001), and 5 year OS (72 vs. 18%; p = 0.006). Other studies have also demonstrated a signiﬁcant impact for HR on local control in this particular subgroup (26, 34). One potential explanation of the missing signiﬁcance in LR rate, LR free survival, DFS 5 year and 5 year overall survival in the HR group in the two other studies might be that these studies were underpowered. Hur et al. included 38 patients in total (15 RFA and 23 HR) and 18 patients (13 RFA and 5 HR) were included in the study conducted by Ko et al., respectively. DISCUSSION Hepatic resection is the treatment of choice for colorectal liver metastases with 5 year OS approaching 60% (40, 41). Ablation techniques have shown great promise in other disease types but this does not mean that it should necessarily be applied to CRLM (16, 42). Indeed, there remains a lack of clarity surrounding the precise role of ablation compared to surgery for CRLM. The American Society of Clinical Oncology (ASCO) guidelines have highlighted the wide variation in overall survival and local recurrence rates after ablation, and suggested that in the absence October 2019 \| Volume 9 \| Article 1052 Frontiers in Oncology \| www.frontiersin.org 7 Ablation or Resection for CRLM Kron et al. study also showed a signiﬁcant advantage for HR in terms of local recurrence free survival over RFA in the treatment of solitary CRLM (34, 37). White et al. showed a lower disease progression rate in the HR group (12 vs. 55%) that was not signiﬁcant in their analysis (34). Ko et al. showed a trend toward an improved DFS and 5 year OS in the HR group, but these diﬀerences were not signiﬁcant, potentially triggered by a very limited number of patients treated: 13 patients were assigned to the RFA group and only ﬁve patients were included in the resection cohort (38). When looking a little bit closer at the subgroup analysis of this study, Ko et al. did show a signiﬁcant advantage for resection compared to RFA for bith DFS and 5 year OS for CRLM > 3 cm despite the very limited number of patients included in this retrospective analysis. This ﬁnding is conﬁrmed by other studies and a tumor size > 3 cm was identiﬁed as an independent negative predictor of outcome following RFA treatment (50). published data on MWA and other interventional ablation approaches in direct comparison with resection, RFA only was chosen as the primary treatment of choice for comparison. Frontiers in Oncology \| www.frontiersin.org CONCLUSIONS So far based on the available evidence resection remains the gold standard in the treatment of CRLM, and cannot be replaced by ablation at present. Although interventional treatment approaches have gained popularity in other tumor entities with promising results in certain subgroups, the available data in this systematic review does not support the use of RFA as a solitary curative treatment in CRLM. We recognize, however, that in the treatment algorithm for CRLM ablation has a role as an adjunct to surgery or as a single treatment option in selected patient subgroups, especially in the treatment of multimorbid patients. Ablation does oﬀer some advantages, including lower post- interventional morbidity, lower complication rates and shorter hospital stays compared to resection but these advantages do not compensate the inferior oncological outcome of RFA compared to resection (22, 51–53). The mentioned short- term beneﬁts of RFA are more a justiﬁcation for RFA as a treatment tool for selected patient subgroups. Summary of Main Findings The potential short term advantages of the percutaneous approach in terms of reduced invasiveness and reduced morbidity do not counterbalance the long-term eﬀects of inferior oncological outcome of this approach (54). When trying A potential explanation of the lack of OS beneﬁt in ﬁve of the studies could be that the investigators were comparing the eﬀects of the two treatment modalities for solitary CRLM with better long-term outcomes (24, 26, 34, 37, 38). Indeed, it could be argued that these data suggest that patients with solitary metastases do equally well whether treated with ablation or resection. This lack of diﬀerence in long term survival is made even more intriguing by the results of Aliyev et al. who showed that the local recurrence rate was signiﬁcantly lower in patients undergoing resection compared to ablation (26). It may therefore be that local lesional recurrence in biologically good prognosis patients has no impact on long term outcome. One additional October 2019 \| Volume 9 \| Article 1052 8 Ablation or Resection for CRLM Kron et al. to assess the eﬃcacy of laparoscopic RFA in comparison to open RFA, results analyzing the outcome following these two treatment modalities are much harder to ﬁnd. In a manuscript published by Mulier et al. the authors showed in their analysis that the local recurrence rate following laparoscopic RFA was higher compared to open RFA even for solitary metastases ≤3 cm (26.3 vs. 1.7%), respectively (57). However, this paper was published in 2008. Improvements in imaging technologies may mean that percutaneous ablation is now better able to accurately target and destroy lesions. It will however, for the moment at least, rely on operator dependability, whatever approach is used. Furthermore, in 11% of the studies included, RFA was performed by surgeons and in 33.3% by radiologists. In 55.6% it is not clearly stated who performed the intervention. If the ablation procedure was not performed by experienced radiologists this might potentially be an additional factor explaining the superior outcome of surgery. a RCT comparing RFA and HR in the treatment of resectable CRLM even in solitary CRLM ≤3 cm since no study, especially in this subgroup analysis could show an advantage for RFA. Outlook So far there is only limited evidence available in matched cohorts comparing the outcome of these two treatment modalities. The results of the HELARC trial as well as the study results from Engstrand et al. in their propensity score matched cohort are eagerly anticipated. Furthermore, with the introduction of new technologies e.g., 3D navigation, multi needle ablation and robotic approaches RFA has further improved. There are promising results showing the eﬃcacy of stereotactic radiofrequency ablation (SRFA) even in the treatment of CRLM up to 13 cm (63). But these new treatment approaches need further testing in direct comparison with HR. pp The thermal ablation of colorectal metastases has been clearly demonstrated to result in complete tumor destruction in experimental models (61). Any diﬀerence in long- term outcomes seen between ablation and resection must therefore be explained by a diﬀerent reason. This may be because occult micrometastases are removed during hepatic resection surgery. Although there is parity for OS, parenchymal sparing resection is also associated with more local recurrence but not inferior in OS due to more repeat resections in order to achieve the OS. This may be the same for RFA (62). Lesional recurrence rates varied in this review, and may explain some of these long- term diﬀerences. However, the EORTC 40004 study of RFA for irresectable CRLM reported a lesional recurrence rate of 10% and so likely better reﬂects contemporary management. More importantly, selection for ablation is likely to represent a biologically worse cohort. The failure of trials of ablation and resection to recruit suggest that surgeons will try and oﬀer surgical resection unless the oncological prognosis is dismal, or patients are unﬁt for surgery. It may therefore be selection for ablation itself reﬂects a worse prognosis disease. Limitations The clear strength of this systematic review is, that it provides a comprehensive picture of all available evidence on RFA vs. resection in the treatment of CRLM. As no RCTs are available on this topic, the results of the included pro-/retrospective studies have to be interpreted with caution, particularly as the majority of the studies reviewed were retrospective. We found that the groups compared in the diﬀerent studies were inhomogeneous in terms of patient characteristics. In the majority of the studies included, patients assigned to RFA were not suitable for resection due to medical ﬁtness or resectability: patients eligible for RFA seemed to be unresectable or too frail for resection, clearly illustrating a selection bias representing a particular subgroup of patients with poor prognosis in the RFA cohorts. Therefore, the level of available evidence is low. In addition, the number of patients included in several studies was limited. These studies might be underpowered to detect any potential signiﬁcance in the outcomes. Recent studies have shown that intraoperative ultrasound is associated with a higher accuracy in detecting CRLM compared to the percutaneous treatment approach (58). The last argument in favor for surgical RFA may be the better access. 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(2018) 18:821. doi: 10.1186/s12885-018-4716-8 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. 46. Gurusamy KS, Ramamoorthy R, Imber C, Davidson BR. Surgical resection versus non-surgical treatment for hepatic node positive patients with colorectal liver metastases. Cochrane Database Syst Rev. (2010) 2010:CD006797. doi: 10.1002/14651858.CD006797.pub2 Copyright © 2019 Kron, Linecker, Jones, Toogood, Clavien and Lodge. 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. 47. Chen MS, Li JQ, Zheng Y, Guo RP, Liang HH, Zhang YQ, et al. A prospective randomized trial comparing percutaneous local ablative therapy and partial hepatectomy for small hepatocellular carcinoma. Ann Surg. (2006) 243:321–8. doi: 10.1097/01.sla.0000201480.65519.b8 48. van Amerongen MJ, Jenniskens SFM, van den Boezem PB, Futterer JJ, de Wilt JHW. Radiofrequency ablation compared to surgical resection for curative October 2019 \| Volume 9 \| Article 1052 Frontiers in Oncology \| www.frontiersin.org 11
https://openalex.org/W1720610081	https://durham-repository.worktribe.com/preview/1438672/15478.pdf	English	null	Journeying through words: <scp>L</scp>es <scp>B</scp>ack reflects on writing with <scp>T</scp>homas <scp>Y</scp>arrow	Journal of the Royal Anthropological Institute	2,014	cc-by	4,304	Tom. I share your sense of the value of writing on the move - on trains, cafes or places along the journey. This is for two reasons. Firstly, the alchemy of Wi-Fi hot spots and the global reach of email make it almost impossible to escape academic responsibilities for longer than the duration of a plane flight or a train journey. Connectivity offers writers a staggering capacity to access information from all over the world and check facts and follow up leads. The price we pay for this resource, that has so quickly been taken for granted, is the exasperation of seemingly endless queries about meetings, essays and deadlines. The academic life has become open access. In order to think and write I find myself seeking out places to disconnect and get off the information super highway, places on the move where the 'connection' is bad. Those places afford writing time. Secondly, sometimes I suffer from an allergic reaction to my desk and need to get out. In the age of the laptop computer and the mobile phone writers are no longer hostage to the immobile typewriter. A desk can be found almost anywhere as long as the battery is charged or there is a compatible mains socket close at hand. I settle down to work in crowded cafes, noisy airport lounges or even parks. Today I am in my current favourite spot Pistachios in the Park Café on Hill Fields, one of south London’s most beautiful – and lesser known – parks. I find it an ideal location to get my laptop out and write. It is located almost exactly halfway between where I live and where I work. It literalises aptly the place of writing in my own life: a vocation that is between what I get paid to do and the rest of my life. Now I am surrounded by the sounds of toddlers crying, young Mums laughing over the absurdities of parenting and dogs barking loudly. ‘Don’t you find it distracting’ asks Fred, the owner. Truth is I don’t. On the odd occasion explosions of mirth from Sixth formers gathered around laptops watching comic YouTube virals disrupt my concentration - but those are exceptional lapses. The visitors to the café are busy getting on with more important things and are not asking for an immediate reply to email inquiry. [Writing on Writing] Journeying Through Words: Les Back reflects on writing with Tom Yarrow [Writing on Writing] Journeying Through Words: [Writing on Writing] Journeying Through Words: Les Back reflects on writing with Tom Yarrow You have written about the importance of taking the reader 'there', as well as on the relationship between what and where we write. The medium of this exchange is email and so the 'there' is distributed in time and space. I am writing this on a train between Durham and York, my daily commute. It takes about an hour and is a space I like to use to write. There is something about the literal movement that generates a sense of momentum. If I get stuck I can look out of the window -- the North York moors on one side, the Dales to the other. It's enough of a distraction to move me on but not so much as to break the flow. The buffet trolley goes by 'Any teas, coffees or refreshments.' I'd like you to start by setting the scene at your end. Tom. Writing is just difficult, plain and simple. The temptation to put off writing is strong as a result. We end up becoming what psychologist Paul J Silvia calls ‘binge writers’ (Silvia 2007: 14). Delaying the moment when we sit down to write means we are then faced with a deadline that can only be met through late night binges at the keyboard. For Silvia the only way out of this pattern is to become a routine writer and treat writing time as a non- negotiable commitment - like teaching a class or attending a department board meeting. His little book entitled How to Write a Lot (2007) is crammed with useful tips on how to foster better literary habits. We all have periods in the day when we are most intellectually awake, which is when we should be writing. I try and write in the mornings because that is when my mind is most agile. Sometimes, like this morning, I wake up at 4.30am and suddenly a link or a connection I was trying to make in something I am working on becomes clear. I find that I have no real control over that process because ideas can't simply be willed to come. Leonard Cohen commented once that he didn't know where the good songs came from because if he did he'd visit that place more often. Imaginative leaps or analytical connections are like that too. They seem to me like unexpected guests that we need to be ready to receive at anytime. I record their arrival with a few scribbles in my notebook. Having said this I do give myself designated periods of time to write. The torture for me is starting. Once I've started I am usually fine but if I get interrupted - if the phone rings, or if something else intervenes and time drifts - then I am really in trouble. I usually give myself a block of time rather than whole days. I find that after 3-4 hours of writing intensely I start to achieve less and less. So, your train journey sounds perfect to me - you start writing when the train pulls away from the platform and stop when it arrives at your destination. The other thing that I try to do is stop a writing session before I have exhausted all of the things I wanted to say. Tom. The middle–aged guy tapping away at his laptop in such a public place nonetheless draws comfort and inspiration from them. It helps counteract inhibitions of authorial self-consciousness, which can be so stifling. It gets me started and helps me keep moving with the work. The noise of the children playing is also a reminder that writing is a profoundly social activity, it connects my thoughts to yours - in short, it let's them travel. is also a reminder that writing is a profoundly social activity, it connects my thoughts to yours - in short, it let's them travel. Les You have described how writing is shaped by the places in which we write. I think there is a more general point here: that good writing involves sufficient detachment from the kinds of distractions that accompany modern academic life; but as you say, it also involves a kind of distraction (a disconnection) from a debilitating over-absorption in ourselves, and from awareness of the technicalities of how we do it (thinking about our thoughts; or about words, sentences and grammar, the task becomes very difficult!). I'd like to follow up my initial question, by picking up on the temporal dimension to writing, which you have touched on in your message. I think part of the reason I find trains good places to write is that they are spaces that segment time in a very specific and finite way: I get to my destination where I have to get off. This thought focuses the mind, and also reduces the fear of failing: whether or not anything good emerges, I have still got to where I needed to be. I'd be interested to hear more about the times in which you write, and also about the sense in which you experience time in relation to your writing. Les You have described some of the ways in which what you write, relates to where and when you do the writing. Your description complicates ideas about authorial autonomy which have been widely deconstructed, but which nonetheless remain powerfully constitutive of the ways in which writing is thought about and practiced. If writers can consciously control the conditions in which ideas emerge but not the ideas themselves, ideas involve a movement that takes us beyond ourselves. Because writing is creative, the journey is not pre-figured. That makes it is difficult but also transformative: it is a slightly different person who starts and ends a text. I'd like to pick up now on this relationship between the authorial 'you' and the words that you write. Adam Reed (2011) has recently written of 'inspiration' as a specific sense in which writers (in his case members of a literary society) imagine themselves as conduits of the voice and actions of specific others. Your writing has a distinctive voice, but I'd be interested in your reflections on where this comes from, which might return us to your earlier observation about the social basis of writing in a slightly different way. I am thinking less about the specific kinds of influence acknowledged in references, and more about whether or how your own voices contains others (personally known or not) in sedimented and habitual ways of thinking and writing. Tom. Always leave an argument or description to be written. So, I never stop writing without knowing what the next point is going to be. It makes picking up the thread the next time easier. stop writing without knowing what the next point is going to be. It makes picking up the thread the next time easier. Tom. Thirdly, I think the writing is often shaped by the critical eyes of trusted readers. I have been very lucky to have educated readers - not always academics - to let me know if I was 'writing it right' or not. I have an old friend who is a bus driver called Pete. We were at school together and he is the most gifted and intelligent person I know, although he didn't get a formal education beyond what we call GCSEs today. I often give him my things to read. I remember I gave Pete a book I had written about football culture and racism. After a few weeks we met up and I asked him what he thought about the book. He said agitatedly, 'It really pissed me off.' I asked him 'why'? He replied 'well, you seem so uncertain, you 'suggest' all the time but you don't tell us what you think!' It cured me of what Clifford Geertz called 'epistemological hypochondria' in a single stroke. I think I had fallen foul of the all too tempting passive literary voice. In Rushdie's terms I know my work has benefitted from the re-writing that my critical readers do when they pass comment on the things I asked them to read for me. So, that voice that you recognise when you read it is shaped by all of those dimensions. In recent times too I have felt a desire to try and make academic writing more artful. This means trying to make our craft a bit more crafty - working with the counter-intuitive and trying to surprise the reader if I can. I'd like to pick up on your point about ethnographic writing as a way of taking us beyond ourselves, and to pose a question about the different ways in which writing can affect that transformative movement in the reader. You have written earlier about the imaginative journey of writing from the author's perspective, and I'd like to ask now how you see the relationship between the journey that you take as a writer, and the journey that the reader takes. Writing, as you have earlier observed, connects the thoughts of a writer to those of a reader and gives the reader space to travel. Tom. Tom. To my mind ethnographic writing has no point at all unless it takes us beyond ourselves. That was the danger of the turn to authorial reflexivity, or what is called derisively in American sociological circles as 'me-search'. The writing we do is about our reasoning with others, our dialogue with them and it should aim to communicate to the readers what we learned and brought back from those encounters. It has to be that otherwise the profoundly sociable nature of the kind of writing that ethnography invites is lost. I still think Salman Rushdie put it rather well: he said writing becomes a collective process 'that both writes as it reads and reads as it writes'. If we think of the process of reading as listening to the voices of others then our own writing is never entirely individual. You wrote kindly that my own writing has a 'distinctive voice'. I don't know what that is, to be honest. Although I trust you as a reader to know that voice when you read it. I know too that my writing is not my own creation, although I would insist it is my sole responsibility. So, where then does it come from? I want to pay your perceptive question the courtesy of a considered and serious answer. So, the first place it comes from is reading the writers I admire. This is reading not just for the content of their ideas but the form of their rhetoric. Here I don't mean rhetoric as hollow sloganeering but the art of persuasive writing. My writing voice is a bricolage of influences drawn from reading great writers without being consciously aware of their imprint. I don't think this is imitation but combination, adaption and re-assembly. It is 'reading as we are writing,' as Rushdie put it. The second place it comes from is the encounter with the ethnographic world and texture of the lives I am listening to. I think our job as writers is to try and take our readers to that place, whether it is is the football grounds of south London or an anti-racist political demonstration in Chinatown. I often listen to those voices and the soundscapes of those worlds as I am writing. That cultural landscape furnishes the texture of my writing as well as the content, be it ethnographic descriptions or quotations from participants. Tom. I am particularly interested to know what your understanding of writing as evocation implies for the way in which you envisage the relationship between the kinds of writing we call 'descriptive' and those we call 'theoretical'. Tom. It seems to me that academic writing - particular in the context of PhD theses - has become increasingly heavy on theory and light on description. We're all so concerned to convey our theoretical sophistication that sometimes theory's referent (i.e. the ethnographic setting) is almost forgotten. The possibility then of theoretically infused descriptions is lost. To be honest, I just don't want to accept the simple separation of theory from description. What we notice and describe is often deeply connected to theoretical commitments and theoretical arguments are often best communicated through a compelling description. I also think there are new opportunities in our time to imagine description differently. In this sense I think James Clifford is right to warn that in order to return to realism you have to leave it in the first place (Clifford 1986: 25). What I am thinking of is how to use digital devices and screen media can create objects that are productive of the social and an appreciation of this productivity – more than this I want to suggest an embrace of this productive/creative dimension – might help enable an encounter with ‘the real’ without a naïve realism slipping in through the back door. The recordings made by the digital sound recorder or camera provide the illusion of ‘being there’. If we leave behind the simple idea that they ‘capture’ the real but instead produce a realist imaginative object then they may provide a different kind of possibility for social understanding or revelation as well as medium for theorising in which the abstract is made to live. Also, those digital facsimiles become props and aids for writing more descriptively about the unfolding of culture. You make an important point about the relation between description and theory. Descriptions are not simply accounts of the pre-existing ‘reality’ from which theoretical arguments are built; they are artifacts of interpretive processes that include the judgments that are made as part of the act of writing itself. Am I right to infer that from this perspective a commitment to the ‘real’ of ethnographic experience is as much about ‘thinning’ as ‘thickening’ description, involving selective erasure as well as layering? Tom. I wonder if you would share my observation that, though anthropological forms of writing routinely combine the two, truth to ethnographic complexity –manifold voices and contexts – points in a slightly different direction to the truth of an argument or conceptual ‘point’? We are indexing Clifford Geertz’s famous notion of ‘thick description’ as a way of describing ethnography and maybe we should make that explicit. One of the things I have always admired about Geertz is his insistence on the idea of a ‘situated observer’. So any ethnographic account needs to acknowledge its own partial nature i.e. produced by a particular observer in a particular place and time. I think that involves ‘thinning down’ the arrogant claim that we know ‘the real’ once and for all, or the sleight of hand involved in what Bourdieu calls ‘view from nowhere’ which writes out the writer. A winnowing of that kind of arrogance and certainty is very welcome from my point of view. I have really tried to think about this differently by asking the simple question - what are we doing when we are writing descriptions? There is a passage in a book I wrote called The Art of Listening (Back 2007: pp. 21-22) that focuses on this question. I have found Hannah Arendt’s essay on Walter Benjamin is very suggestive because she characterizes him as a ‘pearl diver’ who descends to the bottom of the sea not to bring the sea floor to the surface whole because that’s not possible. Rather, he is prying loose the rich and the strange pearls in the depth. Similarly I think the empirical depths that ethnography tries to plumb in life’s surface cannot be described entirely. Well then, what am I doing? I have come to think that what I am doing with writing is to describe fragments of life and enhancing them through description. This is not a simply a kind of facsimile of the ‘real’ but an augmentation of it - turning up the background, enlarging the unremarked upon and making it remarkable. The usefulness of theory is that it can hover above the ethnographic ground and provide a vocabulary for its explication, magnification and enchantment. I have come to realize that is what I am trying to do as I sit down to write in a crowded café. Tom. I have just finished writing a chapter on writing for an edited book by Carol Smart, Jenny Hockey and Alison James called The Craft of Knowledge that will be published later this year. It argues that the words we write are valuable because they enable movements of imagination but also because they provide companionship in further thought. That sense of openness, or trying to write in such a way to facilitate movements of imagination, is what I have been aiming for. Whether or not I achieve it the reader alone will decide. I want to end with a question about ending. I have recently been working with stone masons working on the conservation of Glasgow cathedral (Yarrow and Jones 2014). For them the point at which they stop carving is not a passive ending but emerges as an active achievement: the process requires meticulous, painstaking work, and an ethic of perfectionism, but this in turn can be associated with the problems of over-absorption and loss of perspective. Paradoxically perfectionism can lead to over-working, and less than perfect results -- in this sense the best can be the enemy of the good. They describe the end- point -- the point at which the stone is released as an object for incorporation in the cathedral -- as a moment of 'letting go'. Previously locked together through a process of cutting, stone and mason part company as subject and object. Related but distinct, they go their own way. I think it is quite a good analogy for the writing process, and the issues of ending. So my question to you is about how you work with your own words once they are there. Specifically I am interested in the role of editing in your own work and how, through this, you achieve and experience the point at which you finish. A related question is whether stopping is always the same thing as finishing. In other words, is it always clear to you that you have reached a point of resolution? Or is it inherent in the writing process that questions and doubts linger? That is a lovely analogy. I know Mariam Motemedi-Fraser talks about writing as the process of sculpting words (Motamedi-Fraser 2012: 97). The first thing I do with my own words when drafted is to give them to a reader. I let them go almost immediately. Tom. I think the cruel thing about authorship is that we cannot really judge our own work. I think that’s what we need readers for, to help us cut and hone the writing. We all need a discerning reader that we can trust. Feedback can come in many forms from a PhD supervisor or referee’s reports on a journal article or book manuscript. Being able to take criticism is a real skill because it involves not only being able to learn from critical comments but also to have the capacity to act on them. Stopping writing is not the same as finishing because the words come back for re-shaping time and time again. I think a point is reached where you have to say - I can’t do anymore, I have polished the arguments as much as I can. That is the point to let it go and start something else. I often feel a sense of dissatisfaction and failure. I do not think it is wise to listen to those feelings because we are not the best judges of our own work. Those feelings are only useful as a spur to try and write better next time. People have said to me – ‘oh your have written a lot of books!’ To which I always reply: ‘that’s because I am still trying to write a good one.’ I am not sure I will ever achieve this in my own estimation but I will keep to that endeavour. Back, L. The Art of Listening Oxford: Berg. Becker, H. (2007) Telling About Society Chicago: University of Chicago Press Clifford, J. (1986) ‘Introduction: Part Truths’ in J. Clifford and G. E. Marcus Writing Culture: The Poetics and Politics of Ethnography References Berkeley, Los Angeles & London: University of California Press. Geertz, C. (1988) Works and Lives: The Anthropologist as Author Cambridge: Polity Press (2000) Available Light: Anthropological Reflections on Philosophical Topics ( ) g p Princeton, NJ: Princeton University Press. ( ) g p Princeton, NJ: Princeton University Press. Princeton, NJ: Princeton University Press. Motamedi-Fraser, M. (2012) ‘Once Upon a problem’ in L. Back and N. Puwar eds. Live Methods. Oxford; Wiley Blackwell/ The Sociological Review. pp.84-107. Perec, G. (1997) Species of Spaces and Other Pieces, London: Penguin Books. Reed, A. (2011). Literature and Agency in English Fiction Reading: A Study of the Henry Williamson Society. ??, Manchester University Press. Rushdie, S. (1991) Imaginary Homelands: Essays and Criticism 1981-1991 London: Granta & Penguin. Silvia, P. J. (2007) How to Write A Lot: A Practical Guide to Productive Academic Writing, Washington, DC: American Psychological Association Yarrow, T. and S. Jones (2014). "'Stone is stone': engagement and detachment in the craft of conservation masonry." JRAI 20(2): 256-275. Motamedi-Fraser, M. (2012) ‘Once Upon a problem’ in L. Back and N. Puwar eds. Live Methods. Oxford; Wiley Blackwell/ The Sociological Review. pp.84-107. Perec, G. (1997) Species of Spaces and Other Pieces, London: Penguin Books. erec, G. (1997) Species of Spaces and Other Pieces, London: Penguin Books. Reed, A. (2011). Literature and Agency in English Fiction Reading: A Study of t Williamson Society. ??, Manchester University Press. Rushdie, S. (1991) Imaginary Homelands: Essays and Criticism 1981-1991 London: Granta & Penguin. ushdie, S. (1991) Imaginary Homelands: Essays and Criticism 1981-1991 London: Gran & Penguin. Silvia, P. J. (2007) How to Write A Lot: A Practical Guide to Productive Academic Writing, Washington, DC: American Psychological Association Yarrow, T. and S. Jones (2014). "'Stone is stone': engagement and detachment in the craft of conservation masonry." JRAI 20(2): 256-275. Acknowledgements This conversation has developed from an earlier piece of writing contributed to the Writing Across Boundaries project website (http:/www.dur.ac.uk/writingacrossboundaries/). This project is an ESRC funded Researcher Development Initiative (RES 035 25 0013). Bob Simpson and Robin Humphrey at Durham and Newcastle University lead the project. The initiative is dedicated to supporting social science researchers seeking to engage more effectively with the practical and intellectual issues that arise in the quest to produce texts that are engaging, accurate and analytically insightful. Les Back completed a PhD in Social Anthropology at Goldsmiths, University in 1991. He is part of what some refer to as the ‘Lost Generation’ i.e. anthropologists who left the discipline during the 1980s and 1990s to work in allied fields. His writing retains a strong ethnographic sensibility, albeit now elaborated in the context of cultural studies and sociology. He has conducted fieldwork in London (New Ethnicities and Urban Culture, UCL 1996 & The Art if Listening, Berg 2007), Birmingham (Race, Politics and Social Change with John Solomos, Routledge 1995) and Muscle Shoals and Memphis in the southern United States (Out of Whiteness: Color, Politics and Culture with Vron Ware, University of Chicago Press 2002).
https://openalex.org/W4250863981	https://europepmc.org/articles/pmc4245828?pdf=render	English	null	Leveraging practice-based research networks to accelerate implementation and diffusion of chronic kidney disease guidelines in primary care practices: a prospective cohort study	Implementation science	2,014	cc-by	9,301	Implementation Science Implementation Science Implementation Science Implementation Science Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Mold et al. Implementation Science 2014, 9:169 Abstract Background: Four practice-based research networks (PBRNs) participated in a study to determine whether networks could increase dissemination, implementation, and diffusion of evidence-based treatment guidelines for chronic kidney disease by leveraging early adopter practices. Methods: Motivated practices from four PBRNs received baseline and periodic performance feedback, academic detailing, and weekly practice facilitation for 6 months during wave I of the study. Each wave I practice then recruited two additional practices (wave II), which received performance feedback and academic detailing and participated in monthly local learning collaboratives led by the wave I clinicians. They received only monthly practice facilitation. The primary outcomes were adherence to primary care-relevant process-of-care recommendations from the National Kidney Foundation Kidney Disease Outcomes Quality Initiative Guidelines. Performance was determined retrospectively by medical records abstraction. Practice priority, change capacity, and care process content were measured before and after the interventions. Results: Following the intervention, wave I practices increased the use of ACEIs/ARBs, discontinuation of NSAIDs, testing for anemia, and testing and/or treatment for vitamin D deficiency. Most were able to recruit two additional practices for wave II, and wave II practices also increased their use of ACEIs/ARBs and testing and/or treatment of vitamin D deficiency. Conclusions: With some assistance, early adopter practices can facilitate the diffusion of evidence-based approaches to other practices. PBRNs are well-positioned to replicate this process for other evidence-based innovations. Keywords: Implementation, Diffusion, Primary care, Practice-based research network, Chronic k : Implementation, Diffusion, Primary care, Practice-based research network, Chronic kidney disease tested and refined in Ontario, Canada, the Oklahoma Physicians Resource/Research Network (OKPRN), and in other primary care settings [5-11]. Respectful relationships between the academic detailers and practice facilitators (PFs), and the practice clinicians and staff, appear to be critical to success. This principle has also been the key to the success of Cooperative Extension, in which extension agents develop relationships with farmers and their families to facilitate implementation of innovative farm- ing practices [12]. Leveraging practice-based research networks to accelerate implementation and diffusion of chronic kidney disease guidelines in primary care practices: a prospective cohort study Leveraging practice-based research networks to accelerate implementation and diffusion of chronic kidney disease guidelines in primary care practices: a prospective cohort study James W Mold1, Cheryl B Aspy1, Paul D Smith2, Therese Zink3, Lyndee Knox4, Paula Darby Lipman5, Margot Krauss5, D Robert Harris5, Chester Fox6, Leif I Solberg7 and Rachel Cohen5 © 2014 Mold 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/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access Correspondence: james-mold@ouhsc.edu 1Department of Family and Preventive Medicine, University of Oklahoma Health Sciences Center, 900 NE 10th Street, Oklahoma City, OK 73104, USA Full list of author information is available at the end of the article Study design This was a cohort study involving primary care practices enrolled in two waves. Data on practice enrollment, prac- tice characteristics, and practice change component scores were captured concurrently. Practice performance data were collected retrospectively after each wave using med- ical records abstraction. Additional information was pro- vided by concurrent facilitator field notes and retrospective clinician interviews. Four PBRNs participated in the pro- ject: OKPRN, the Los Angeles Practice-Based Research Network (LA Net), the Minnesota Academy of Family Phy- sicians Research Network (MAFPRN), and the Wisconsin Research and Education Network (WREN). A coordinating center, Westat, managed the project activities and con- ducted the data management and analysis. The Agency for Healthcare Quality and Research wanted to determine whether PBRNs could speed dissemination and implementation of evidence-based practices. We were one of the several groups to receive funding for this initia- tive. We chose to address chronic kidney disease (CKD) guidelines, since member practices indicated that this was an area of both weakness and interest. Our goal was to try to help a group of early-adopter practices implement the guidelines using standard implementation strategies, then see whether those practices could recruit and train add- itional practices using LLCs and less intensive facilitation. Each PBRN was asked to recruit eight member prac- tices (N = 32) to participate in wave I of the study. Since each network handled this differently, we have no data on the number of practices who declined to participate. At the time of enrollment, a clinician from each of these practices agreed to help the investigators recruit two additional practices (N = 64) and help to facilitate the LLC strategy during wave II. Each PBRN provided a local project coordinator, an academic detailer, and PFs (1 full-time equivalent) to work directly with the practices, collect performance data, and, during wave II, organize the monthly LLC meetings. In addition to practice per- formance data, surveys completed by a lead clinician from each practice were used to collect information about the impact of the implementation and diffusion strategies on three components of practice change: priority for change, change process capability, and care process content [28]. Between the 1988–1994 and the 2003–2006 National Health and Nutrition Examination Surveys, the preva- lence of CKD in individuals aged 60 and older increased from 18.8% to 24.5% [18]. Background Many practice-based research networks (PBRNs) func- tion as learning communities that strive to improve quality of care in member practices [1-4]. Some provide resources to support dissemination and implementation, such as performance feedback, identification and spread of best indigenous practices, academic detailing, and prac- tice facilitation, evidence-based strategies that have been * Correspondence: james-mold@ouhsc.edu 1Department of Family and Preventive Medicine, University of Oklahoma Health Sciences Center, 900 NE 10th Street, Oklahoma City, OK 73104, USA Full list of author information is available at the end of the article While this approach to dissemination and implemen- tation has been successful in practices that volunteer to Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 2 of 11 participate, questions remain about how to engage other practices: whether the rate of diffusion can be increased and whether the cost of implementation assistance can be reduced. Evidence that peer-to-peer learning can mo- tivate practice improvement, especially when enhanced by competition, has led to the development and use of learning collaboratives [13-15]. These collaboratives typic- ally involve large numbers of practices and include peri- odic conferences between periods of local improvement activities. In OKPRN, researchers working with small rural practices have adapted this approach, holding shorter more frequent meetings with a smaller number of prac- tices [16]. These local learning collaboratives (LLCs) are less costly and probably better-accepted by clinicians and staff than the larger, more formal collaboratives [17]. outcome measures for this aim were changes in priority for improvement of CKD care, change process capability, and care process content, concepts proposed by Solberg [27]. Study design CKD is associated with a 5-year all-cause mortality rate of 24% and a 20% 5-year require- ment for transplant or dialysis [19]. Evidence-based guide- lines for the management of patients with CKD have been available to practicing physicians since 2002 [20], and there is some evidence to suggest that guideline-based care can delay CKD progression and reduce mortality [13,21-24]. However, dissemination, implementation, and diffusion of guideline recommendations have been sub- optimal [16,25,26]. Practices were directed to focus on the eight processes of care emphasized in the National Kidney Foundation Kidney Disease Outcomes Quality Initiative Guidelines: 1) making and documenting the diagnosis, 2) ordering appro- priate tests based upon severity of disease, 3) discontinuing potentially harmful medications, 4) starting potentially beneficial medications, 5) managing diabetes and cardio- vascular disease risk factors, 6) educating patients about vein preservation, 7) giving appropriate immunizations, and 8) referring patients with advanced disease to a neph- rologist [20]. Specific recommendations within each of these categories were consolidated into a one-page, lami- nated decision-support tool, which was provided to the practices and discussed in the academic detailing sessions. The primary aim of the present study was to deter- mine the extent to which ‘early adopter’ practices could facilitate recruitment of other practices and, using the experience gained during their own implementation ef- forts, help the new practices to implement guideline rec- ommendations. Success was measured by numbers of wave II practices enrolled; numbers of LLCs formed, held, and attended by wave I practice representatives; and im- provements in performance by wave II practices. In other words, we wanted to determine if we could speed up the diffusion of ‘best practices’ by leveraging the relationships and resources of practice-based research networks. Maintenance of Certification (MOC) Part IV credit was provided through the American Boards of Family Medicine and Internal Medicine, which was a significant motivator The secondary aim was to determine whether the change processes used by wave II practices would be the same as or different from those used by the early adopters. The Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 3 of 11 for clinician participation. Continuing Medical Education (CME) credit was also provided through the American Academy of Family Physicians. Practice performance measurement After completion of each wave, the PFs screened medical records of diabetic patients between 50 and 84 years of age to identify at least 30 patients with two estimated glomerular filtration rates (eGFR) <60, at least 3 months apart for the next phase of abstraction. If 30 patients meeting these criteria could not be identified, PFs were instructed to search the records of patients between 50 and 84 years of age with a diagnosis of hypertension. If 30 patients still could not be identified, PFs were instructed to identify patients already diagnosed with CKD. The PFs then abstracted the medical records of patients who had evidence of CKD to collect information relevant to prac- tice performance during the specified time periods of interest. Data on the following were abstracted for every visit during the qualifying time period: 1) CKD diagnosis defined as an eGFR staying below 60 for more than 3 months; 2) A1c measurement at least annually, 3) LDL cholesterol measurement at least annually, 4) microalbu- min measurement at least annually, 5) Hgb measurement annually if eGFR <45, 6) ACEI or ARB initiation, 7) dis- continuation of NSAIDS, 8) vitamin D measurement and/ or initiation of vitamin D supplements if eGFR <45, and 9) referral to a nephrologist for any eGFR <30. We also examined changes in LDL, A1c, and the mean of the last three diastolic (DBP) and systolic blood pressures (SBP) before and after the intervention. Training and standardization Training and standardization Training was provided for the academic detailers and PFs prior to the interventions. The academic detailer training focused on the guideline recommendations and suggestions regarding implementation. The PF training addressed QI concepts, practice facilitation and group fa- cilitation skills, chart auditing and feedback, the Chronic Care Model, the nature and behavior of complex adaptive systems, MOC and CME logistics, and the CKD guidelines (including the toolkit and implementation suggestions) and project-related data collection and data transfer. Prior to wave II, PFs received additional training on the facilita- tion of LLCs and were provided a sample LLC agenda to further standardize the experience for the participating cli- nicians. PFs also participated in training on collection of the retrospective medical record data upon which these primary analyses are based. Interventions Wave I At the start of wave I, practices received performance evaluation and feedback reports and academic detailing, followed by weekly practice facilitation for 6 months. Academic detailing included a review of the guideline recommendations, a discussion of the one-page decision- support tool (included as an attachment), a forum to dis- cuss questions and concerns, a review of the practice’s current methods and performance, and guidance from the methods used in high performing practices, culminating in an agenda for improvement. The PFs were expected to spend 2–4 h per week in each practice helping clinicians and staff implement the agreed-upon changes in their care processes, teaching them how to do quality improvement (QI) (e.g., plan-do-study-act cycles, QI teams, staff meet- ings, etc.), and collecting ‘unofficial’ baseline and monthly performance data for their use. The PBRNs worked with these practices to recruit and enroll two additional prac- tices in close geographic proximity to the wave I practices, and typically belonging to similar health systems or using an electronic health record (EHR) from the same vendor. Study design the assistance provided by wave I practices during the LLC meetings would reduce the need for PF visits and that monthly, rather than weekly, visit would be sufficient. This was based only upon our collective judgment, not on spe- cific data. The project was approved by the Institutional Review Boards at the University of Oklahoma Health Sciences Center, the University of Wisconsin at Madison, the University of Minnesota, the University of Southern California Health Sciences Center, and Westat. Practice contexts and measures of change in priorities and processes Data on organizational characteristics (context) were col- lected from practices upon enrollment. Priority for im- proving CKD care was measured on a visual analog scale (CKD priority scale). The instructions read, ‘Considering all the priorities your clinic has over the next year (e.g., EHR, financial goals, QI of various conditions, physician recruitment), what is the priority for your clinic to im- prove CKD care (on a scale of 0–10, where 0 = not a prior- ity, 5 = medium priority, and 10 = highest priority of all)?’ The Change Process Capability Questionnaire (CPCQ), developed and tested by Solberg [27], was used to meas- ure organizational factors associated with readiness of the practice to manage changes needed to implement guideline recommendations. The instrument includes 30 items measured on a five-point Likert scale, from strongly agree to strongly disagree. Three domain scores under organizational factors include ‘previous history of change’, ‘plans for organizational refinement’, and ‘ability to initiate and sustain change’. The strategies (‘strategy count’) are specific approaches to managing change, such as periodic measurement of performance or delegating physician work to non-physician staff. Data on organizational characteristics (context) were col- lected from practices upon enrollment. Priority for im- proving CKD care was measured on a visual analog scale (CKD priority scale). The instructions read, ‘Considering all the priorities your clinic has over the next year (e.g., EHR, financial goals, QI of various conditions, physician recruitment), what is the priority for your clinic to im- prove CKD care (on a scale of 0–10, where 0 = not a prior- ity, 5 = medium priority, and 10 = highest priority of all)?’ To account for possible correlation of repeated mea- sures within patients, a generalized estimating equation (GEE) approach was used to fit repeated measures models to assess the pre- to post-intervention change in the im- plementation of the guideline recommendations. Except for Hgb and vitamin D measurements, the modeling ana- lyses were restricted to patients with evidence of CKD prior to the intervention (at least 3 months of eGFR <60 or documentation of CKD in the problem list). For Hgb and vitamin D measures, the modeling was further re- stricted to those subjects with an eGFR <45 in both periods. Only those patients with outcome information available during both the pre- and post-intervention pe- riods contributed to the models. Practice contexts and measures of change in priorities and processes Changes in implementa- tion of the CKD guidelines were modeled separately for wave I and wave II practices. y, p y, g p y ) The Change Process Capability Questionnaire (CPCQ), developed and tested by Solberg [27], was used to meas- ure organizational factors associated with readiness of the practice to manage changes needed to implement guideline recommendations. The instrument includes 30 items measured on a five-point Likert scale, from strongly agree to strongly disagree. Three domain scores under organizational factors include ‘previous history of change’, ‘plans for organizational refinement’, and ‘ability to initiate and sustain change’. The strategies (‘strategy count’) are specific approaches to managing change, such as periodic measurement of performance or delegating physician work to non-physician staff. Since preliminary analyses indicated that CKD recog- nition (CKD reported in the problem list in the medical record) might have influenced the implementation of the other guideline recommendations, the models were fit to the data for each study outcome to assess the effect of the intervention, adjusted for any influence of when CKD was recognized and the interaction of the intervention with CKD recognition, if any (Tables 1 and 2). A signifi- cant main effect of intervention would indicate that imple- mentation of the guideline changed significantly over time, independent of CKD recognition or the timing of CKD recognition. Assuming the change to be in the de- sired direction, the result would suggest that the interven- tion was effective in helping the practices to implement the guideline. No other variables were included in the models for this feasibility study. The Physician Practice Connections—Patient-Centered Medical Home Research Version (PPC-PCMH-R), devel- oped by the National Committee for Quality Assurance, assessed the implementation of key practice systems for chronic illness management, including health systems (e.g., QI processes), information systems (e.g., registries), deci- sion support (e.g., structured visits), delivery system design (e.g., teamwork), and patient self-management support. Survey data were collected at the beginning and end of each wave including the priority for improving CKD care and CPCQ data from the lead clinician, and one PPC-PCMH-R from each practice. The CPCQ measures readiness to change and use of various change process strategies, while the PPC-PCMH-R measures actual prac- tice systems put into place to achieve better performance through the use of that readiness and those strategies. The initial modeling of the wave II data included all study practices. Wave II Practices in wave II also received performance evalu- ation and feedback and academic detailing. The three practices in each cluster (one wave I, two wave II) were expected to participate in a 1-h LLC meeting each month to review each other’s performance data, discuss lessons learned, and share EHR templates, order sets, and other tools and materials they had discovered or created (e.g., patient education handouts). The basic agendas for the LLC sessions were preset, including review of interim per- formance data in each participating practice and discus- sion of successful and unsuccessful approaches tried and obstacles and opportunities encountered. The first one or two LLCs often focused on the methods adopted by the wave I practices, with arrangements for sharing checklists, templates, and order sets. The PFs generally handled ad- ministrative aspects of the LLCs, and the wave I clinicians tended to lead and facilitate the discussions. During this wave, the PFs spent 2–4 h per month helping wave II practices implement desired changes in their care processes, teaching them about QI, and abstract- ing performance data for QI purposes. Our hope was that The abstraction windows for wave I practices were the 12 months prior to the beginning of the intervention (pre-intervention) and the 12 months after the start of Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 4 of 11 Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 the intervention (post-intervention). The abstraction windows for wave II practices were the 12 months prior to the beginning of the intervention (pre-intervention) and the 9 months after the beginning of the intervention (post-intervention). compared to the proportion with CKD on the problem list post-intervention but not pre-intervention. Confi- dence intervals (CI) were calculated. The proportions of patients with eGFR <30 who were referred to a neph- rologist before and after the interventions were similarly compared. The McNemar test was used to assess changes in guideline implementation with respect to prescribing ACEI/ARB, discontinuing NSAIDS and A1c, Hgb, and vitamin D testing (or supplementation), while the paired t test was used to assess changes in A1c, SBP and DBP, and LDL test results. Practice contexts and measures of change in priorities and processes However, there were a number of wave II prac- tices that attended none or very few of the LLC sessions. These practices would not be expected to benefit from the diffusion strategy, possibly leading to underestimation of the effect of the intervention when included. Consequently, a sub-analysis was conducted on those 30 practices that participated in at least five LLC sessions to assess the stabil- ity of the model estimates (sensitivity analysis). Analytic methods Except as noted below, all analyses were preplanned. Descriptive statistics (frequencies and proportions) were used to describe the wave I and wave II practice charac- teristics; comparisons between waves were made using Fisher’s exact test. The proportion of patients with CKD on the problem list in the pre-intervention period was Differences between measures of priority and change capacity and care processes between baseline and post- Page 5 of 11 Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Table 1 Wave I modeling of intervention effect, adjusted for timing of recognition of CKD diagnosis in the medical record Model effects CKD guideline HbA1c measured ACEI/ARB prescribed Taking NSAIDS Hgb measured Vitamin D measured Alb/Cr ratio measured OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) Intervention effect (post- vs. pre-intervention) NA 1.3 (1.1–1.6) 0.7 (0.4–1.0) 1.7 (1.1–2.6) 0.9 (0.7–1.3) 2.6 (1.7–3.8) NA p value 0.001 0.030 0.014 0.68 <0.0001 CKD recognition: NA NA Recognized pre-intervention vs. not recognized 0.9 (0.5–1.6) 0.9 (0.3–2.6) 2.9 (0.9–9.5) 1.1 (0.6–1.9) 0.9 (0.2–3.4) Recognized post-intervention vs. not recognized 0.8 (0.4–1.6) 0.8 (0.2–2.6) 2.5 (0.7–8.6) 1.6 (0.9–3.1) 1.0 (0.3–3.9) Recognized pre-intervention vs. recognized post-intervention 1.1 (0.7–1.7) 1.2 (0.5–2.7) 1.2 (0.6–2.2) 0.7 (0.4–1.1) 0.9 (0.5–1.7) p value 0.84 0.92 0.30 0.18 0.95 Interaction of intervention with CKD recognition: Post- vs. pre-intervention for CKD recognized pre-intervention 1.4 (1.0–1.9) 1.6 (1.1–2.1) Post- vs. pre-intervention for CKD recognized post-intervention 1.1 (0.8–1.6) 1.2 (0.7–1.9) Post- vs. pre-intervention for CKD not recognized 0.6 (0.3–1.0) 0.5 (0.3–0.9) p value 0.037 0.004 NA not applicable (The main effects contributing to an interaction have different interpretations in the presence of the interaction). deling of intervention effect, adjusted for timing of recognition of CKD diagnosis in the medical Table 1 Wave I modeling of intervention effect, adjusted for timing of recognition of CKD diag record NA not applicable (The main effects contributing to an interaction have different interpretations in the presence of the interaction). intervention were estimated using the paired t test. All analyses were carried out using SAS software system, version 9.2 (SAS Institute Inc., Cary, NC, USA). Imput- ation for missing values was not considered nor was there any correction made in the analyses for multiple comparisons. and was moved to wave II, leaving 31 practices that par- ticipated in wave I. Analytic methods All wave I practices had been mem- bers of one of the four PBRNs prior to this project. Following the wave I implementation intervention, the 31 wave I practices were able to help the research team recruit and enroll a total of 58 wave II practices (mean: 1.9 per practice). Wave II practices were statistically less likely to be a member of a PBRN and to have participated in a QI project before, but they were otherwise similar to wave I practices with regard to the characteristics exam- ined. A comparison of wave I and wave II practices, with Practice participation and characteristics Practice participation and characteristics Across the four PBRNs, 32 practices were recruited for wave I. One practice had to delay entry into the project Table 2 Wave II modeling of intervention effect adjusted for timing of recognition of CKD diagnosis in the medical record Model effects CKD guideline HbA1c measured ACEI/ARB prescribed Taking NSAIDS Vitamin D measured OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) Intervention effect (post- vs. pre-intervention) 1.0 (0.8–1.2) 1.4 (1.2–1.6) 0.9 (0.7–1.2) 2.2 (1.7–2.9) p value 0.84 <0.001 0.35 <0.0001 CKD recognition: recognized pre-intervention vs. not recognized 1.4 (0.9–2.2) 1.2 (0.8–1.8) 0.8 (0.4–1.6) 2.3 (0.8–7.0) Recognized post-intervention vs. not recognized 1.5 (0.9–2.5) 1.3 (0.8–2.0) 1.0 (0.5–2.3) 2.5 (0.8–7.9) Recognized pre-intervention vs. recognized post-intervention 0.9 (0.6–1.4) 1.0 (0.7–1.3) 0.8 (0.4–1.4) 0.9 (0.6–1.5) p value 0.24 0.51 0.67 0.30 ble 2 Wave II modeling of intervention effect adjusted for timing of recognition of CKD diagnosis in the medical cord odeling of intervention effect adjusted for timing of recognition of CKD diagnosis in the medical Table 2 Wave II modeling of intervention effect adjusted for timing of recognition of CKD diagno Mold et al. Practice participation and characteristics Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 6 of 11 Table 3 Practice characteristics by study wave Characteristic Wave I Wave II p valuec (N = 31a) (N = 58b) Outer setting Practice type: n (%) Academic practice 3 (10) 5 (10) 0.4 Federally designated health center or rural clinic 9 (30) 20 (39) Hospital outpatient practice 5 (17) 6 (12) Private practice 8 (27) 19 (37) Other (Free Clinics, etc.) 5 (16) 2 (4) Missing 1 6 Practice location: n (%) Rural 11 (38) 15 (29) 0.7 Suburban 6 (21) 14 (27) Urban 12 (41) 23 (44) Missing 2 6 Clinician owned: n (%) Yes 8 (27) 13 (25) 1.0 No 22 (73) 40 (75) Missing 1 5 PBRN member Yes 31 (100) 39 (67) 0.0002 No 0 19 (33) Inner setting Medical record type: n (%) EHR 28 (93) 43 (81) 0.2 Paper 2 (7) 10 (19) Missing 1 5 Number of full-time clinicians: n (%) 1 4 (14) 8 (16) 0.7 2 5 (18) 7 (14) 3–5 10 (36) 12 (24) ≥6 9 (32) 22 (45) Missing 3 9 Mid-level practitioners: n (%) Yes 19 (63) 35 (66) 0.8 No 11 (37) 18 (34) Missing 1 5 Ever in QI project: n (%) Yes 25 (89) 31 (65) 0.0292 No 3 (11) 17 (35) Missing 3 10 aOne wave I practice did not provide any baseline information. bOne wave II practice did not provide any baseline information. cp values obtained from Fisher’s exact test. EHR electronic health record, eGFR estimated glomerular filtration rate, QI quality improvement, PF practice facilitator, LLC local learning collaborative. Table 3 Practice characteristics by study wave characteristics categorized as suggested by Damshroeder et al., can be found in Table 3 [29]. Twenty of the expected 30 LLCs actually met as planned. The predominantly inner-city safety net practices in Los Angeles found it impossible to schedule LLCs and instead held monthly conference calls to which all partici- pating practices were invited. In OKPRN, MAFPRN, and WREN, eight, seven, and seven LLCs were formed and met a total of 121 times. Wave I clinicians attended 82% of the LLC meetings. Wave I practice performance Wave I practice performance data were obtained from the medical records of 711 patients. The majority were female (62%), born prior to 1/1/45 (54%) and thus were Medicare eligible, and white (78%). Among the 271 patients with evidence of CKD during the pre-intervention period, 39% (95% CI: 33%–45%) had CKD in the problem list com- pared to 66% (95% CI: 62%–70%) of 461 meeting CKD criteria during the post-period. Among the 105 patients with eGFR <30 during the pre-intervention period, 66% (95% CI: 56%–75%) had a re- ferral to a nephrologist, compared to 71% (95% CI: 62%– 79%) of 124 with eGFR <30 during the post-intervention period. In the unadjusted analysis, prescription of ACE/ ARBs and testing for Hgb and vitamin D (or supple- mentation) increased significantly from the pre- to post- intervention period (Table 4). The interaction of the intervention with CKD recogni- tion contributed significantly to predicting the probability of A1c measurement in the adjusted modeling (p = 0.037), indicating that the effect of the intervention varied accord- ing to whether or not CKD was recognized in the patient and the timing of such (Table 1). Among participants with CKD recognized in the pre-intervention period, there was a significantly increased probability of having HbA1c mea- sured in the post-intervention period compared to the pre-intervention period (odds ratio (OR) = 1.4, 95% CI: 1.0–1.9). This was also true for the measurement of microalbumin, with the interaction contributing signifi- cantly to the model (p = 0.004); participants with CKD recognized in the pre-intervention period were signifi- cantly more likely to have microalbumin measured in the post-intervention period compared to the pre-intervention period (OR = 1.6, 95% CI: 1.1–2.1). The modeling results indicate that the probability of being prescribed ACEIs/ARBs and having Hgb and vita- min D measured (or vitamin D supplements given) in- creased significantly from pre- to post-intervention (OR = 1.3, 95% CI: 1.1–1.6, p = 0.001; OR = 1.7, 95% CI: 1.1–2.6, p = 0.014; and OR = 2.6, 95% CI: 1.7–3.9, p <0.0001, re- spectively), but that the probabilities did not differ accord- ing to whether or not CKD was recognized or the timing of such (p ≥0.30). The odds of being on NSAIDS was One wave I practice did not provide any baseline information. bOne wave II practice did not provide any baseline information. cp values obtained from Fisher’s exact test. Wave I practice performance values obtained from Fisher’s exact test. EHR electronic health record, eGFR estimated glomerular filtration rate, QI quality improvement, PF practice facilitator, LLC local learning collaborative. Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 7 of 11 Page 7 of 11 Table 4 CKD performance outcome measures for wave I and wave II practices Performance measure Wave I Wave II Pre-intervention period Post-intervention period p value* Pre-intervention period Post-intervention period p value* If eGFR <60 N = 374 N = 660 ACE or ARB prescribed: n (%) 232 (63%) 255 (69%) 0.002 354 (55%) 407 (63%) <0.001 Taking NSAIDs: n (%) 25 (7%) 18 (5%) 0.10 51 (8%) 45 (7%) 0.44 A1c testing performed: n (%) 288 (77%) 297 (79%) 0.25 492 (89%) 487 (88%) 0.65 Urine microalbumin test performed: n (%) 158 (42%) 175 (47%) 0.16 NA NA NA LDL test performed: n (%) 309 (83%) 305 (82%) 0.75 NA NA NA A1c test results: Mean (SD) 7.1% (1.4) 7.2% (1.5) 0.78 7.4% (1.6) 7.3% (1.5) 0.07 Systolic BP measure (mmHg): mean (SD) 132.5 (16.3) 132.8 (16.0) 0.72 132.0 (15.9) 131.4 (14.9) 0.39 Diastolic BP measure (mmHg): mean (SD) 72.8 (9.1) 72.7 (9.0) 0.93 72.0 (9.4) 71.7 (8.5) 0.48 LDL test result (mg/dl): mean (SD) 89.8 (36.6) 90.2 (32.8) 0.84 96.3 (38.1) 92.0 (34.5) 0.004 If eGFR <45 N = 138 N = 233 Hemoglobin test performed: n (%) 84 (61%) 100 (72%) 0.02 NA NA NA Vitamin D testing (or supplement): n (%) 51 (37%) 83 (60%) <0.001 87 (37%) 132 (57%) <0.001 SD standard deviation, NA not applicable because it was an annual recommendation and in wave II, the post-intervention period was only 9 months. McNemar’s test was used to generate p values for categorical-scaled outcomes and the paired t test for continuous-scaled outcome measures. Table 4 CKD performance outcome measures for wave I and wave II practices SD standard deviation, NA not applicable because it was an annual recommendation and in wave II, the post-intervention period was only 9 months. McNemar’s test was used to generate p values for categorical-scaled outcomes and the paired t test for continuous-scaled outcome measures. licable because it was an annual recommendation and in wave II, the post-intervention period was only 9 months. rate p values for categorical-scaled outcomes and the paired t test for continuous-scaled outcome measures. Wave I practice performance significantly lower in the post-intervention period com- pared to the pre-intervention period (OR = 0.7, 95% CI: 0.4–0.95; p = 0.030) but did not differ according to whether or not CKD was recognized or the timing of such (p = 0.92). to have Hgb measured in the post-intervention period compared to pre-intervention period than those with CKD recognized in the post-intervention period (OR = 0.4, 95% CI: 0.2–0.8; p = 0.006). The probability of being prescribed ACEIs/ARBs in- creased significantly from pre- to post-intervention (OR = 1.4, 95% CI: 1.2–1.6; p <0.0001), as did measurement of vitamin D (or vitamin D supplements given) (OR = 2.2, 95% CI: 1.7–2.9; p <0.0001) (Table 2). The probability of implementation of these guidelines did not differ accord- ing to whether or not CKD was recognized or the timing of such (p ≥0.3). The probability of having microalbumin measured increased from the pre- to post-intervention period, but the change was of only borderline significance (OR = 1.2, 95% CI: 1.0–1.5; p = 0.069). CKD recognition did not contribute to predicting the change in microalbu- min measurement (p = 0.36). Wave II practice performance For Wave II, data were abstracted on 1,179 patients. The majority were female (63%), Medicare eligible (60%) and white (78%), similar to wave I patients. Among the 466 pa- tients with evidence of CKD during the pre-intervention period, 41% (95% CI: 37%–45%) had CKD in the problem list compared to 58% (95% CI: 54%–62%) of 664 with CKD evidence during the post-intervention period. Among the 129 patients with any eGFR <30 during the pre-intervention period in wave II, 60% (95% CI: 51%– 68%) had a referral to a nephrologist, compared to 55% (95% CI: 47%–63%) of 176 patients with eGFR <30 first documented during the post-intervention period. The pre- scription of ACEI/ARBs and testing for vitamin D (or sup- plementation) among those with eGFRs <45 increased significantly post-intervention (Table 4). In the adjusted modeling, the probability of having Hgb measured did not differ significantly from zero (no change pre- to post-intervention) (OR = 0.8, 95% CI: 0.6–1.2, p = 0.38), but did differ in relation to CKD recog- nition (p = 0.009) (Table 2). Patients with CKD recognized in the pre-intervention period were significantly less likely Among the 129 patients with any eGFR <30 during the pre-intervention period in wave II, 60% (95% CI: 51%– 68%) had a referral to a nephrologist, compared to 55% (95% CI: 47%–63%) of 176 patients with eGFR <30 first documented during the post-intervention period. The pre- scription of ACEI/ARBs and testing for vitamin D (or sup- plementation) among those with eGFRs <45 increased significantly post-intervention (Table 4). Sub-analyses were conducted on those 30 practices that attended at least five LLC sessions; ten practices attended five (all included wave I staff members) and 20 practices attended six (12 wave I practice staff members attended six; eight attended five). These analyses included 387 sub- jects with information available during the pre- and post- intervention periods; this number was further reduced to 125 subjects for the Hgb and vitamin D modeling which required eGFR to be <45. As was true for the original wave II modeling, the prescribing of ACEIs/ARBs increased significantly for the post-intervention period compared to In the adjusted modeling, the probability of having Hgb measured did not differ significantly from zero (no change pre- to post-intervention) (OR = 0.8, 95% CI: 0.6–1.2, p = 0.38), but did differ in relation to CKD recog- nition (p = 0.009) (Table 2). Discussion pre-intervention period for this subset of practices (OR = 1.3, 95% CI: 1.1–1.5, p = 0.005), as did measurement of vitamin D (OR = 2.4, 95% CI: 1.7–3.4, p <0.0001). Meas- urement of A1c (p = 0.81), Hgb (p = 0.84), and microalbu- min (p = 0.67) did not differ significantly according to the study period in either modeling or did the use of NSAIDS (p = 0.68). In their comprehensive review of innovation diffusion, Greenhalgh et al. identified a number of unanswered ques- tions including,‘Is there a role for a central agency, resource center, or officially sanctioned demonstration programs? and What is (or could be) the role of professional organiza- tions and informal inter-professional networks in spreading innovation among health care organizations?’ [30] The re- sults of this study suggest that diffusion, which is generally considered to be a passive process, can be facilitated by PBRN researchers and member practices using a combin- ation of assistance and incentives. Whether PBRNs should assume this role or whether a broader extension system similar to cooperative extension in agriculture should be developed for this purpose remains open [31,32]. In either case, PBRNs are likely to be a source of early adopter prac- tices and possibly clinician opinion leaders. Changes in eGFR No significant changes were seen in mean eGFRs be- tween the pre- and post-intervention periods for patients in either wave. Mean (SD) eGFRs for wave I patients were 42.3 (11.9) pre-intervention and 42.1 (12.9) post- intervention (p = 0.71). Mean eGFRs for wave II patients were 43.9 (11.1) pre-intervention and 43.2 (11.9) post- intervention (p = 0.06). Our results also speak to a second question posed by the Greenhalgh review, ‘What is the nature of the net- works, of different players… [and] How do these networks serve as channels for …embedding of complex service in- novations?’ Most of the early adopters chose to recruit practices with similar characteristics—either members of the same health system or those that used the same EHR. Reducing the complexity of the implementation setting is likely to facilitate adoption; furthermore, health system ad- ministrators often must approve efforts such as these that require templates and order sets. Wave II practice performance Patients with CKD recognized in the pre-intervention period were significantly less likely Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 8 of 11 Page 8 of 11 g p ation, CPCQ Change Process Capability Questionnaire, PPC-PCMH Physician Practice Connections—Patient-Centered Medical Home standard deviation, CPCQ Change Process Capability Questionnaire, PPC-PCMH Physician Practice Connections—Patient-Centered Medi earch Version. p g p SD standard deviation, CPCQ Change Process Capability Questionnaire, PPC-PCMH Physician Practice Connections—Patient-Centered Medical Home Research Version. Practice process changes Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Page 9 of 11 and implementation researchers. PBRNs could be helpful in this role. and care of patients with CKD in motivated practices. We demonstrated improvements in the use of ACEIs and ARBs, measurement of Hgb, and vitamin D testing or sup- plementation. Since diabetes care had received a great deal of attention already, baseline adherence to those measures was high in many practices. Likewise, rates of usage of NSAIDs were already low. It is also important to remem- ber that 100% adherence is not an appropriate expect- ation, since guidelines do not apply to all patients with CKD, and we did not exclude patients with, for example, terminal illness, dementia, or contraindications to particu- lar strategies. The interventions resulted in development of both new strategies (techniques) and new processes of care (system level changes). This study has several limitations. It was designed to determine whether experienced practices could facilitate improvements in other practices. A separate control group was not included. Therefore, a secular trend toward better care of CKD patients could have contributed to observed performance improvements. However, practices engaged in the project uniformly indicated that they had done little to improve their care of patients with CKD prior to the pro- ject, and we know of no attempts by others in the involved states to address this problem during the project. Likewise, we are unaware of changes in medication cost or availabil- ity that might have resulted in increased prescribing of ACEIs or ARBs, though that is certainly a possibility. Wave II practices showed improvements in care of their CKD patients similar to wave I practices with less PF assistance. This is important since PF support is rela- tively expensive. As in wave I, the interventions resulted in the development of new strategies, but there was no significant increase in new processes of care. However, wave II practices had less time (9 vs. 12 months) to change, and their baseline process scores were nearly as high as the post-intervention process scores for wave I practices, leaving less room for improvement. This could have, in part, been due to cross-contamination within health systems that chose to implement system wide changes prior to wave II. Practice process changes For wave I practices, priority for improving care of pa- tients with CKD remained relatively high, with no significant change from pre- to post-intervention, and there was no significant change in subscales designed to measure organizational factors associated with practice change capacity (i.e., history of change, continuous re- finement, and sustaining change), but the use of change strategies increased (Table 5). A similar pattern of find- ings occurred in wave II, including a significant increase in strategy count. There was, however, no statistically significant increase in practice systems in wave II prac- tices, but their baseline scores were also higher. As expected, the combination of performance feed- back (baseline and monthly), academic detailing, and weekly PF for 6 months resulted in improved recognition Table 5 Pre- and post-intervention priority, change capacity, and change process content scores Instrument Wave I Wave II n Pre-intervention Post-intervention p value* n Pre-intervention Post-intervention p value* Mean (SD) Mean (SD) Mean (SD) Mean (SD) Priority for improving CKD care 28 6.1 (2.4) 6.4 (2.1) 0.46 39 6.0 (2.1) 6.4 (1.90) 0.27 CPCQ: Organizational factors 26 14.5 (11.4) 16.8 (9.0) 0.29 39 13.2 (9.8) 15.0 (7.9) 0.25 Previous history of change 28 2.4 (3.1) 3.2 (2.3) 0.25 39 2.4 (2.3) 2.5 (2.5) 0.95 Plans for organizational refinement 29 3.6 (2.6) 4.2 (1.7) 0.21 40 3.0 (2.7) 3.7 (2.0) 0.08 Ability to initiate and sustain change 26 8.3 (6.7) 9.4 (6.2) 0.43 40 8.0 (5.9) 9.1 (4.8) 0.22 Strategy count 25 3.3 (4.1) 18.6 (4.5) <0.0001 37 5.5 (7.8) 16.7 (7.9) <0.0001 PPC-PCMH-R: Health systems 28 45.8 (32.3) 50.0 (30.8) 0.27 28 41.1 (28.9) 40.5 (31.2) 0.90 Clinical information system 28 48.8 (16.0) 56.3 (16.5) 0.016 28 53.2 (16.2) 58.9 (20.2) 0.12 Decision support 28 45.8 (17.1) 63.3 (19.6) 0.0001 28 52.8 (18.4) 60.5 (24.5) 0.14 Delivery system design 25 25.0 (14.8) 34.8 (22.7) 0.013 28 32.8 (18.6) 34.4 (20.0) 0.73 Patient self-management support 28 21.8 (12.5) 32.1 (16.8) 0.005 28 26.5 (17.4) 30.8 (18.6) 0.23 *The paired t test was used to generate the p values. SD standard deviation, CPCQ Change Process Capability Questionnaire, PPC-PCMH Physician Practice Connections—Patient-Centered Medical Home vention priority, change capacity, and change process content scores Table 5 Pre- and post-intervention priority, change capacity, and change process content scores Table 5 Pre- and post-intervention priority, change capacity, and change p Mold et al. Conclusions After receiving implementation assistance, early adopter practices were able to help to recruit and support add- itional practices with implementation of the National Kidney Foundation Kidney Disease Outcomes Quality Initiative CKD guidelines through monthly LLCs, per- formance feedback (baseline and monthly), academic de- tailing, and monthly practice facilitation over a 6-month period. This may be an important way to disseminate, im- plement, and diffuse other evidence-based innovations. We found evidence that putting CKD on the problem list is a critical first step in providing evidence-based care for patients with CKD. Guideline producers should consider developing both practice-friendly summaries and suggestions regarding implementation sequencing. This would require collaboration with end-users (practices) Practice process changes Due to delays in recruitment and enrollment of wave II practices and the prohibition on no-cost extensions imposed by the American Recovery and Reinvestment Act, the length of the wave II post-intervention period was shortened from 12 months (as in wave I) to 9 months. As a result, improvement in measures of guideline adher- ence requiring annual testing (e.g., measurement of lipids, anemia tests, and microalbumin) may be underestimated. Wave II practices also had less time to implement sys- tem level changes which could have reduced the impact of the intervention. We anticipated abstracting data on 30 patients from each participating practice. Unfortunately, practices var- ied widely in their ability to identify patients with two eGFRs <60 at least 3 months apart. Therefore, the num- ber of patients from practices eligible for these analyses ranged from 1 to 27, making it impossible to adjust for variation among practices. Although the smaller than ex- pected sample reduced the statistical power of our ana- lyses, we were nevertheless able to demonstrate significant improvements in guideline implementation by both wave I and wave II practices. We are optimistic that many of the new processes im- plemented during this project will be sustained since they were typically accompanied by the creation of tem- plates, order sets, and other modifications to the EHRs. The automatic calculation of eGFR by the labs used by participating practices prior to or as a result of the pro- ject, and the increased awareness of the importance of eGFR created by the project are also likely to persist. However, at this point, we have no data upon which to assess sustainability. Despite the availability of EHR options like templates and order sets, the one-page summary of guideline recommendations organized by CKD stage and substage proved to be very helpful to practices as they developed strategies for improvement (see Additional file 1). Many practices reported putting them in exam rooms and referring to them routinely. The one-page summary was also provided to several practices not involved in the project upon request after having heard about it from colleagues. This we found interesting, since, in theory, all of this could be pro- grammed into an EHR. Additional file 1: Summary of CKD guideline recommendations for primary care (updated). Authors’ contributions 9. Nagykaldi Z, Mold JW, Robinson A, Niebauer L, Ford A: Practice facilitators and practice-based research networks. J Am Board Fam Med 2006, 19(5):506–510. JWM served as the principal investigator (PI) for the project and was involved in all aspects or planning, conducting, analyzing, and disseminating the results. CBA served as the project coordinator and site PI for Oklahoma and was heavily involved in training and supervising the PFs and in the various organizational aspects of the project. PDS (site PI in Wisconsin), TZ (site PI in Minnesota), and LK (site PI in Los Angeles) were involved in directing recruitment and enrollment of practices, training and supervising the ADs and PFs, collecting and transferring the data, and participating in the overall project management and decision-making. PDL served as the site PI at Westat, organizing the data collection and transfer processes, scheduling conference calls, and tracking decisions and timelines. MK was responsible for the data collection and management, for assembling the data into an analyzable form, and for the training of PFs to assure data accuracy. DRH was the statistician for the project conducting all analyses and contributing those pieces of the manuscript. CF was the project’s content expert, assuring that guideline recommendations were properly taught and clinician questions and concerns were addressed. LIS served as the project’s expert in quality improvement methods and evaluation of the components of his change model. RC assisted with the data management and the analysis. All authors were given the opportunity to read and approve the final manuscript. 10. Nagykaldi Z, Mold JW: The role of health information in the translation of research into practice. J Am Board Fam Med 2002, 20(2):188–195. 11. Nagykaldi Z, Mold JW: Diabetes patient tracker, a personal digital assistant-based diabetes management system for primary care practices in Oklahoma. Diabetes Technol Ther 2003, 5(6):997–1001. 12. Rogers EM: Change agents. In Diffusion of Innovations. 5th edition. Edited by Rogers. New York, NY: The Free Press; 2003:168–218. 13. Lineker SC, Bell MJ, Boyle J, Badley EM, Flakstad L, Fleming J, Lyddiatt A, MacDonald J, McCarthy J, Zummer M: Implementing arthritis clinical practice guidelines in primary care. Med Teach 2009, 31(3):230–237. 14. Ayers LR, Beyea SC, Godfrey MM, Harper DC, Nelson EC, Batalden PB: Quality improvement learning collaboratives. Qual Manag Health Care 2005, 14(4):234–247. 15. Funding h This research was supported by a grant from the Agency for Healthcare Research and Quality (1R18HS019945). This research was supported by a grant from the Agency for Healthcare Research and Quality (1R18HS019945). 17. Aspy CB, Mold JW, Thompson DM, Blondell RD, Landers PS, Reilly KE, Wright-Eakers L: Integrating screening and interventions for unhealthy behaviors into primary care practices. Am J Prev Med 2008, 35(5 Suppl): S373–S380. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Authors’ contributions McMullen CK, Schneider J, Firemark A, Davis J, Spofford M: Cultivating engaged leadership through a learning collaborative: lessons from primary care renewal in Oregon safety net clinics. Ann Fam Med 2013, 11(Suppl 1):S34–S40. 16. Mangione-Smith R, Schonlau M, Chan KS, Keesey J, Rosen M, Louis TA, Keeler E: Measuring the effectiveness of a collaborative for quality improvement in pediatric asthma care: does implementing the chronic care model improve processes and outcomes of care? Ambul Pediatr 2005, 5(2):75–82. Abbreviations l b 4. Lipman PD, Lange CJ, Cohen RA, Peterson KA: A mixed methods study of research dissemination across practice-based research networks. J Ambul Care Manage 2014, 37(2):179–188. A1c: Hemoglobin A1c; ACEIs: Angiotensin converting enzyme inhibitors; ARBs: Angiotensin receptor blockers; CI: Confidence intervals; CKD: Chronic kidney disease; CME: Continuing medical education; CPCQ: Change process capability questionnaire; DBP: Diastolic blood pressure; eGFR: Estimated glomerular filtration rates; EHR: Electronic health record; GEE: Generalized estimating equations; Hgb: Hemoglobin; LDL: Low-density lipoprotein; LLCs: Local learning collaboratives; microalbumin: Urine albumin/creatinine ratio; MOC: Maintenance of certification; NSAIDS: Non-steroidal anti- inflammatory medications; OKPRN: Oklahoma physicians resource/research network; OR: Odds ratio; PBRN: Practice-based research networks; PFs: Practice facilitators; PPC-PCMH-R: Physician practice connections – patient-centered medical home research version; QI: Quality improvement; SBP: Systolic blood pressure 5. Aspy CB, Enright M, Halstead L, Mold JW: Improving mammography screening using best practices and practice enhancement assistants: an Oklahoma Physicians Resource/Research Network (OKPRN) study. J Am Board Fam Med 2008, 21(4):326–333. 6. Hogg W, Baskerville N, Lemelin J: Cost savings associated with improving appropriate and reducing inappropriate preventive care: cost consequences analysis. BMC Health Serv Res 2005, 5(1):20. 7. Mold JW, Aspy CA, Nagykaldi Z: Implementation of evidence-based preventive services delivery processes in primary care: an Oklahoma Physicians Resource/Research Network (OKPRN) study. J Am Board Fam Med 2008, 21(4):334–344. PFs: Practice facilitators; PPC-PCMH-R: Physician practice connections – patient-centered medical home research version; QI: Quality improvement; SBP: Systolic blood pressure. patient-centered medical home research version; QI: Quality improvement; SBP: Systolic blood pressure. 8. Mold JW, Fox C, Wisniewski A, Darby Lipman P, Krauss MR, Harris RD, Aspy C, Cohen RA, Elward K, Frame P, Yawn B, Solberg L, Gonin R: Helping primary care practices implement asthma guidelines: a randomized controlled trial comparing practice facilitation and local learning collaboratives. Ann Fam Med. in press Author details 1 1Department of Family and Preventive Medicine, University of Oklahoma Health Sciences Center, 900 NE 10th Street, Oklahoma City, OK 73104, USA. 2University of Wisconsin School of Medicine and Public Health, 1100 Delaplaine Court, Madison, WI 53715, USA. 3Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA. 4Los Angeles Practice-Based Research Network (LA Net), 3940-B East Broadway, Long Beach, CA 90803, USA. 5Westat, 1600 Research Boulevard, Rockville, MD 20850, USA. 6State University of New York at Buffalo, 1315 Jefferson Avenue, Buffalo, NY 14208, USA. 7HealthPartners Institute for Education and Research, Mail Stop 23301, P.O. Box 1524, Minneapolis, MN 55440-1524, USA. 18. Kidney Disease Statistics for the United States. [http://kidney.niddk.nih. gov/kudiseases/pubs/kustats/KU_Diseases_Stats_508.pdf] 19. Keith DS, Nichols GA, Guillion CM, Brown JB, Smith DH: Longitudinal follow-up and outcomes among a population with chronic kidney disease in a large managed care organization. Arch Intern Med 2004, 164(6):659–663. 20. National Kidney Foundation: K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2002, 39(2 Suppl 1):S1–S266. Education and Research, Mail Stop 23301, P.O. Box 1524, Minneapolis, MN 55440-1524, USA. 21. Jafar TH, Stark PC, Schmid CH, Strandgaard S, Kamper AL, Maschio G, Becker G, Perrone RD, Levey AS: The effect of angiotensin-converting-enzyme inhibitors on progression of advanced polycystic kidney disease. Kidney Int 2005, 67(1):265–271. Received: 14 May 2014 Accepted: 5 November 2014 Received: 14 May 2014 Accepted: 5 November 2014 22. Gooch K, Culleton BF, Manns BJ, Zhang J, Alfonso H, Tonelli M, Frank C, Klarenbach S, Hemmelgarn BR: NSAID use and progression of chronic kidney disease. Am J Med 2007, 120(3):e1-7. Additional file Additional file 1: Summary of CKD guideline recommendations for primary care (updated). Page 10 of 11 1. Mold JW, Peterson KA: Primary care practice-based research networks: working at the interface between research and quality improvement. Ann Fam Med 2005, 3(Suppl 1):S12–S20. Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 Mold et al. Implementation Science 2014, 9:169 http://www.implementationscience.com/content/9/1/169 25. Schonlau M, Mangione-Smith R, Chan KS, Keesey J, Rosen M, Louis TA, Wu SY, Keeler E: Evaluation of a quality improvement collaborative in asthma care: does it improve processes and outcome of care? Ann Fam Med 2005, 3(3):200–208. 26. Hulscher ME, Wensing M, van Der Weijden T, Grol R: Interventions to implement prevention in primary care. Cochrane Database Syst Rev 2001, 1:CD000362. 27. Solberg LI, Crain AL, Jaeckels N, Ohnsorg KA, Margolis KL, Beck A, Whitebird RR, Rossom RC, Crabtree BF, Van de Ven AH: The DIAMOND initiative: implementing collaborative care for depression in 75 primary care clinics. Implement Sci 2013, 8:135. 28. Solberg LI: Improving medical practice: a conceptual framework. Ann Fam Med 2007, 5(3):251–256. 29. Damschroder LJ, Aron DC, Keith RE, Kirsh SR, Alexander JA, Lowery JC: Fostering implementation of health services research findings into practice: a consolidated framework for advancing implementation science. Implement Sci 2009, 4:50. 30. Greenhalgh T, Robert G, Macfarlane F, Bate P, Kyriakidou O: Diffusion of innovations in service organizations: systematic review and recommendations. Milbank Q 2004, 82(4):581–629. 31. Grumbach K, Mold JW: A health care cooperative extension service: transforming primary care and community health. JAMA 2009, 301(24):2589–2591. 32. Phillips RL Jr, Kaufman A, Mold JW, Grumbach K, Vetter-Smith M, Berry A, Burke BT: The primary care extension program: a catalyst for change. Ann Fam Med 2013, 11(2):173–178. 32. Phillips RL Jr, Kaufman A, Mold JW, Grumbach K, Vetter-Smith M, Berry A, Burke BT: The primary care extension program: a catalyst for change. Ann Fam Med 2013, 11(2):173–178. doi:10.1186/s13012-014-0169-x Cite this article as: Mold et al.: Leveraging practice-based research networks to accelerate implementation and diffusion of chronic kidney disease guidelines in primary care practices: a prospective cohort study. Implementation Science 2014 9:169. References 1. Mold JW, Peterson KA: Primary care practice-based research networks: working at the interface between research and quality improvement. Ann Fam Med 2005, 3(Suppl 1):S12–S20. 23. Gouva C, Nikolopoulos P, Ioannidis JP, Siamopoulos KC: Treating anemia early in renal failure patients slows the decline of renal function: a randomized controlled trial. Kidney Int 2004, 66(2):753–760. pp 2. Riley WT, Glasgow RE, Etheredge L, Abernethy AP: Rapid, responsive, relevant (R3) research: a call for a rapid learning health research enterprise. Clin Transl Med 2013, 2(1):1–6. 24. Shepherd J, Kastelein JJ, Bittner V, Deedwania P, Breazna A, Dobson S, Wilson DJ, Zuckerman A, Wenger NK: Intensive lipid lowering with atorvastatin in patients with coronary heart disease and chronic kidney disease: the TNT (Treating to New Targets) study. J Am Coll Cardiol 2008, 51(15):1448–1454. p 3. Williams RL, Rhyne RL: No longer simply a practice-based research network (PBRN) health improvement networks. J Am Board Fam Med 2011, 24(5):485–488. Page 11 of 11 Mold et al. 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chr tkus ds Ik’pkr~ Hkh tutkrh; lekt dh vf/kdka’k vkcknh xjhch] ruko] 'kks"k.k] fgalk] vyxko] fuj{kjrk] ySafxd fo"kerk] O;fDrxr Lora=rk dk guu] lkekftd U;k; ij dqBkjk?kkr] iztkrh; HksnHkko] vf/kdkjksa dh oapuk vkfn vusd leL;kvksa ds chp viuk thou O;rhr djus dks foo’k gSA bu lHkh leL;kvksa dk fujkdj.k ekuokf/kdkjksa dh izkfIr ls gh laHko gSA bl n`f"V ls ekuo vf/kdkj dh vo/kkj.kk ,d vk’kk dh fdj.k ds :i esa n`f"Vxr gksrh gS tks ekuo fodkl dks izHkkfor djrh gSA Key words : vuqlwfpr tutkfr] ekuo vf/kdkj] fodkl] lkr n’kd] Fkk:] lekt] leL;k,aA ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag vflLVsaV izksQslj] jktuhfr foKku foHkkx jkuh /keZ dqaoj jktdh; egkfo\|ky; nYykokyk] [kkuiqj ¼gfj}kj½] mRrjk[k.M Email - dr.sksinghpolsc@gmail.com Received : 01/03/2023 1st BPR : 07/03/2023 2nd BPR : 16/03/2023 Accepted : 22/03/2023 larks"k dqekj flag vflLVsaV izksQslj] jktuhfr foKku foHkkx jkuh /keZ dqaoj jktdh; egkfo\|ky; nYykokyk] [kkuiqj ¼gfj}kj½] mRrjk[k.M Email - dr.sksinghpolsc@gmail.com Received : 01/03/2023 1st BPR : 07/03/2023 2nd BPR : 16/03/2023 Accepted : 22/03/2023 Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 331 dh n`f"V ls fiNMs+ bu ekuo lewg dks fofHkUu laKk 'kCnksa tSls& tutkfr] vkfne tkfr] vkfnoklh] ouoklh] oU;tkfr] fxfjtu vkfn ls lEcksf/kr fd;k x;k gSaA tutkfr dh ifjHkk"kk nsrs gq, fxfyu vkSj fxfyu us dgk gS fd] LFkkuh; tutkrh; lewgksa dk ,slk lewnk; tutkfr dgk tkrk gS tks ,d lkekU; {ks= esa fuokl djrk gS rFkk ftldh ,d lkekU; laLd`fr gSA etwenkj ds 'kCnks esa] tutkfr ifjokjksa ;k ifjokjksa ds lewg dk ,d ladyu gksrk gS ftlds lnL; ,d fuf’pr Hkw&Hkkx ij jgrs gS] lkekU; Hkk"kk cksyrs gS vkSj fookg] O;olk; ;k m\|ksx ds fo"k; esa dqN fu"ks/kksa dk ikyu djrs gSa vkSj ,d fuf’pr ,oa mi;ksxh ijLij vknku&iznku dh O;oLFkk dk fodkl djrs gSaA mijksDr ifjHkk"kkvksa ds vk/kkj ij ;g dgk tk ldrk gS fd tutkfr ,d ,slk lewg gS ftldh laLFkk,a rFkk fu;e ijLij tqM+s gksrs gSa rFkk ,d gh izdkj ds lkekftd O;oLFkk ij vk/kkfjr gksrs gSaA ,d fof’k"V tutkfr ds lkaLd`frd rRoksa esa ,d:irk ikbZ tkrh gS ftlls mUgsa vU; leqnk;ksa ls ,d ne vyx fd;k tk ldrk gSA Hkkjr 2020 okf"kZd lanHkZ xzFk ds vuqlkj] vuqlwfpr tutkfr ds :i esa fdlh leqnk; dks fofufnZ"V djus ds fy, vkeRkkSj ls viuk, tkus okys ekin.M gS % ¼d½ izkjfEHkd y{k.kksa ds ladsr ¼[k½ fo’ks"k laLd`fr ¼x½ leqnk; ds lkFk laidZ cukus esa ladksp vkSj ¼?k½ HkkSxksfyd :i ls vyx&Fkyx ;kuh fiNM+kiuA tux.kuk 2011 ds vuqlkj] ns’k esa vuqlwfpr tutkfr;ksa dh tula[;k 10-45 djksM+ gSA vuqlwfpr tutkfr dh tula[;k ns’k dh dqy tula[;k dk 8-60 izfr’kr gS rFkk dqy xzkeh.k tula[;k dk 11-30 izfr’kr gSA vuqlwfpr tutkfr ds iq:"kksa dh tula[;k 5-25 djksM+ rFkk efgykvksa dh tula[;k 5-20 djksM+ gSA vuqlwfpr tutkfr;ksa ds lEcU/k esa fyax vuqikr 990 gS tks 943 ds jk"Vªh; vkSlr ls vf/kd gSA tux.kuk 2011 ds vkdM+ks ds vuqlkj] Hkkjr esa vuqlwfpr ds fy, lk{kjrk nj o"kZ 2001 esa 47-10 izfr’kr ls 2011 esa 59-00 izfr’kr rd lq/kkj gqvk gSA bu o"kksZ ds nkSjku vuqlwfpr tutkfr ds iq:"kksa esa lk{kjrk nj 59-20 izfr’kr ls c<+dj 68-50 rd c<+h gS rFkk vuqlwfpr tutkfr dh efgykvksa esa lk{kjrk nj 34-80 ls 49-40 rd c<+h gSA laiw.kZ tula[;k esa lk{kjrk nj esa o"kZ 2001 esa 64-80 izfr’kr ls o"kZ 2011 esa 73-00 rd dh c<+ksrjh gqbZ gSA Hkkjrh; lafo/kku ds vuqPNsn 342 ¼1½ ds vuqlkj] jk"Vªifr fdlh jkT; ;k dsUnz ’kkflr izns’k ds lanHkZ esa vkSj jkT; ds ekeys esa jkT;iky ls ijke’kZ ds ckn tutkfr;ka ;k tutkrh; leqnk;ksa ;k muds leqgksa dks vuqlwfpr tutkfr;ksa ds :i esa vf/klwfpr dj ldrs gSaA Hkkjr ds lafo/kku ds vuqPNsn 342 ds varxZr vf/klwfpr 700 ls vf/kd tutkfr;ka gS tks ns’k ds fofHkUUk jkT;ksa vkSj la?k jkT; {ks=ksa esa QSyh gqbZ gSaA vusd tutkfr;ka ,d ls vf/kd jkT;ksa esa ekStwn gSaA vuqlwfpr tutkfr;ksa ds :i esa vf/klwfpr leqnk;ksa dh lcls vf/kd la[;k 62 mM+hlk esa gSA gfj;k.kk vkSj iatkc rFkk dsUnz ’kkflr paMhx<+] fnYyh vkSj iqnqpsjh esa fdlh Hkh leqnk; dks vuqlwfpr tutkfr ds :i esa vf/klwfpr ugha fd;k x;k gSA s s q q q q w w 'kks/k {ks= mRrjk[k.M jkT; dh LFkkiuk 9 uoEcj 2000 dks gqbZA ;g ns’k dk 27 oka jkT; gS ftls mRrj izns’k ls i`Fkd djds fd;k x;k gSA mRrjk[k.M esa ikap tutkfr;ka&HkksfV;k] tkSulkjh] Fkk:] cqDlk vkSj jkth fuokl djrh gSaA 'kks/k v/;;u ds fy, Fkk: tutkfr dk p;u fd;k x;k gSA mRrjk[k.M esa Fkk: tutkfr dk fuokl {ks= dsoy Å\|eflag uxj tuin ds [kVhek vkSj flrkjxat fodkl [k.M esa gSA bl dkj.k buds fuokl {ks= dks FkMqokV uke ls Hkh tkuk tkrk gSA tux.kuk 2011 ds vuqlkj] [kVhek fodkl [k.M esa Fkk: tutkfr dh dqy tula[;k 50371 gS ftlesa 25023 iq:"k vkSj 25348 efgyk,a gSaA blh izdkj flrkjxat fodkl[k.M esa Fkk: tutkfr dh dqy tula[;k 36992 gS ftlesa 18618 iq:"k vkSj 18374 efgyk,a gSaA bl izdkj bu nksuks fodkl [k.M esa Fkk: tutkfr dh dqy tula[;k 87363 gSA ’kks/k {ks= esa [kVhek fodkl [k.M ds 5 xkaoksa&xqj[kqMk] me:dyka] lqft;k] ckuqlk vkSj dqavk[ksMk dks ’kkfey fd;k x;kA nwljh rjQ flrkjxat fodkl [k.M ds 5 xkaoksa& rqdkZfrlkSj] VqdM+h] lqUnjiqj] fcpqok vkSj uohuxj dk p;u fd;k x;kA izR;sd xkao ls 5 mRrjnkrkvksa dks pqurs gq, 50 mRrjnkrkvksa ¼25 mRrjnkrk efgyk vkSj 25 mRrjnkrk iq:"k½ ls rF; ,df=r fd;k x;k ftlls Fkk: tutkfr esa ekuokf/kdkj ls lEcfU/kr fofHkUu igyqvksa dk irk py lds vkSj mlh vk/kkj ij mldk vkadyu fd;k tk ldsA Tkutkrh; ifjn`’; Lkekftd] vkfFkZd] jktuhfrd] lkaLd`frd] vkSj izkfof/kd fodkl ds fofHkUu Lrjksa dk izfrfuf/kRo djus okys tutkrh; lewgksa dh lkaLd`fr;ka vkt Hkh vusd n`f"V;ksa ls viuk fo’ks"k egRo j[krh gSaA Hkkjr esa tgka ,d vksj fofHkUu HkkSxksfyd {ks=ksa dk lekos’k gS ogha nwljh vksj fofHkUu iztkrh; rRoksa dk Hkh lekos’k gSA Hkkjr ds bu fofHkUu {ks=ks esa vusd ,sls ekuo lewg fuokl djrs gSa tks vkt Hkh lH;rk ds vkfnHk Lrj ij gSa vkSj vR;f/kd fiNMs+ gq, gSaA lH;rk https://recentjournals.in & https://ijorr.in Periodicity : Quarterly, Language : English & Hindi [ 49 ] ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 3318 \| Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 3318 \| lek/kku <+w<+us dk iz;kl fd;k tkrk gSA ;ax dk ekuuk gS fd] lekftd ’kks/k dk izeq[k mn~ns’; fudV vFkok nwjxkeh lekftd thou dks le>uk vkSj mlds }kjk lkekftd O;ogkj ij csgrj fu;a=.k izkIr djuk gSA izLrqr ’kks/k i=] ekuo vf/kdkj ,oa tutkrh; lekt % lkr n’kd dk fo’ys"k.k ds vk/kkj ij ’kks/kkFkhZ us mRrjk[k.M dh Fkk: tutkfr dk p;u fd;k gSA 'kks/kkFkhZ us ’kks/k dk mn~ns’; fuEu vk/kkj ij cuk;k gS& 1- Fkk: tutkfr esa ekuokf/kdkjksa ds izfr tkx:drk dk vkadyu djukA - Fkk: tutkfr esa ekuokf/kdkjksa ds izfr tkx:drk dk vkadyu djukA 2- Fkk: tutkrh; lekt ds xSj tutkrh; lekt dh rqyuk esa fiNM+s gksus ds dkj.kksa dk v/;;u d 2- Fkk: tutkrh; lekt ds xSj tutkrh; lekt dh rqyuk esa fiNM+s gksus ds dkj.kksa dk v/;;u djukA h a S f f sa s f s f Sa h h 2- Fkk: tutkrh; lekt ds xSj tutkrh; lekt dh rqyuk es fiNM+s gksus ds dkj.kks dk v/;;u djukA 3- Fkk: tuTkkrh; lekt laoS/kkfud vf/kdkjksa ls fdrus ykHkkfUor gq, gSa] bldh tkudkjh izkIr djukA 4- fofHkUUk ljdkjh ;kstukvksa dk ykHk D;k Fkk: tutkrh; leqnk; dks fey jgk gS] bldk irk yxkukA 5- Fkk: tutkrh; lekt esa ekuokf/kdkj ds guu dh fLFkfr dk vkadyu djukA 2- Fkk: tutkrh; lekt ds xSj tutkrh; lekt dh rqyuk es fiNM+s gksus ds dkj.kks dk v/;;u djukA 3- Fkk: tuTkkrh; lekt laoS/kkfud vf/kdkjksa ls fdrus ykHkkfUor gq, gSa] bldh tkudkjh izkIr djukA f f h s sa h s f S 4- fofHkUUk ljdkjh ;kstukvksa dk ykHk D;k Fkk: tutkrh; leqnk; dks fey jgk gS] bldk irk yxkukA 5 Fkk: tutkrh; lekt esa ekuokf/kdkj ds guu dh fLFkfr dk vkadyu djukA 4- fofHkUUk ljdkjh ;kstukvksa dk ykHk D;k Fkk: tutkrh; leqnk; dks fey jgk gS] bldk irk yxkukA s s 'kks/k dk mn~ns’; fdlh Hkh ’kks/kkFkhZ }kjk vius 'kks/k fo"k; dk p;u djus ds lkFk&lkFk v/;;u dk mn~ns’; fu/kkZfjr djuk 'kks/k dk;Z dh vk/kkjf’kyk ekuh tkrh gSA 'kks/k ds mn~ns’; ls gh vusd leL;kvksa dk irk pyrk gS ftlds vk/kkj ij mudk Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in [ 50 ] ’kks/k vfHkdYi izLrqr 'kks/k ds fy, 'kks/kkFkhZ us 'kks/k vfHkdYi ds varxZr nSo fun’kZu i)fr dk lgkjk fy;k gSA bl i)fr esa lexz esa ls dqN bdkb;ksa dks pquko djus dk volj izkIr jgrk gSA i{kikriw.kZ fun’kZu pquko dks nwj djus ds fy, bl fof/k dk iz;ksx fd;k tkrk gSA xqMs vkSj gkV ds 'kCnksa esa] fun’kZu tSlk fd uke }kjk gh izrhr gksrk gS fdlh o`gn~ lewg dk ,d vis{kkd`r y?kq izfrfuf/k gSA Periodicity : Quarterly, Language : English & Hindi ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag lkexzh ¼lead½ ds L=ksr lkexzh ¼led½ d L=kr Kkr vFkok miyC/k rF;ksa] vkdM+ksa vkSj lwpuk dks lkexzh ¼lead ;k MkVk½ dgk tkrk gSA lead ds L=ksr dks izkIr djus ds fy, “kks/kkFkhZ us fuEu ek/;e viuk;k gS& w djus ds fy, “kks/kkFkhZ us fuEu ek/;e viuk;k gS& 1- izkFkfed L=ksr izkFkfed L=ksr dk vfHkizk; ,slh lwpukvksa] rF;ksa ;k vkadM+ksa ls gS ftldk laxzg 'kks/kkFkhZ Lo;a igyh ckj 'kks/k {ks= esa tk dj ,d= djrk gSA 'kks/kkFkhZ mRrjnkrkvksa ls lk{kkRdkj djds ;k Lo;a fujh{k.k djds ;k vuqlwph ;k iz’ukoyh ds ek/;e ls rF;ksa dk laxzg djrk gSA jkor ds 'kCnksa esa] izkFkfed L=ksr os ekSfyd lwwpuk,a rFkk vkdM+s gksrs gS] ftudk ladyu izFke ckj ladfyr fd, tkus ds dkj.k gh bUgs izkFkfed lead dgk tkrk gSA 2- f}rh;d L=ksr f}rh;d L=ksr dk vfHkizk; ,slh lwpukvksa] rF;ksa ;k vkdM+ksa ls gksrk gS ftldk ladyu igys ls gh fdlh vU; O;fDr ;k laLFkk }kjk fd;k x;k gSA Mk;jh] laLEkj.k] vkRedFkk] ik.Mqfyfi] i=] ljdkjh xtV] 'kks/k fjiksVZ] ljdkjh izfrosnu] iqLrdsa vkfn dks f}rh;d L=ksr ds varxZr j[kk tkrk gSA f=ikBh vkSj JhokLro ds vuqlkj] f}rh;d lkexzh dk ladyu igys ls gh gks pqdk jgrk gSA dsoy vuqla/kkudrkZ mUgsa vius iz;ksx esa ykrk gSA og Lo;a laxzg.k dk;Z ugha djrkA fdlh vU; mn~ns’; ds fy, ladfyr lkexzh dks vius iz;ksx esa ykrk gSaA ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u ekuo vf kdkj ,o tutkrh; lekt ,d fo’y kukRed v ;;u vktknh ds 75 o"kZ ls vf/kd le; chr tkus ds Ik’pkr~ Hkh tutkfr;ksa dk ,d cM+k fgLlk ¼60 izfr’kr ls Hkh vf/kd½ igkM+ksa vkSj taxyksa esa fuokl dj jgk gSA tutkrh; leqnk; ds yksxksa esa vius vf/kdkjksa dks ysdj tnnkstgn py jgh gSA Hkkjrh; tutkfr;ksa ds chp xjhch] vf’k{kk vkSj csjkstxkjh ,d xaHkhj leL;k dk :i ys pqdk gSA ;g budh =klnh gS fd vktknh ls igys vkSj vktknh ds ckn Hkh oU; miknkuksa vkSj [kfut L=ksrksa dk tks bLrseky gqvk gS mlls tutkfr;ksa dk nnZ c<+k gh gSaA ledkyhu ifjn`’; esa ;g loZfofnr gS fd tutkrh; lekt ds yksx gtkjksa o"kZ ls ,d i`Fkd thou O;rhr djrs vk jgs gSaA lekt dh eq[;/kkjk ls ges’kk ls os vyx&Fkyx jgs gSaA vaxzstksa us Hkh bUgsa fodkl dh eq[;/kkjk ls tksM+us ds fy, vius 'kkludky esa dksbZ fo’ks"k dksf’k’k ugha dh cfYd ml 'kksf"kr vkSj misf{kr lewg dks mlh gky esa NksM+ fn;k vkSj QyLo:i muds 'kks"k.k dk flyflyk iwoZor~ pyrk jgk gSA mRrjk[k.M esa fuokl djus okyh Fkk: tutkfr Hkh bUk lHkh leL;kvksa ls xzLr gSA s Z s S s s s b gA 'kks/kkFkhZ }kjk ;g tkuus dk iz;kl fd;k x;k gS fd Fkk: tutkfr ds O;fDr fdu&fdUk vf/kdkjksa dks izkIr dj jgsa gS] ftlls muds ekuokf/kdkj lqjf{kr jg ldsaA bl ifjizs{; esa Fkk: leqnk; ds yksx vius vf/kdkjksa ds izfr fdrus ltx gSa] bldk Hkh ewY;kadu djus dk iz;kl fd;k x;k gSA 'kks/kkFkhZ }kjk bl lanHkZ esa fuEu iz;kl fd, x, gS& rkfydk&1 % D;k vkius ekuokf/kdkjksa ds ckjs esa lquk gS ;k tkurs gS \ s 'kks/kkFkhZ }kjk ;g tkuus dk iz;kl fd;k x;k gS fd Fkk: tutkfr ds O;fDr fdu&fdUk vf/kdkjksa dks izkIr dj jgsa gS] ftlls muds ekuokf/kdkj lqjf{kr jg ldsaA bl ifjizs{; esa Fkk: leqnk; ds yksx vius vf/kdkjksa ds izfr fdrus ltx gSa] bldk Hkh ewY;kadu djus dk iz;kl fd;k x;k gSA 'kks/kkFkhZ }kjk bl lanHkZ esa fuEu iz;kl fd, x, gS& rkfydk&1 % D;k vkius ekuokf/kdkjksa ds ckjs esa lquk gS ;k tkurs gS \ Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in [ 51 ] q dFku n`f"Vdks.k ekuokf/kdkjksa ds ckjsa esa tkudkjh gka ugha irk ugha ;ksx 16 ¼32%½ 32 ¼64%) 02 ¼4%½ 50 ¼100%½ [ 51 ] Periodicity : Quarterly, Language : English & Hindi ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 3318 \| rkfydk&1 ls Li"V gS fd 64 izfr’kr mRrjnkrkvksa us ;g dgk gS fd mUgsa ekuokf/kdkjksa dh tkudkjh ugha gS] tcfd 32 izfr’kr mRrjnkrkvksa us dgk gS fd mUgsa ekuokf/kdkjksa dh tkudkjh gSA nwljh rjQ 4 izfr’kr mRrjnkrkvksa us bl lUnHkZ esa irk ugha n`f"Vdks.k izdV fd;k gSA bl izdkj gesa ;g Kkr gksrk gS fd brus vf/kd mRrjnkrkvksa dks ekuokf/kdkjksa ds ckjs esa tkudkjh u gksuk ,d fpark fo"k; gSA rkfydk&2 D;k vki ekurs gS fd f’k{kk ekuokf/kdkjksa dks izHkkfor djrh gS\ rkfydk&2 D;k vki ekurs gS fd f’k{kk ekuokf/kdkjksa dks izHkkfor djrh gS\ dFku n`f"Vdks.k f’k{kk ekuokf/kdkjksa dks izHkkfor djrh gS \ gka Ukgha irk ugha ;ksx 38 (76%) 08 (16%) 04 (8%) 50 (100%) rkfydk&2 n’kkZrk gS fd 76 izfr’kr mRrjnkrkvksa dks ekuuk gS fd f’k{kk ekuokf/kdkjksa dks izHkkfor djrh gS] tcfd 16 izfr’kr mRrjnkrkvksa us bldk mRrj ugha fn;k gSA nwljh rjQ 8 izfr’kr mRrjnkrkvksa bl laUnHkZ esa irk ugha n`f"Vdks.k izLrqr djrs gSA bl izdkj ;g ekuk tk ldrk gS fd mRrjnkrkvksa esa f’k{kk ds izfr laosnu’khyrk gSA rkfydk 3 % D;k vki ewyHkwr lqfo/kkvksa ¼Hkkstu] oL=] vkokl] f’k{kk] fpfdRlk vkfn½ dk m 3 % D;k vki ewyHkwr lqfo/kkvksa ¼Hkkstu] oL=] vkokl] f’k{kk] fpfdRlk vkfn½ dk miHkksx dj ik jgs gSa \ dFku n`f"Vdks.k ewyHkwr lqfo/kkvksa dk miHkksx dj ikuk gka ugha irk ugha ;ksx 15 (30%) 34 (68%) 01 (2%) 50 (100%) rkfydk&3 ls Li"V gS fd 68 izfr’kr mRrjnkrkvksa dk ekuuk gS fd ge ewyHkwr lqfo/kkvksa dk miHkksx ugha dj ik jgs gS tcfd 30 izfr’kr mRrjnkrkvksa us ;g Lohdkj fd;k fd ge ewyHkwr lqfo/kkvksa dk miHkksx dj jgs gSaA nwljh rjQ 2 izfr’kr mRrjnkrkvksa us ewyHkwr lqfo/kkvksa ds miHkksx ds ckjs esa irk ugha n`f"Vdks.k vafdr fd;k gSA ;s rF; tutkfr;ksa esa ekuo vf/kdkj ds lkFk&lkFk ekuo fodkl dh U;wu fLFkfr dks bafxr djrs gSaA rkfydk 4 % D;k vkids leqnk; ds vf/kdka’k yksx Hkwfeghurk ds f’kdkj gSa \ rkfydk 4 % D;k vkids leqnk; ds vf/kdka’k yksx Hkwfeghurk ds f’kdkj gSa \ dFku n`f"Vdks.k Hkwfeghurk ds f’kdkj gka ugha irk ugha ;ksx 46 (92%) 02 (4%) 02 (4%) 50 (100%) rkfydk& 4 ls ;g irk pyrk gS fd Fkk: leqnk; esa 92 izfr’kr mRrjnkrkvksa us ;g Lohdkj fd;k gS fd gekjs lekt esa vf/kdka’k O;fDr Hkwfeghurk ds f’kdkj gq, gS tcfd bl laUnHkZ esa 4 izfr’kr mRrjnkrkvksa us ugha vkSj brus gh vFkkZr~ 4 izfr’kr mRrjnkrkvksa us irk ugha n`f"Vdks.k iznf’kZr fd;k gSA vr% ;g dgk tk ldrk gS fd Fkk: leqnk; ds yksxksa ds ewy vf/kdkjksa dk guu gks jgk gS tks fdlh Hkh yksdrkaf=d 'kklu iz.kkyh ds fy, mfpr ugha gSA rkfydk&5 % D;k vkids ifjokj@lekt esa ySafxd HksnHkko fd;k tkrk gS \ rkfydk&5 % D;k vkids ifjokj@lekt esa ySafxd HksnHkko fd;k tkrk gS \ Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in [ 52 ] rkfydk&5 % D;k vkids ifjokj@lekt esa ySafxd HksnHkko fd;k tkrk gS \ dFku n`f"Vdks.k ySafxd HksnHkko dh fLFkfr gka ugha irk ugha ;ksx 34 (68%) 15 (30%) 01 (2%) 50 (100%) rkfydk&5 % D;k vkids ifjokj@lekt esa ySafxd HksnHkko fd;k tkrk gS \ dFku n`f"Vdks.k ySafxd HksnHkko dh fLFkfr gka ugha irk ugha ;ksx 34 (68%) 15 (30%) 01 (2%) 50 (100%) Periodicity : Quarterly, Language : English & Hindi [ 52 ] Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 3318 \| rkfydk& 5 ls izrhr gksrk gS fd 68 izfr’kr mRrjnkrkvksa us ;g ekuk gS fd muds ifjokj@lekt esa ySafxd HksnHkko gksrk gS tcfd 30 izfr’kr mRrjnkrkvksa us ;g dgk gS fd gekjs lekt@ifjokj esa ySafxd HksnHkko ugha fd;k tkrk gSaA nwljh rjQ bl lanHkZ esa 2 izfr’kr mRrjnkrkvksa us irk ugha n`f"Vdks.k dks izdV fd;k gSA bl izdkj ;g dgk tk ldrk gS Fkk: lekt esa ySafxd HksnHkko dh fLFkfr fpark dk fo"k; gSaA 6 % D;k vkids ifjokj@lekt esa gksus okys lkekftd] vkfFkZd vkSj jktuhfrd dk;ksZ esa efgykvksa dh jk; gS \ rkfydk&6 % D;k vkids ifjokj@lekt esa gksus okys lkekftd] vkfFkZd vkSj jktuhfrd dk;ksZ esa efgykvksa dh jk; yh tkrh gS \ dFku n`f"Vdks.k Lkekftd] vkfFkZd] jktuhfrd fØ;kdykiksa esa efgykvksa dh jk; gka ugha irk ugha ;ksx 14 (28%) 33 (66%) 03 (6%) 50 (100%) rkfydk& 6 ls Kkr gksrk gS fd 66 izfr’kr mRrjnkrkvksa dk ekuuk gS fd lkekftd] vkfFkZd] jktuhfrd fØ;k dykiksa esa efgykvksa dh jk; ugha yh tkrh tcfd bl lanHkZ esa 28 izfr’kr mRrjnkrkvksa us ugha vkSj 6 izfr’kr mRrjnkrkvksa us irk ugha n`f"Vdks.k izLrqr fd;k gSA bl izdkj ;g Li"V gksrk gS fd Fkk: tutkfr esa efgykvksa dh fLFkfr fuEu gSA rkfydk& 7 % D;k fodkl ls lEcfU/kr ljdkjh ;kstukvksa dh lwpuk vketu rd igqap ikrh gS\ rkfydk& 7 % D;k fodkl ls lEcfU/kr ljdkjh ;kstukvksa dh lwpuk vketu rd igqap ikrh gS\ dFku n`f’Vdks.k ljdkjh ;kstukvksa dh lwpuk dk vketu rd igqap ikuk gka ugha irk ugha ;ksx 06 (12%) 40 (80%) 04 (8%) 50 (100%) rkfydk& 7 ls Li"V gS fd 80 izfr’kr mRrjnkrkvksa dk ekuuk gS fd ljdkjh ;kstukvksa dh lwpuk vketu rd ugha igaqp ikrh gS tcfd 12 izfr’kr mRrjnkrkvksa dk ekuuk gS fd ljdkjh ;kstukvksa dh lwpuk vketu rd igqap ikrh gSA nwljh rjQ 8 izfr’kr mRrjnkrkvksa us bl lUnHkZ esa irk ugha n`f"Vdks.k fn;k gSA bl izdkj ;g izrhr gksrk gS fd Fkk: lekt esa Lo;a dh vKkurk gksus ls ljdkjh ;kstukvksa dh lwpuk mu rd ugha igqap ikrh gSaaA rkfydk&8 % D;k ljdkjh ;kstukvksa@uhfr;ksa@dk;ZØeksa ls vkidh vkfFkZd fLFkfr lqn`<+ gqbZ gS\ dFku n`f"Vdks.k ljdkjh ;kstukvksa ls vkfFkZd fLFkfr dk lqn`<+ gksuk gka ugha irk ugha ;ksx 26 (52%) 16 (32%) 08 (16%) 50 (100%) rkfydk&8 ls irk pyrk gS fd 52 izfr’kr mRrjnkrkvksa us ;g Lohdkj fd;k gS fd ljdkjh ;kstukvksa ls mudh vkfFkZd fLFkfr lqn`<+ gqbZ gS tcfd 32 izfr’kr mRrjnkrkvksa us bl laUnHkZ esa ugha n`f"Vdks.k fn;k gSA nwljh rjQ 16 izfr’kr mRrjnkrkvksa us ljdkjh ;kstukvksa ls mudh vkfFkZd faLFkfr lqn`<+ gksus ds ckjs esa irk ugha n`f"Vdks.k vafdr fd;k gSA bl izdkj ;g dgk tk ldrk gS fd ljdkjh ;kstukvksa@uhfr;ksa dk;ZØeksa ds fØ;kUo;u ls mudh vkfFkZd fLFkfr esa /khjs&/khjs lq/kkj gks jgk gSA rkfydk& 9 % D;k vki pquko ds le; ernku esa lfØ; Hkwfedk fuHkkrs gS \ Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in [ 53 ] rkfydk& 9 % D;k vki pquko ds le; ernku esa lfØ; Hkwfedk fuHkkrs gS \ dFku n`f"Vdks.k ernku esa lfØ; Hkwfedk fuHkkuk gka ugha irk ugha ;ksx 39 (68%) 08 (16%) 03 (6%) 50 (100%) [ 53 ] Periodicity : Quarterly, Language : English & Hindi ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag rkfydk & 13 ls ;g Li"V gksrk fd lokZf/kd 56 izfr’kr mRrjnkrkvksa us vf'k{kk dks ekuokf/kdkj ds mYya?ku dks lcls cM+k ftEesnkj ekuk gSA blds Ik”pkr~ 24 izfr’kr mRrjnkrk ljdkjh ra= vkSj 20 izfr’kr mRrjnkrk iqfyl dks ekuokf/kdkj ds mYya?ku dk ftEesankj ekurs gSaA vr% ;g dgk tk ldrk gS fd vf’k{kk ekuokf/kdkj ds le{k lcls cM+h pqUkkSrh gSA rkfydk& 14 % Fkk: tutkrh; lekt vU; tutkfr;ksa ls fiNM+h gqbZ D;ksa gS \ dFku dkj.k dqy çfr”kr Fkk: tutkrh; lekt dk fiNM+kiu vf’k{kk 28 56% Cksjkstxkjh 08 16% xjhch 07 14% _.kxzLrrk 07 14% dqy;ksx 50 100% rkfydk& 14 % Fkk: tutkrh; lekt vU; tutkfr;ksa ls fiNM+h gqbZ D;ksa gS \ rkfydk& 14 ls ;g Li"V gS fd 56 izfr’kr mRrnkrkvksa us ;g Lohdkj fd;k gS fd vf’k{kk ds dkj.k gh ge vU; tutkfr;ksa ls fiNM+ jgs gSA blds Ik’pkr~ 16 izfr’kr mRrjnkrkvksa us csjkstxkjh] 14 izfr’kr mRrjnkrkvksa us xjhch vkSj brus gh vFkkZr~ 14 izfr’kr mRrjnkvksa us _.kxzLrrk dks vU; tutkfr;ksa ls fiNM+siu dk dkj.k crk;k gSA bl izdkj ;g dgk tk ldrk gS fd vf’k{kk gh og egRoiw.kZ dkj.k gS ftlls O;fDr vius vf/kdkjksa ds izfr tkx:d curk gSA rkfydk& 14 ls ;g Li"V gS fd 56 izfr’kr mRrnkrkvksa us ;g Lohdkj fd;k gS fd vf’k{kk ds dkj.k gh ge vU; tutkfr;ksa ls fiNM+ jgs gSA blds Ik’pkr~ 16 izfr’kr mRrjnkrkvksa us csjkstxkjh] 14 izfr’kr mRrjnkrkvksa us xjhch vkSj brus gh vFkkZr~ 14 izfr’kr mRrjnkvksa us _.kxzLrrk dks vU; tutkfr;ksa ls fiNM+siu dk dkj.k crk;k gSA bl izdkj ;g dgk tk ldrk gS fd vf’k{kk gh og egRoiw.kZ dkj.k gS ftlls O;fDr vius vf/kdkjksa ds izfr tkx:d curk gSA Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 33 rkfydk& 9 ls ;g Kkr gksrk gS fd 68 izfr’kr mRrjnkrkvksa us ernku esa lfØ; Hkwfedk dks ekuk gS tcfd 16 izfr’kr mRrjnkrkvksa us ernku esa lfØ; Hkwfedk dks ugha ekuk gSA nwljh rjQ 6 izfr’kr mRrjnkrkvksa us bl lanHkZ esa irk ugha n`f"Vdks.k izdV fd;k gSA bl izdkj ;g dgk tk ldrk gS fd Fkk: tutkfr pquko ds le; ernku esa lfØ; Hkwfedk fuHkkrh gSA rkfydk& 10 % D;k vki xzke lHkk@iapk;r lfefr dh cSBdksa esa fu;fer Hkkx ysrs gS\ kfydk& 10 % D;k vki xzke lHkk@iapk;r lfefr dh cSBdksa esa fu;fer Hkkx ysrs gS\ rkfydk& 10 % D;k vki xzke lHkk@iapk;r lfefr dh cSBdksa esa fu;fer Hkkx ysrs gS\ dFku n`f’Vdks.k xzke lHkk@iapk;r lfefr dh cSBd esa fu;fer Hkkx gka ugha irk ugha ;ksx 26 (52%) 22 (44%) 02 (4%) 50 (100%) rkfydk& 10 ls ;g izdV gksrk gS fd 52 izfr’kr mRrjnkrkvksa us xzke lHkk@iapk;r lfefr dh cSBdksa esa fu;fer Hkkx ysrs gS tcfd 44 izfr’kr mRrjnkrkvksa us fu;fer Hkkx ysus ds ckjs esa ugha n`f"Vdks.k fn;k gSA nwljh rjQ 4 izfr’kr mRrjnkrkvksa us bl lanHkZ esa irk ugha n`f"Vdks.k vafdr fd;k gSA rkfydk& 11 % vkids {ks= esa fdlh O;fDr@lewg ds ekuokf/kdkjksa dk guu gksus ij D;k mlds f[kykQ vkanksyu fd;k gS \ rkfydk& 11 % vkids {ks= esa fdlh O;fDr@lewg ds ekuokf/kdkjksa dk guu gksus ij D;k mlds f[kykQ vkanksyu fd;k gS \ dFku n`f"Vdks.k ekuokf/kdkjksa ds guu gksus ij mlds f[kykQ vkanksyu djuk gka ugha irk ugha ;ksx 20 (40%) 27 (54%) 03 (6%) 50 (100%) rkfydk& 11 ls Kkr gksrk gS fd 40 izfr’kr mRrjnkrkvksa us Lohdkj fd;k gS fd mUgksaus vius ekuokf/kdkjksa ds guu ij mlds f[kykQ vkanksyu fd;k gS tcfd 54 izfr’kr mRrjnkrkvksa us ekuokf/kdkj ds guu gksus ij mlds f[kykQ vkanksyu djus dks vLohdkj fd;k gSA nwljh rjQ 6 izfr’kr mRrjnkrkvksa us bl lanHkZ esa irk ugha n`f"Vdks.k izLrqr fd;k gSA vr% Li’V gS fd vf/kdka’k mRrjnkrk vius vf/kdkjksa ds guu ij mlds f[kykQ vkanksyu ugha djrs gSaA rkfydk& 11 ls Kkr gksrk gS fd 40 izfr’kr mRrjnkrkvksa us Lohdkj fd;k gS fd mUgksaus vius ekuokf/kdkjksa ds guu ij mlds f[kykQ vkanksyu fd;k gS tcfd 54 izfr’kr mRrjnkrkvksa us ekuokf/kdkj ds guu gksus ij mlds f[kykQ vkanksyu djus dks vLohdkj fd;k gSA nwljh rjQ 6 izfr’kr mRrjnkrkvksa us bl lanHkZ esa irk ugha n`f"Vdks.k izLrqr fd;k gSA vr% Li’V gS fd vf/kdka’k mRrjnkrk vius vf/kdkjksa ds guu ij mlds f[kykQ vkanksyu ugha djrs gSaA rkfydk&12 % D;k vkidks lkekftd lqj{kk vf/kfu;e ¼TkSls& ngst fujks/kd vf/kfu;e@izlwfr izlqfo/kk vf/kfu;e@ ?kjsyw fgalk fu"ks/k vf/kfu;e vkfn½ dh tkudkjh gS \ rkfydk&12 % D;k vkidks lkekftd lqj{kk vf/kfu;e ¼TkSls& ngst fujks/kd vf/kfu;e@izlwfr izlqfo/kk vf/kfu;e@ ?kjsyw fgalk fu"ks/k vf/kfu;e vkfn½ dh tkudkjh gS \ dFku n`f"Vdks.k lkekftd lqj{kk gka ugha irk ugha ;ksx rkfydk&12 % D;k vkidks lkekftd lqj{kk vf/kfu;e ¼TkSls& ngst fujks/kd vf/kfu;e@izlwfr izlqfo/kk vf/kfu;e@ ?kjsyw fgalk fu"ks/k vf/kfu;e vkfn½ dh tkudkjh gS \ dFku n`f"Vdks.k lkekftd lqj{kk vf/kfu;e dh tkudkjh gksuk gka ugha irk ugha ;ksx 23 (46%) 26 (52%) 01 (2%) 50 (100%) dFku n`f"Vdks.k lkekftd lqj{kk vf/kfu;e dh tkudkjh gksuk gka ugha irk ugha ;ksx 23 (46%) 26 (52%) 01 (2%) 50 (100%) rkfydk& 12 ls Li"V gS fd 46 izfr’kr mRrjnkrk lkekftd lqj{kk vf/kfu;e dh tkudkjh j[krs gS tcfd 52 izfr’kr mRrjnkrkvksa us lkekftd lqj{kk vf/kfu;e dh tkudkjh ds ckjs esa ugha n`f"Vdks.k fn;k gSA nwljh rjQ 2 izfr’kr mRrjnkrkvksa us bl lanHkZ esa irk ugha n`f"Vdks.k izdV fd;k gSA vr% vf/kdka’k mRrjnkrkvksa esa visf{kr tkx:drk dk vHkko gSA rkfydk& 13 % ekuokf/kdkjksa ds mYya?ku dk mRrjnkf;Ro fdl ij gS \ dFku ço`fr;ka dqy çfr’kr ekuokf/kdkj ds mYya?ku dk mRrjnkf;Ro vf’k{kk 28 56% iqfyl 10 20% ljdkjh ra= tSls& U;k;ky; jk’Vªh;@jkT; ekuovf/kdkj vk;ksx vkfn 12 24% dqy ;ksx 50 100% rkfydk& 13 % ekuokf/kdkjksa ds mYya?ku dk mRrjnkf;Ro fdl ij gS \ Periodicity : Quarterly, Language : English & Hindi [ 54 ] Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in ewY;kadu gekjs lafo/kku fuekZrkvksa us Hkkjrh; lafo/kku esa lkekftd&vkfFkZd&jktuhfrd U;k; dh ladYiuk dh Fkh vkSj blh U;k; dh izkfIr ds fy, lafo/kku esa egRoiw.kZ izko/kku fd, x,A lafo/kku fuekZrkvksa us Lora=rk izkfIr ds Ik’pkr~ lafo/kku esa vkfFkZd vkSj jktuSfrd lerk ds lkFk lkekftd lekurk dk y{; j[kkA vr% lkekftd n`f"V ls fiNMs+ gq,] vNwr yksxksa vkSj vyx&Fkyx iM+h tutkfr;ksa dh lkekftd lgHkkxhnkjh c<+kus ds fy, vusd laoS/kkfud mica/k fd, x, gSaA tSls&vuPqNsn 14] 15] 16] 16¼4½] 19¼5½] 23] 29] 46] 164] 243¼3½ ]242] 244¼1½] 244¼2½] 275¼1½] 330] 332] 336] 339] 342] 342¼1½] 342¼2½ vkSj 366¼25½ eq[; gSaA ekuo vf/kdkj ds mYya?ku dks jksdus ds fy, jk’Vªh; ekuo vf/kdkj vk;ksx ¼1993½ vkSj vuqlwfpr tutkfr vk;ksx ¼2003½ ,oa jk"Vªh; efgyk vk;ksx ¼1992½ dk xBu fd;k x;kA blds vfrfjDr tutkrh; dk;Z ea=ky; ¼1999½ dh LFkkiuk dh xbZ ftldk mn~ns”; ,d lefUor vkSj lqfu;ksftr rjhds ls Hkkjrh; lekt ds vR;Ur oafpr oxZ vFkkZr~ vuqlwfpr tutkfr;ksa ds lesfdr lkekftd&vkfFkZd fodkl ij /;ku dsfUnzr djuk gS] ftlls os viuk xfjeke;h thou O;rhr dj ldsaA vuqlwfpr tutkfr;ksa dh leL;kvksa dks ysdj Hkkjr ljdkj Lora=rk ds Ik’pkr~ ls gh le;≤ ij leh{kk djrh jgh gSA buds fodkl ds fy, vusd lfefr;ka xfBr dh xbZ ftuesa dkysydj lfefr ¼1955½] js.kqdk jk; lfefr ¼1959½] osfj;j ,fYou lfefr ¼1960½] oscj lfefr ¼1961½] “khyw vkvks lfefr ¼1966½] yksdqj lfefr ¼1965½] BDdj lfefr ¼1971½] vIiw lfefr ¼1971½] nwcs lfefr ¼1972½] Hkwfj;k lfefr ¼2014½ eq[; gSaA blds lkFk&LkkFk iapo’khZ; ;kstukvksa ds ek/;e ls Hkh vusd dY;k.kdkjh ;kstuk,a fØ;kfUor dh xbZA blds vykok Hkh tutkrh; {ks=ksa esa iz’kklfud lq/kkj gsrq dbZ jkT;Lrjh; lfefr;ka ,oa milfefr;ka dk xBu fd;k ftlls tutkrh; leqnk; ds thou esa xq.kkRed ifjoZru vk ldsA bruk lc gksus ds ckotwn Hkh tutkfr;ksa dk visf{kr fodkl ugha gks ldkA ;g lky vktknh dk 75 oka lky gS vkSj Hkkjr vktknh dk ve`r egksRlo euk jgk gS fdarq vkt Hkh ns’k ds taxy izns’k ds fuoklh viuh nqnZ’kk vkSj vHkko ds fujkdkj.k ds mik; dkxth fodkl ;kstukvksa esa ryklus esa O;Lr gSaA tutkrh; lekt dh vf/kdka’k vkcknh xjhch] ruko] 'kks"k.k] fgalk] vyxko] vkfFkZd ladV] ySafxd fo"kHkrk] O;fDrxr Lora=rk dk guu] lkekftd U;k; ij dqBkjk?kkr] tkrh; ,oa iztkrh; HksnHkko] vf/kdkjksa dh oapuk] uDlyokn vkfn leL;kvksa ls tw> jgk gSA bl izdkj ;g Lohdkj fd;k tk ldrk gS fd ekuo vf/kdkj dh vo/kkj.kk bl fn’kk esa valfnX/k izrhr gksrh gSA vktknh ds 75 lky ckn dh =klnh ;g gS fd tutkrh; dY;k.k ls lEcfU/kr vusd laoS/kkfud izko/kku] lfefr] uhfr;ka] ;kstuk,a bl tutkrh; {ks= ds fy, ,d feFkd lkfcr gqbZ gS D;ksafd fodkl ds uke ij muds izkd`frd lalk/ku ns[krs&ns[krs gkFk ls fQlyrs pys x,A tutkrh; lekt dks iznRr ekuokf/kdkjksa dk Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in [ 55 ] https://recentjournals.in & https://ijorr.in fØ;kUo;u ukSdj’kkgh ds gkFkksa esa jgk gS ysfdu ukSdj’kkgh esa visf{kr laosnu’khyrk ,oa ikjnf’kZrk ds vHkko ds dkj.k mudk thou gkf’k, tSlk gks x;k gSA Fkk: tutkrh; lekt dh ekuo vf/kdkj lEcU/kh leL;k,a vU; tutkfr;ksa ls fHkUu ugha gSA vr% vkt lcls cM+h vko”;drk tutkfr;ksa dh fLFkfr lq/kkjus dh gS] ftlls muds vf/kdkj lqjf{kr jg ldsA bl lanHkZ esa fuEu lq>ko fn, tk ldrs gSa& 1- ty] taxy vkSj tehu ij tutkfr;ksa ds vf/kdkjksa dks v{kq..k j[krs gq, muds vf/kdkjksa dh j{kk dh tk,A blds fy, ;g vko’;d gS fd tutkrh; {ks=ksa dk iz’kklu dk dk;Z mUgha yksxksa ds gkFkksa esa lksaik tkuk pkfg,A 2- vuqlwfpr tutkfr;ksa ds fgrksa ds cpko ds fy, i`Fkd :Ik ls jk"Vªh; iquokZl uhfr cukbZ tkuh pkfg, D;ksafd Hkwfe&gLrkarj.k ek= LokfeRo ds gLrkUrj.k dk ekeyk ugha gS cfYd bls vkfFkZd vf/kdkjksa dh gkfu ds :i esa ns[kk tkuk pkfg,A 3- vuqlwfpr tutkfr vkSj vU; ijEijkxr ¼ou vf/kdkj dh ekU;rk½ fu;ekoyh ¼2012½ dks xzkl:V Lrj ij ykxw fd, tk,a ftlls tutkfr;ksa ds vf/kdkjksa dh j{kk gks ldsaA 4- fodkl ;kstukvksa dh mi;ksfxrk dks /;ku esa j[krs gq, {ks=okj losZ{k.k gksus pkfg, vkSj ;kstuk,a {ks= dh ekax ds vuqlkj fØ;kfUor dh tkuh pkfg,A ;kstukvksa dk le;≤ ij vkadyu gksuk vko”;d gS ftlls ;g Kkr gks ldsa fd ykHkkfUorksa dks mldk iw.kZ ykHk izkIr gks jgk gS ;k ughaA 4- fodkl ;kstukvksa dh mi;ksfxrk dks /;ku esa j[krs gq, {ks=okj losZ{k.k gksus pkfg, vkSj ;kstuk,a {ks= dh ekax ds vuqlkj fØ;kfUor dh tkuh pkfg,A ;kstukvksa dk le;≤ ij vkadyu gksuk vko”;d gS ftlls ;g Kkr gks ldsa fd ykHkkfUorksa dks mldk iw.kZ ykHk izkIr gks jgk gS ;k ughaA 5- tutkrh; dY;k.k ls lEcfU/kr ;kstuk,a ftls dsUnz ljdkj ;k jkT; ljdkj fufHkZr djrh gS mldh tkudkjh ;Fkk’kh?kz nh tkuh pkfg, ftlls vketu mudk vf/kd ls vf/kd ykHk mBk ldsaA 6- tutkfr;ksa ds 'kSf{kd ikB~;Øe esa vkSipkfjd f’k{kk dk vHkko gS ftl dkj.k tutkrh; {ks=ksa esa f’k{kk dk Lrj fuEu gSA vr% ,d ,sls ikB~;Øe dh lajpuk gksuh pkfg, tks tutkrh; ijEijkvksa] LFkkuh; vko”;drkvksa vkSj jk"Vªh; f’k{kk ;kstuk ls larqyu LFkkfir gks ldsA 7- tutkrh; lekt esa f’k{kk dk Lrj U;wu gS ftldk eq[; dkj.k xjhch vkSj _.kxzLrrk gSA tutkfr;ksa dks ljdkj ds ek/;e ls fn, x, _.kksa dk yphyk vkSj nh?kZdkyhu cuk;k tkuk pkfg, ftlls mudh vkfFkZd fLFkfr esa lq/kkj gks lds vkSj os _.k okil djus esa l{ke cu ldsaA 8- f’k{kk vkSj lk{kjrk ds varxZr ikB~;Øeksa dks bl izdkj cuk;k tkuk pkfg, ftlls mudh jksth&jksVh dk lek/kku gks ldsA 9- f’kf{kr vkSj vf’kf{kr csjkstxkj ;qokvksa dks jkstxkj ds volj iznku djus ds fy, LFkkuh; lalk/kuksa ij vk/kkfjr y?kq vkSj e/;e ntsZ ds m\|ksxksa dks fodflr fd, tkus pkfg,A 10- lekpkj i=] if=dk,a] Vh-oh- vkfn ds ek/;e ls tkx:drk vfHk;ku dks fujarj pyk;k tkuk pkfg, ftlls tutkrh; lekt tkx:d gks ldsa vkSj viuh vf/kdkjksa dks Hkyh&Hkkafr le> ldsaA 10- lekpkj i=] if=dk,a] Vh-oh- vkfn ds ek/;e ls tkx:drk vfHk;ku dks fujarj pyk;k tkuk pkfg, ftlls tutkrh; lekt tkx:d gks ldsa vkSj viuh vf/kdkjksa dks Hkyh&Hkkafr le> ldsaA dsUnz ljdkj@jkT; ljdkj ekuo vf/kdkjksa dh j{kk ds izfr dfVc) gSA blds fy, ljdkj us vusdksa ,sls lkFkZd ,oa l’kDr dne mBk, gSa ftlls fd ns’k esa ekuo vf/kdkjksa dk mfpr ,oa izHkkoh laj{k.k gks ldsA fQj Hkh] tutkfr;ksa ds 'kks"k.k ,oa muds ekuokf/kdkjksa ds guu dh ?kVuk,a vuk;kl vkt Hkh fn[krs ,oa lqukbZ nsrs gSaA tc rd buds ekuo vf/kdkjksa dks iw.kZ:is.k bZekunkjh ,oa dBksjrk ls ykxw ugha fd;k tk ldrk rc rd gekjs lH; lekt ij loky mBrs jgsaxsA vkt tutkfr;ka viuh Lo;a dh vf’k{kk] tkx:drk dk vHkko vkSj orZeku foLFkkiu ds dkj.k gkf’k, dk thou thus dks foo’k gSaA ljdkj ds pkjksa LrEHk&O;oLFkkfidk dk;Zikfydk] U;k;ikfydk vkSj ehfM;k ds LkkFk&LkkFk lekt ds izR;sd oxZ dks Hkh laosnu’khy cuuk gksxk rHkh tkdj tutkfr;ksa ds ekuo vf/kdkjksa dh lqj{kk gks ldsxhA Periodicity : Quarterly, Language : English & Hindi https://recentjournals.in & https://ijorr.in [ 56 ] Periodicity : Quarterly, Language : English & Hindi ekuo vf/kdkj ,oa tutkrh; lekt % ,d fo’ys"kukRed v/;;u larks"k dqekj flag International Refereed, Blind Peer-Reviewed Multidisciplinary & Open Access Research Journal Issue: 1 \| Vol.: 10 \| Jan.-Feb.-Mar.-2023 \| Pages: 49–57 \| RRSSH \| ISSN: 2348 – 3318 \| lanHkZ ln k 1- yokfu;k] ,e- ,e- vkSj tSu] “k’kh ds] ^^Hkkjr esa tutkfr;ksa dk lekt’kkL= fjlpZ ifCyds’kUl] t;iqj] 2012- 1- yokfu;k] ,e- ,e- vkSj tSu] “k’kh ds] ^^Hkkjr esa tutkfr;ksa dk lekt’kkL= fjlpZ ifCyds’kUl] t;iqj] 2012- 2- ehuk] 'khry] ^^tutkfr;ksa ds ekuokf/kdkj ,oa ekuo fodkl] iksbUVj ifCy’klZ] t;iqj] 2012- 3- nks’kh] ,l- ,y- vkSj tSu] ih- lh-] ^^tutkrh; lekt’kkL=] jkor ifCyds’kUl] t;iqj] 2020- 2- ehuk] 'khry] ^^tutkfr;ksa ds ekuokf/kdkj ,oa ekuo fodkl] iksbUVj ifCy’klZ] t;iqj] 2012- 3 nks’kh ,l ,y vkSj tSu ih lh ^^tutkrh; lekt’kkL= jkor ifCyds’kUl t;ij 2020 2- ehuk] 'khry] ^^tutkfr;ksa ds ekuokf/kdkj ,oa ekuo fodkl] iksbUVj ifCy’klZ] t;iqj] 2012- 3- nks’kh] ,l- ,y- vkSj tSu] ih- lh-] ^^tutkrh; lekt’kkL=] jkor ifCyds’kUl] t;iqj] 2020- 4 f=ikBh lR;sUn vkSj JhokLro vfuy dekj ^^lkekftd vula/kku ,oa lkaf[;dh jkor ifCyds’kUl t;ij 2- ehuk] 'khry] tutkfr;k d ekuokf/kdkj ,o ekuo fodkl] ikb Vj ifCy’kl] t;iqj] 2012- 3- nks’kh] ,l- ,y- vkSj tSu] ih- lh-] ^^tutkrh; lekt’kkL=] jkor ifCyds’kUl] t;iqj] 2020- 4- f=ikBh] lR;sUnz vkSj JhokLro] vfuy dqekj] ^^lkekftd vuqla/kku ,oa lkaf[;dh] jkor ifCyds’kUl] t;iqj] 2017- b q 3- nks’kh] ,l- ,y- vkSj tSu] ih- lh-] ^^tutkrh; lekt’kkL=] jkor ifCyds’kUl] t;iqj] 2020- 4- f=ikBh] lR;sUnz vkSj JhokLro] vfuy dqekj] ^^lkekftd vuqla/kku ,oa lkaf[;dh] jkor ifCyds’kUl] t;iqj] 2017- q 4- f=ikBh] lR;sUnz vkSj JhokLro] vfuy dqekj] ^^lkekftd vuqla/kku ,oa lkaf[;dh] jkor ifCyds’kUl] t;iqj] 2017- 5- nkl] Mh- ds-] ^^lkekftd ’kks/k] jkor ifCyds’kUl] t;iqj] 2017 5- nkl] Mh- ds-] ^^lkekftd ’kks/k] jkor ifCyds’kUl] t;iqj] 2017 6- ,uqvy fjiksVZ ¼2021&22½] fefuLVªh vkWQ Vªkbcy vQs;lZ] xouZesaV vkQ bafM;k] U;w nsgyh] 2022- 7- Hkkjr 2022 okf’kZd lanHkZ&xzaFk] izdk’ku foHkkx] lwpuk vkSj izlkj.k ea=ky;] Hkkjr ljdkj] ubZ fnYyh] 2022- 8- flag] vuqjk/kk ¼laik-½] vkfnoklh bfrgkl vkSj laLd`fr^^] ok.kh izdk’ku] ubZ fnYyh] 2023- af h f Z Z a af f h f a 5- nkl] Mh- d-] lkekftd ’kk k] jkor if yd’k l] t;iqj] 2017 6- ,uqvy fjiksVZ ¼2021&22½] fefuLVªh vkWQ Vªkbcy vQs;lZ] xouZesaV vkQ bafM;k] U;w nsgyh] 2022- 7- Hkkjr 2022 okf’kZd lanHkZ&xzaFk] izdk’ku foHkkx] lwpuk vkSj izlkj.k ea=ky;] Hkkjr ljdkj] ubZ fnYyh] 2022- q 6- ,uqvy fjiksVZ ¼2021&22½] fefuLVªh vkWQ Vªkbcy vQs;lZ] xouZesaV vkQ bafM;k] U;w nsgyh] 2022- Z Z S Z 6- ,uqvy fjiksVZ ¼2021&22½] fefuLVªh vkWQ Vªkbcy vQs;lZ] xouZesaV vkQ bafM;k] U;w nsgyh] 2022- 7- Hkkjr 2022 okf’kZd lanHkZ&xzaFk] izdk’ku foHkkx] lwpuk vkSj izlkj.k ea=ky;] Hkkjr ljdkj] ubZ fnYyh] 2022- 8- flag] vuqjk/kk ¼laik-½] vkfnoklh bfrgkl vkSj laLd`fr^^] ok.kh izdk’ku] ubZ fnYyh] 2023- q w 7- Hkkjr 2022 okf’kZd lanHkZ&xzaFk] izdk’ku foHkkx] lwpuk vkSj izlkj.k ea=ky;] Hkkjr ljdkj] ubZ fnY 8- flag] vuqjk/kk ¼laik-½] vkfnoklh bfrgkl vkSj laLd`fr^^] ok.kh izdk’ku] ubZ fnYyh] 2023- 7- Hkkjr 2022 okf’kd lnHk&xzFk] izdk’ku foHkkx] lwpuk vkj izlkj.k e=ky;] Hkkjr ljdkj] ub fnYyh] 2022- 8- flag] vuqjk/kk ¼laik-½] vkfnoklh bfrgkl vkSj laLd`fr^^] ok.kh izdk’ku] ubZ fnYyh] 2023- Z Z 8- flag] vuqjk/kk ¼laik-½] vkfnoklh bfrgkl vkSj laLd`fr^^] ok.kh izdk’ku] ubZ fnYyh] 2023- af h f Z Z a af f h f a q ¼ ½ b ` b 9- lkaf[;dh; if=dk 2021] dk;kZy; vFkZ ,oa lkaf[;kf/kdkjh&Å/ke flag uxj] 2022- Periodicity : Quarterly, Language : English & Hindi [ 57 ]
https://openalex.org/W2098394600	https://europepmc.org/articles/pmc3016857?pdf=render	English	null	Molecular Cloning, Expression Profile and 5′ Regulatory Region Analysis of Two Chemosensory Protein Genes from the Diamondback Moth,<i>Plutella xylostella</i>	Journal of insect science	2,010	cc-by	6,284	nal of Insect Science: Vol. 10 \| Article 143 Gong Molecular cloning, expression profile and 5 regulatory region analysis of two chemosensory protein genes from the diamondback moth, Plutella xylostella Liang Gonga, Guo-Hua Zhongb, Mei-Ying Huc, Qian Luod, Zhen-Zhen Rene Gong et al. Journal of Insect Science: Vol. 10 \| Article 143 Liang Gonga, Guo-Hua Zhongb, Mei-Ying Huc, Qian Luod, Zhen-Zhen Rene Key Laboratory of Pesticide and Chemical Biology, Ministry of Education of P.R. China. South China Agricultural University, Guang Zhou, 510642, Guangdong, China Abstract Chemosensory proteins play an important role in transporting chemical compounds to their receptors on dendrite membranes. In this study, two full-length cDNA codings for chemosensory proteins of Plutella xylostella (Lepidoptera: Plutellidae) were obtained by RACE-PCR. Pxyl- CSP3 and Pxyl-CSP4, with GenBank accession numbers ABM92663 and ABM92664, respectively, were cloned and sequenced. The gene sequences both consisted of three exons and two introns. RT-PCR analysis showed that Pxyl-CSP3 and Pxyl-CSP4 had different expression patterns in the examined developmental stages, but were expressed in all larval stages. Phylogenetic analysis indicated that lepidopteran insects consist of three branches, and Pxyl- CSP3 and Pxyl-CSP4 belong to different branches. The 5regulatory regions of Pxyl-CSP3 and Pxyl-CSP4 were isolated and analyzed, and the results consist of not only the core promoter sequences (TATA-box), but also several transcriptional elements (BR-C Z4, Hb, Dfd, CF2-II, etc.). This study provides clues to better understanding the various physiological functions of CSPs in P. xylostella and other insects. Keywords: cDNA cloning, genomic structure, transcription factor recognition site analysis Abbreviations: BR-C Z4, Broad-Complex Z4; CSP, Chemosensory protein; CF2-II, Zinc finger domain; Dfd, Deformed; Hb, Hunchback; ORF, open reading frames; Pxyl, Plutella xylostella; RACE, rapid amplification of cDNA ends; UTR, untranslated region. Correspondence: a 125314689@qq.com, b guohuazhong@scau.edu.cn, c* humy@scau.edu.cn, d 13929346@qq.com, e 542438779@qq.com, Corresponding author Associate Editor: Zhijian Jake Tu was editor of this paper. Received: 14 December 2008, Accepted: 9 June 2009 Copyright : This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 \| Vol. 10, Number 143 Cite this paper as: Gong L, Zhong GH, Hu MY, Luo Q, Ren ZZ. 2010. Molecular cloning, expression profile and 5 regulatory region analysis of two chemosensory protein genes from the diamondback moth, Plutella xylostella. Journal of Insect Science 10:143 available online: insectscience.org/10.143 Keywords: cDNA cloning, genomic structure, transcription factor recognition site analysis Abbreviations: BR-C Z4, Broad-Complex Z4; CSP, Chemosensory protein; CF2-II, Zinc finger domain; Dfd, Deformed; Hb, Hunchback; ORF, open reading frames; Pxyl, Plutella xylostella; RACE, rapid amplification of cDNA ends; UTR, untranslated region. Correspondence: a 125314689@qq.com, b guohuazhong@scau.edu.cn, c humy@scau.edu.cn, d 13929346@qq.com, e 542438779@qq.com, Corresponding author Associate Editor: Zhijian Jake Tu was editor of this paper. Received: 14 December 2008, Accepted: 9 June 2009 Copyright : This is an open access paper. al of Insect Science: Vol. 10 \| Article 143 Gong et a Molecular cloning, expression profile and 5 regulatory region analysis of two chemosensory protein genes from the diamondback moth, Plutella xylostella Liang Gonga, Guo-Hua Zhongb, Mei-Ying Huc, Qian Luod, Zhen-Zhen Rene al of Insect Science: Vol. 10 \| Article 143 Gong et a Molecular cloning, expression profile and 5 regulatory region analysis of two chemosensory protein genes from the diamondback moth, Plutella xylostella Liang Gonga, Guo-Hua Zhongb, Mei-Ying Huc*, Qian Luod, Zhen-Zhen Rene Insects P. xylostella pupae were collected from an insecticide-free cabbage field and taken to the laboratory for rearing. Larvae were allowed to feed on cabbage leaves in the insect growth room with conditions set at 25 ± 1° C, 16:8 L:D, and 70-85% RH until pupation. Introduction expression in different insect stages can reveal the possible extent of activity of these specific genes in the physiology of the different stages. In recent years, the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) has become the most destructive insect of cruciferous plants throughout the world, and the annual cost for its management is estimated to be US $1 billion (Talekar 1993). In order to find the solution, differentially expressed genes from this insect should be identified, cloned, and studied. In this regard, the study of chemosensory proteins of P. xylostella will be helpful in providing critical information about their behavioral characteristics and relative physiological processes. In the last two decades, insect chemosensory proteins have been studied extensively for their structural properties, various physiological functions, affinity to small molecular ligands, expression pattern in insects, and subcellular localization, but little research has been reported on the analysis of the 5-regulatory sequence of the chemosensory protein gene. In this study, the full-length cDNA was cloned for two chemosensory protein genes (Pxyl-CSP3 and Pxyl-CSP4) in P. xylostella, using rapid amplification of cDNA ends (RACE). It was followed by the genome walking method to obtain the 5-upstream regulatory sequence of Pxyl-CSP3 and Pxyl-CSP4. The results revealed not only the core promoter sequences (TATA-box), but also several transcriptional elements (BR-C Z4, Hb, Dfd, CF2-II etc). Insect chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) are believed to be involved in chemical communication and perception, and these two soluble proteins belong to different classes. OBPs have the size of approximately 150 amino acid residues, out of which six highly conserved cysteines are paired to form three disulfide bridges. It has been experimentally demonstrated that OBPs are involved in the binding of pheromones and odorant molecules (Vogt 1881; Kruse 2003; Andronopoulou 2006). CSPs are small proteins of about 110 amino acids that contain four cysteines forming two disulfide bridges (McKenna 1994; Pikielny 1994; Jansen 2007). In comparison to OBPs, which are specifically reported in olfactory sensilla (Vogt and Riddiford 1981; Steinbrecht 1998), the CSPs are expressed more extensively in various insect tissues such as the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, pheromone glands, wing disks, and compound eyes, suggesting that CSPs are crucial for multiple physiological functions of insects (Gong 2007). Similarly, the study of gene Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. Abstract We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 \| Vol. 10, Number 143 Cite this paper as: Gong L, Zhong GH, Hu MY, Luo Q, Ren ZZ. 2010. Molecular cloning, expression profile and 5 regulatory region analysis of two chemosensory protein genes from the diamondback moth, Plutella xylostella. Journal of Insect Science 10:143 available online: insectscience.org/10.143 Journal of insect Science \| www.insectscience.org 1 Journal of Insect Science: Vol. 10 \| Article 143 Journal of Insect Science: Vol. 10 \| Article 143 Cloning of Pxyl-CSP3 and Pxyl-CSP4 Cloning of Pxyl-CSP3 and Pxyl-CSP4 Two degenerate primers were designed by alignment of published CSP-like transcripts from distantly related species. The 3 RACE forward primers of Pxyl-CSP3 and Pxyl-CSP4 are 5-(C/T)AC(A/G)GA(T/C)AA(A/G)CA (C/G)GAA(A/G)C(C/A)(A/T)GCCGTGA-3 and 5-GAA(A/G)ACCA(C/T)C(C/T)GCGG CAA (G/C/A)TGCA -3, respectively, and oligod (T)18 was used as the reverse primer. The PCR reaction was performed with the following conditions: one cycle (94° C, 2 min); 35 cycles (94° C, 1 min; 55° C, 1 min; 72° C, 1 min); and a last cycle 72° C for 10 min. The PCR product was then cloned into a pMD-20-T vector (TaKaRa), and positive clones were sequenced. RNA preparation and synthesis of first- strand cDNA Total RNA was extracted from adults of P. xylostella using the Trizol reagent (Invitrogen, www.invitrogen.com) according the protocol provided by the manufacturer. First-strand cDNA was synthesized from the total RNA with reverse transcriptase AMV and oligod (T)18 (TaKaRa, www.takara-bio.com). 5- and Journal of Insect Science \| www.insectscience.org 2 2 Table1. Specific primers (SP1, SP2, and SP3) were designed for (first, second, and third round) genome walking, respectively, which is based on the genome sequence of Pxyl-CSP3 and Pxyl-CSP4. Primers of CSP3-sqPCR and CSP4-sqPCR were designed for RT-PCR. Name Primer sequence(5-3) Fragment size CSP3-SP1 GGTAAATAGACAAGGTACCTTCGGG 25bp CSP3-SP2 ATCCGCTCACTACTGGTTAAACCAG 25bp CSP3-SP3 ATTTGAGTTCCTTAGCCTCGGGCGA 25bp CSP4-SP1 TCGAGTATAATTCACCTGGCACCCA 25bp CSP4-SP2 CGTCGGAGAAACAACAGATAAGTTG 25bp CSP4-SP3 CGCCTCTTTAGAGCACCCAATCAAG 25bp CSP3-sqPCR(F) GAACGTGGACGAGATCCTGGCTAATG 26bp CSP3-sqPCR(R) TGGTCCTCGTACTGGGCGGTGTATTTC 27bp CSP4-sqPCR(F) GTGCAGGGCCCCTTGCTCAGGAAGCACT 28bp CSP4-sqPCR(R) CTGGTGCGAGTCGTGGTGAAGGGCT 25bp Journal of Insect Science: Vol. 10 \| Article 143 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. 5-RACE nest primer of Pxyl-CSP4 are 5- CTTGGCGAAGGAGTCCTTGTACTCTCC- 3 and 5-TCAGAAGATGTCATCTAAGT TC-3, respectively. The first PCR conditions were as follows: one cycle (94° C, 2 min); 5 cycles (94° C, 30 s; 72° C, 1 min ); 5 cycles (94° C, 30 s; 72° C, 1 min ); 25cycles (94° C, 30 s; 66° C, 30 s; 70° C, 1 min); and a last cycle of 72° C for 10 min. Full-length cDNA of Pxyl-CSP3 and Pxyl-CSP4 was obtained by overlapping the two cDNA fragments. 3-RACE-ready cDNA were prepared according to the instructions of the Gene RacerTM Kit protocol (Catalog #: L1500-01, Invitrogen). 3-RACE-ready cDNA were prepared according to the instructions of the Gene RacerTM Kit protocol (Catalog #: L1500-01, Invitrogen). Genomic DNA isolation and DNA sequence amplification The PCR amplification was performed using the following thermal cycle conditions: one cycle (94° C, 2min); 27 cycles (94° C, 30 s; 60° C, 45 s; 72° C,1 min) and a last cycle 72° C for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis. AACCTTGTTACGACTT-3. The cDNA (1-2 RT- PCR analysis RT-PCR was used to measure gene expression at different developmental stages. The cDNA samples from male and female adults, from all stages of larvae and from pre-pupae and pupae, were prepared using the plant RNA kit (Catalog #: R6827, Omega, www.omega.com) and reverse transcriptase AMV (TaKaRa). http://www.cbs.dtu.dk/services/SignalP-3.0/. p g The phylogenetic tree was constructed using MEGA 3.0 software (Kumar 2004) using the neighbour joining method, and it was reconstructed with 1000-replicate bootstrap analysis. The gene-specific primer was designed from the cDNA sequences of Pxyl-CSP3 and Pxyl- CSP4, named CSP3-sqPCR and CSP4-sqPCR, respectively (Table 1). The 18S rRNA gene of P. xylostella was used as the reference with the following primers: 18S-F: 5- CCGATTGAATGATTTAGTGAGGTCTT- 3; 18S-R: 5-TCCCCTACGGA Isolation of genomic 5- upstream region of Pxyl-CSP3 and Pxyl-CSP4 Genomic DNA of P. xylostella was prepared as above. In order to obtain the 5–upstream regulatory sequences of the chemosensory protein genes, the genome walking approach was performed according to the introductions of the kit (TaKaRa). The PCR principle of the genome walking approach is thermal asymmetric interlaced PCR (Tail-PCR). The specific reverse primers were designed according to 5-terminal nucleotide sequence of Pxyl-CSP3 and Pxyl-CSP4 (Table 1), and the forward primers were supported by the kit. The conditions for the were PCR reaction were set according to the kit’s introductions. The PCR fragments obtained through the genome walking approach were detected using 1.5% agarose gel electrophoresis and purified for sequencing using SP3 specific primer. Genomic DNA isolation and DNA sequence amplification Genomic DNA was extracted from P. xylostella according to the instructions from the TIANamp Genomic DNA kit protocol (Tiangen, www.tiangen.com). Genomic DNA was precipitated with ddH2O, and agarose gel electrophoresis was carried out to determine its quality. It was shown on a single band. The specific primers were designed to amplify the genomic DNA corresponding to the cDNA code region of Pxyl-CSP3 and Pxyl-csp4. In order to clone the genomic sequence of Pxyl- CSP3, the sense primer was 5-ATGAA CTCCTTGGTACTAGTATGCCTTG-3, and the antisense primer was 5-TACGCCT TGACAGCGCGCAGTTGGTCC-3. For Pxyl-CSP4, the sense primer was 5- ATGCAGACCGTGACTCTCCTATGCCTG T-3, and the antisense primer was 5- TTAATCAGATCCTTCGAGGAACTTGGC G-3. The PCR reaction was performed with According to the CSP-like transcript fragment amplified from P. xylostella by 3 RACE degenerate primers, the 5-RACE specific nest primers were designed and used to amplify the full-length cDNA of Pxyl-CSP3 and Pxyl- CSP4. The 5-RACE primer and 5-RACE nest primer of Pxyl-CSP3 are 5-CCTCC ACTCCGCGGGCTTGTGGTTGAT-3 and 5-TACGCCTTGACAGCGCGCAGTTGGT CC-3, respectively. The 5-RACE primer and Table1. Specific primers (SP1, SP2, and SP3) were designed for (first, second, and third round) genome walking, respectively, which is based on the genome sequence of Pxyl-CSP3 and Pxyl-CSP4. Primers of CSP3-sqPCR and CSP4-sqPCR were designed for RT-PCR. Name Primer sequence(5-3) Fragment size CSP3-SP1 GGTAAATAGACAAGGTACCTTCGGG 25bp CSP3-SP2 ATCCGCTCACTACTGGTTAAACCAG 25bp CSP3-SP3 ATTTGAGTTCCTTAGCCTCGGGCGA 25bp CSP4-SP1 TCGAGTATAATTCACCTGGCACCCA 25bp CSP4-SP2 CGTCGGAGAAACAACAGATAAGTTG 25bp CSP4-SP3 CGCCTCTTTAGAGCACCCAATCAAG 25bp CSP3-sqPCR(F) GAACGTGGACGAGATCCTGGCTAATG 26bp CSP3-sqPCR(R) TGGTCCTCGTACTGGGCGGTGTATTTC 27bp CSP4-sqPCR(F) GTGCAGGGCCCCTTGCTCAGGAAGCACT 28bp CSP4-sqPCR(R) CTGGTGCGAGTCGTGGTGAAGGGCT 25bp Journal of Insect Science \| www.insectscience.org 3 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. the following conditions: one cycle (94° C, 2 min); 35 cycles (94° C, 30 s; 68° C, 45 s; 72° C, 1 min) and a last cycle 72° C for 10 min. The amplified DNA was sequenced. AACCTTGTTACGACTT-3. The cDNA (1-2 μl) was used for amplification, and the final volume of the reaction mixture was 50 μl. The PCR amplification was performed using the following thermal cycle conditions: one cycle (94° C, 2min); 27 cycles (94° C, 30 s; 60° C, 45 s; 72° C,1 min) and a last cycle 72° C for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis. AACCTTGTTACGACTT-3. The cDNA (1-2 μl) was used for amplification, and the final volume of the reaction mixture was 50 μl. Bioinformatics analysis Amino acid sequences of CSPs (n = 27) were retrieved from an NCBI protein search using the keywords “chemosensory protein” and “lepidopteran”. Molecular mass and isoelectric point was predicted using the software, ExPASy (http://www.expasy.ch/). Multiple sequence alignment was carried out with the online service at http://bioinfo.genotoul.fr/multalin/multalin.ht ml (Corpet 1988). Promoter prediction and characterization were carried out using the Neural Network Promoter Prediction (NNPP) server (http://www.fruitfly.org/seq_tools/promoter.ht ml) (Reese 2001). Sequence analysis seeking transcriptional regulation response elements was carried out with TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH. html) (Heinemeyer 1998). The signal peptide was predicted using SignalP 3.0 (Nielsen 1997) at 5 upstream regulatory region analysis of Pxyl-CSP3 and Pxyl-CSP4 Using the genome working approach, the 5 regulatory regions of Pxyl-CSP3 and Pxyl- CSP4 were isolated and had 2242 bp and 533 bp, with the Genebank Numbers FJ948816 and FJ948817, respectively. Nucleotide sequence alignment of the isolated genomic sequence with the full-length Pxyl-CSP4 cDNA showed that the nucleotide sequence of 264 bp was isolated from the 5 UTR of Pxyl- CSP4, including a part of the intron sequence. Nucleotide sequence alignment of the isolated genomic clone with the full-length Pxyl-CSP3 cDNA revealed that the 5 UTR (Figure 1A) was interrupted by an intron of 323 bp, and thus was split in two exons of 61 and 75 bp, respectively. This intron also is in line with the GT-AG rule. The Pxyl-CSP3 5 upstream region of 1921 bp was analyzed to predict the transcription factor binding site, using the online server of TFSEARCH. The results of Figure 1A showed that the 5 upstream region of Pxyl-CSP3 included not only the core promoter sequences (TATA-box), but also several transcriptional elements (BR-C Z4, Hb, Dfd, CF2-II, etc.). An 864 bp cDNA of Pxyl-CSP4 (Figure 2) was obtained by RACE-PCR using the degenerate primers. The cDNA included a 54 bp 5 untranslated region (UTR), a 429 bp 3 UTR, with an AATAAA box and 23 bp poly (A) tail, and a 381 bp open reading frame (ORF) that encodes 126 amino acids. It exhibited significant similarity to CSP6 of Papilio xuthus (68%), CSP8 of Bombyx mori (52%) and CSP4 of Choristoneura fumiferana (46%), as revealed by Blast database research. The deduced protein has a computed molecular mass of 14.0 kDa and a predicted isoelectric point of 8.25. Results Gene cloning of Pxyl-CSP3 and Pxyl-CSP4 A 526 bp cDNA of Pxyl-CSP3 (Figure 1B) was obtained by RACE-PCR using the Journal of Insect Science \| www.insectscience.org 4 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. degenerate primers. The cDNA included a 62 bp 5 untranslated region (UTR), a 108 bp 3 UTR, with an AATAAA box and 25 bp poly (A) tail, and a 381 bp open reading frame (ORF) that encodes 126 amino acids. It exhibited significant similarity to CSP5 of Bombyx mori (59%), CSP3 of Bombyx mandarina (58%), and CSP3 of Mamestra brassicae (58%), as revealed by Blast database research. The deduced protein has a computed molecular mass of 14.1 kDa and a predicted isoelectric point of 8.79. Expression profile of Pxyl-CSP3 and Pxyl- CSP4 PCR amplification of genomic DNA with primers designed corresponding to the cDNA of Pxyl-CSP3 and Pxyl-CSP4 resulted in products of about 1452 bp and 1268 bp, respectively. By comparing their genomic sequence and cDNA sequence, it was found that Pxyl-CSP3 and Pxyl-CSP4 included one intron, and the intron began with 'GT', ended with 'AG', and had 926 bp and 404 bp, respectively. The sequences of the exon/intron-splicing junctions of Pxyl-CSP3 and Pxyl-CSP4 are shown in Figure 1B and Figure 2, respectively. RT-PCR was used to investigate the expression at different developmental stages. The results showed that Pxyl-CSP3 and Pxyl- CSP4 have different expression patterns in examined developmental stages. Pxyl-CSP3 (Figure 3A) was highly expressed in the first instar larva, second instar larva, third instar larva, fourth instar larva , fifth instar larva , pre-pupa , and pre-pupa , but no expression was obtained in pupa or pupa . Lower expression was observed in adult and adult . In the case of Pxyl-CSP4 (Figure 3B), higher expression was found in first Journal of Insect Science \| www.insectscience.org 5 5 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 6 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 7 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. Gong et al. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 8 Journal of Insect Science \| www.insectscience.org Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. and it was the common spacing pattern within the CSP family. Pxyl-CSP3 and Pxyl-CSP4 have only 38% similarity. Pxyl-CSP3 showed high similarity to CSP3 of Mamestra brassicae (56%), but Pxyl-CSP4 showed higher similarity to CSP of Papilio xuthus (69%), suggesting that CSPs from the species of P. xylostella are more similar to CSPs from other species than to that of some members of its own. instar larva, second instar larva, third instar larva, fourth instar larva , and fifth instar larva , while pre-pupa , pre-pupa , adult , and adult expressed lower expression, and no expression was found in pupa or pupa . Homology and phylogenetic analysis The evolutionary relationships among the two P. xylostella CSPs and 25 lepidopteran insect homologs that are reported so far were investigated. An unrooted neighbor-joining tree (Figure 4) was constructed to represent the relationship among selected CSPs. One CSP of Daphnia pulex was used for the out- group. The results obtained from the phylogenetic analysis showed that lepidopteran insects consist of three branches, and Pxyl-CSP3 and Pxyl-CSP4 belong to different branches as well. It provides clues about the diversification of these proteins in this insect order. Discussion Insect chemosensory proteins (CSPs) have been supposed to transport chemical stimuli from air to olfactory receptors. However, CSPs are expressed in various insect tissues including non-sensory tissues, suggesting that these proteins are also vital for other physiological processes. In this study, two full-length cDNA coding for chemosensory proteins of P. xylostella (Pxyl-CSP3 and Pxyl- CSP4) were obtained by RACE-PCR, and the GenBank accession numbers are ABM92663 and ABM92664, respectively. Amino acid sequence alignment from selected lepidopteran CSPs revealed that the conserved Cys spacing pattern was CX6CX18CX2C, Figure 3. RT-PCR analysis showed the expression of Pxyl-CSP3 (A) and Pxyl-CSP4 (B) at different developmental stages. The horizontal data represents from 1 to 11: first instar larva, second instar larva, third instar larva, fourth instar larva , fourth instar larva , pre-pupa , pre-pupa , pupa , pupa , adult , and adult . High quality figures are available online. Figure 3. RT-PCR analysis showed the expression of Pxyl-CSP3 (A) and Pxyl-CSP4 (B) at different developmental stages. The horizontal data represents from 1 to 11: first instar larva, second instar larva, third instar larva, fourth instar larva , fourth instar larva , pre-pupa , pre-pupa , pupa , pupa , adult , and adult . High quality figures are available online. Figure 3. RT-PCR analysis showed the expression of Pxyl-CSP3 (A) and Pxyl-CSP4 (B) at different developmental stages. The horizontal data represents from 1 to 11: first instar larva, second instar larva, third instar larva, fourth instar larva , fourth instar larva , pre-pupa , pre-pupa , pupa , pupa , adult , and adult . High quality figures are available online. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 9 Journal of Insect Science: Vol. 10 \| Article 143 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. C, respectively. This conserved splice site is considered to be a general characteristic of the CSP gene family, so it is evident that these clones belong to this family. The majority of CSP genes in insects have an intron; only three Anopheles gambiae and four Drosophila CSP genes lack introns; the intron splice site is always located on one nucleotide after a conserved lysine (Lys) codon, and its position is indicated by dark cycle (Figure 5). These results are accordant with the findings of Wanner (2004), as the intron splice sites of Pxyl-CSP3 and Pxyl-CSP4 are after the nucleotide acids AAA (Lys) T and AAA (Lys) Insect CSP genes are not only expressed in the olfactory tissues but also in non-olfactory tissues, including the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, and pheromone glands (Gong 2007; Lu 2007). Figure 4. A phylogenetic tree of CSPs from 2 Plutella xylostella CSPs and 25 lepidopteran insect homologs. Protein Ids are indicated. Abbreviations: Pcan, Pieris canidia; Prap, Pieris rapae; Hzea, Helicoverpa zea; Harm, Helicoverpa armigera; Pxut, Papilio xuthus; Mbra, Mamestra brassicae; Hvir, Heliothis virescens; Bmor, Bombyx mori; Cfum, Choristoneura fumiferana; Sexi, Spodoptera exigua; Slit, Spodoptera litura; Dpul, Daphnia pulex. High quality figures are available online. Figure 4. A phylogenetic tree of CSPs from 2 Plutella xylostella CSPs and 25 lepidopteran insect homologs. Protein Ids are indicated. Abbreviations: Pcan, Pieris canidia; Prap, Pieris rapae; Hzea, Helicoverpa zea; Harm, Helicoverpa armigera; Pxut, Papilio xuthus; Mbra, Mamestra brassicae; Hvir, Heliothis virescens; Bmor, Bombyx mori; Cfum, Choristoneura fumiferana; Sexi, Spodoptera exigua; Slit, Spodoptera litura; Dpul, Daphnia pulex. High quality figures are available online. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 10 of Insect Science: Vol. 10 \| Article 143 Gong et Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. Gong et al. Journal of Insect Science \| www.insectscience.org Journal of Insect Science: Vol. 10 \| Article 143 This study succeeded in isolating the 5 regulatory region of Pxyl-CSP3 and is the first report about the 5 upstream regulatory sequence of the insect chemosensory protein gene. This data revealed that the 5 regulatory region of Pxyl- CSP3 have a lot of specific transcription factor binding sites including BR-C Z4, Hb, Dfd, CF2-II, etc. The transcription factor binding site of BR-C Z4 has appeared many times in this regulatory region, which may play an important role for duplication and expression of Pxyl-CSP3. It has been reported that BR-C Z4 directly mediates the formation of the steroid hormone ecdysone for Drosophila melanogaster larvae metamorphosis (Kalm 1994). However, there is no direct evidence for the role of CSPs in insect metamorphosis, but some scientists reported that CSPs are expressed in the pheromonal gland of M. brassicae and the ejaculatory duct of D. melanogaster (Jacquin- Joly 2001; Sabatier 2003). A recent report also showed that the CSP homologue of Agrotis segetum has upregulation expression in the insect-pheromone binding domain; this CSP has also been reported to be the same as juvenile hormone binding protein (Strandh 2008). These findings are in line with the data from the transcription factor binding site analysis, as well as the high expression in the larval stage, which may implicate a function of Pxyl-CSP3 for steroid hormone production or transport in this insect larval stage. Chemosensory protein association with insect development has been confirmed by many scientists, especially in embryo development. For example, CSP5 of Apis mellifera is an ectodermal gene involved in embryonic integument formation (Maleszka 2007). In the CSPs are widely distributed in insect species and so far have been identified in 10 insect orders, including Lepidoptera (Maleszka 1997; Robertson 1999; Nagnan-L Meillour 2000; Picimbon 2000), Diptera (McKenna 1994; Pikielnyl 1994), Hymenoptera (Danty 1998; Briand 2002), Orthoptera (Angeli 1999), Phasmatodea (Tuccini 1996), Blattoidea (Kitabayashi 1998), Hemiptera (Jacobs 2005), Phthiraptera (Zhou 2006), Trichoptera (Zhou 2006), and Coleoptera (Zhou 2006). A CSP-like protein has been reported in a non-insect arthropod, the brine shrimp Artemia franciscana, suggesting that CSPs might be present across the arthropods (Pelosi 2006). But CSPs belong to a conserved protein family, and CSPs in different insect orders have shared common characteristics such as: conserved Cys residues spacing pattern; aromatic residues at positions 27, 85, and 98 that are also highly conserved; and a novel type of -helical structure with six helices connected by - loops. Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. Gong e Gene promoter sequence and transcription factor recognition site analysis are important for understanding regulation and feedback mechanisms in specific physiological processes. This study succeeded in isolating the 5 regulatory region of Pxyl-CSP3 and is the first report about the 5 upstream regulatory sequence of the insect chemosensory protein gene. This data revealed that the 5 regulatory region of Pxyl- CSP3 have a lot of specific transcription factor binding sites including BR-C Z4, Hb, Dfd, CF2-II, etc. The transcription factor binding site of BR-C Z4 has appeared many times in this regulatory region, which may play an important role for duplication and expression of Pxyl-CSP3. It has been reported that BR-C Z4 directly mediates the formation of the steroid hormone ecdysone for Drosophila melanogaster larvae metamorphosis (Kalm 1994). However, there is no direct evidence for the role of CSPs in insect metamorphosis, but some scientists reported that CSPs are expressed in the pheromonal gland of M. brassicae and the ejaculatory duct of D. melanogaster (Jacquin- Joly 2001; Sabatier 2003). A recent report also showed that the CSP homologue of Agrotis segetum has upregulation expression in the insect-pheromone binding domain; this CSP has also been reported to be the same as juvenile hormone binding protein (Strandh 2008). These findings are in line with the data from the transcription factor binding site analysis, as well as the high expression in the larval stage, which may implicate a function of Pxyl-CSP3 for steroid hormone production or transport in this insect larval stage. Chemosensory protein association with insect development has been confirmed by many scientists, especially in embryo development. For example, CSP5 of Apis mellifera is an ectodermal gene involved in embryonic integument formation (Maleszka 2007). In the This wide tissue expression pattern may indicate that CSPs have olfactory and non- olfactory functions. The data here shows that Pxyl-CSP3 and Pxyl-CSP4 have different expression profiles in different developmental stages and that they were all expressed in larval stage. So, it is suggested that Pxyl-CSP3 and Pxyl-CSP4 have important functions for early development of P. xylostella, but the detailed physiological role is still unknown. Gene promoter sequence and transcription factor recognition site analysis are important for understanding regulation and feedback mechanisms in specific physiological processes. Journal of Insect Science \| www.insectscience.org 11 Journal of Insect Science: Vol. 10 \| Article 143 Acknowledgments Gong DP, Zhang HJ, Zhao P, Lin Y, Xia QY, Xiang ZH. 2007. Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology 37: 266- 277. Gong DP, Zhang HJ, Zhao P, Lin Y, Xia QY, Xiang ZH. 2007. Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology 37: 266- 277. The work was supported by grants from the China National Nature Science Foundation (No. 30671387 and 30770291) and Foundation for the Author of National Excellent Doctoral Dissertation of P R China (FANEDD, No.200461). Gogos JA, Hsu T, Bolton J, Kafatos, FC. 1992. Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc finger domain. Science 257: 1951-1955. Journal of Insect Science: Vol. 10 \| Article 143 This data (Figure 5) corresponds to those sequence and structure characteristics as confirmed by multiple sequence alignment. Homology and phylogenetic tree analysis indicated that CSPs from the species of P. xylostella are more similar to CSPs from other species than to some members of its own, suggesting evolutionary divergence in CSPs of P. xylostella. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 12 Journal of Insect Science: Vol. 10 \| Article 143 Gong et al. chemosensory protein (ASP3c) from honeybee (Apis mellifera L) as a brood pheromone carrier. European Journal of Biochemistry 269: 4586-4596. cockroach Periplaneta americana, the CSP p10 increases transiently during limb regeneration at the larval stages (Kitabayashi 1998).The transcription factor binding sites of Hb, Dfd, and CF2-II have been shown to be involved in developmental regulation; for instance, Hb regulates gene expression in the development of the thoracic region of Drosophila embryos (McGregorl 2001), and CF2 may potentially regulate distinct sets of target genes during development (Gogos 1992). This study will provide clues to better understand the function of CSPs in insect development. Corpet F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Research 16: 10881-10890. Danty E, Arnold G, Huet JC, Huet D, Masson C, Pernollet JC. 1998. Separation, characterization and sexual heterogeneity of multiple putative odorant-binding proteins in the honeybee Apis mellifera L. (Hymenoptera: Apidea). Chemical Senses 23: 83-91. 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Diversity of odorant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae. Insect Biochemistry and Molecular Biology 8: 501- 518. Zhou JJ, Kan YC, Antoniw J, Pickett JA, Field LM. 2006. Genome and EST analyses and expression of a gene family with putative functions in insect chemoreception. Chemical Senses 31: 453-465. Sabatier L, Jouanaguy E, Dostert C. 2003. of Insect Biochemistry and Physiology 66: 135-145. Pherokine-2 and -3 two Drosophila molecules related to pheromone/odor-binding proteins induced by viral and bacterial infections. European Journal of Biochemistry 270: 3398- 3407. Sabatier L, Jouanaguy E, Dostert C. 2003. Pherokine-2 and -3 two Drosophila molecules related to pheromone/odor-binding proteins induced by viral and bacterial infections. European Journal of Biochemistry 270: 3398- 3407. Strandh M, Johansson T, Ahrén D. 2008. Transcriptional analysis of the pheromone gland of the turnip moth, Agrotis segetum (Noctuidae), reveals candidate genes involved in pheromone production. Insect Molecular Biology 17: 73-85. Strandh M, Johansson T, Ahrén D. 2008. Transcriptional analysis of the pheromone gland of the turnip moth, Agrotis segetum (Noctuidae), reveals candidate genes involved in pheromone production. Insect Molecular Biology 17: 73-85. Steinbrecht RA. 1998. Odorant-binding proteins: Expression and function. Annals of the New York Academy of Sciences 855: 323- 332. Tuccini A, Maida R, Rovero P, Mazza M, Pelosi P. 1996. Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea). Insect Biochemistry and Molecular Biology 26: 19- 24. Steinbrecht RA. 1998. Odorant-binding proteins: Expression and function. Annals of the New York Academy of Sciences 855: 323- 332. Steinbrecht RA. 1998. Odorant-binding proteins: Expression and function. Annals of the New York Academy of Sciences 855: 323- 332. Tuccini A, Maida R, Rovero P, Mazza M, Pelosi P. 1996. Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea). Insect Biochemistry and Molecular Biology 26: 19- 24. Tuccini A, Maida R, Rovero P, Mazza M, Pelosi P. 1996. Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea). Insect Biochemistry and Molecular Biology 26: 19- 24. Journal of Insect Science \| www.insectscience.org Journal of Insect Science \| www.insectscience.org 15

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